The additional value of first pass myocardial perfusion imaging during peak dose of dobutamine stress cardiac MRI for the detection of myocardial ischemia

Purpose of this study was to assess the additional value of first pass myocardial perfusion imaging during peak dose of dobutamine stress Cardiac-MR (CMR). Dobutamine Stress CMR was performed in 115 patients with an inconclusive diagnosis of myocardial ischemia on a 1.5 T system (Magnetom Avanto, Siemens Medical Systems). Three short-axis cine and grid series were acquired during rest and at increasing doses of dobutamine (maximum 40 μg/kg/min). On peak dose dobutamine followed immediately by a first pass myocardial perfusion imaging sequence. Images were graded according to the sixteen-segment model, on a four point scale. Ninety-seven patients showed no New (Induced) Wall Motion Abnormalities (NWMA). Perfusion imaging showed absence of perfusion deficits in 67 of these patients (69%). Perfusion deficits attributable to known previous myocardial infarction were found in 30 patients (31%). Eighteen patients had NWMA, indicative for myocardial ischemia, of which 14 (78%) could be confirmed by a corresponding perfusion deficit. Four patients (22%) with NWMA did not have perfusion deficits. In these four patients NWMA were caused by a Left Bundle Branch Block (LBBB). They were free from cardiac events during the follow-up period (median 13.5 months; range 6–20). Addition of first-pass myocardial perfusion imaging during peak-dose dobutamine stress CMR can help to decide whether a NWMA is caused by myocardial ischemia or is due to an (inducible) LBBB, hereby preventing a false positive wall motion interpretation.
Introduction
Dobutamine stress Cardiac MRI (CMR) is used to detect myocardial ischemia of the left ventricle by means of wall motion analysis during the infusion of high-dose dobutamine [1–4]. Previous studies have reported a broad range of sensitivity (83–91%) and specificity (80–86%) of dobutamine stress CMR for the detection of myocardial ischemia [1–3, 5]. It has been proven to be more accurate than dobutamine stress echocardiography [2]. The addition of myocardial tagging even further increased the sensitivity (96%) of dobutamine stress CMR [4]. False positive dobutamine stress CMR’s were described for left bundle branch block (LBBB) or an incidental low interpretability [4]. Means to overcome for these false-positive dobutamine stress CMR’s could increase specificity even further.
Assessment of myocardial perfusion is used to provide information on the hemodynamic significance of a coronary artery stenosis. Segments with New Wall Motion Abnormalities (NWMA) detected with dobutamine stress CMR should also show perfusion deficits, since perfusion abnormalities precede wall motion abnormalities in the ischemic cascade [6].
Normal stress perfusion SPECT-results predict a less than 1% annual risk of cardiac death or myocardial infarction, thereby yielding a high negative predictive value [7–10]. High negative predictive values are also reported for MR perfusion imaging combined with MRI cine-angiography (100%) in a small study of 15 patients [11].
The addition of a perfusion sequence on peak-dose dobutamine may further enhance the interpretation of dobutamine stress CMR, by ruling out false positive findings, through the combined strength of both methods.
The purpose of this study is to assess whether the addition of perfusion imaging to dobutamine stress CMR at peak-dose dobutamine reduces the number of false-positive dobutamine stress CMR examinations.
Materials and methods
Patient population
Between September 2004 and April 2006, 124 consecutive patients were referred from the department of Cardiology for a dobutamine stress CMR. The study was approved by the local ethical committee. Informed consent was obtained prior to the study, after the nature of the procedure had been explained. All patients had chest pain and an inconclusive diagnosis of coronary artery disease by means of history, ECG recordings at rest and, if performed, during a bicycle exercise test. Patients with an acute coronary syndrome, atrial fibrillation, severe arterial hypertension (>220/120), CMR-incompatible metallic implants or known claustrophobia were not eligible.
Protocol for dobutamine stress CMR with myocardial perfusion on peak-dose dobutamine
To ensure cardiac response to dobutamine, all anti-anginal medication was stopped 4 days before the dobutamine stress CMR examination. After the patient was positioned on the scanning table, intravenous access was established via an anticubital vein. ECG monitoring leads, a phased-array surface coil covering the heart, and a brachial blood pressure cuff were applied. A single lead ECG was continuously monitored on the MRI-console. Systolic and diastolic blood pressures were recorded using an automatic device (Welch-Allyn, Emro-medical) at baseline and every 3 min throughout the procedure. Blood pressure and heart rate were recorded. The imaging methodology of dobutamine stress CMR has been described in detail previously [4]. Dobutamine stress CMR was performed on a 1.5 T system (Magnetom Avanto, Siemens Medical Systems, Erlangen, Germany). Three short-axis cine breath-hold CMR images of the left ventricle, with and without myocardial tagging, were acquired at rest and during incremental dosage of dobutamine up to 40 μg/kg/min. An ECG-triggered segmental gradient-echo pulse sequence was used: TrueFisp: TR 57.64, ms; TE, 1.1 ms; α, 59°; FOV, 284 × 350 mm; slice-thickness 6 mm; and matrix 125 × 192; iPAT 2. Tagging was performed with a standard FLASH grid-sequence: TR, 46 ms; TE, 3.8 ms; α, 14°; FOV, 284 × 350 mm; slice-thickness 6 mm and matrix 141 × 256. The basal plane was taken 1.5 cm below the mitral valves. The midventricular and apical short-axis views were divided equally over the remaining part of the left ventricle. When a wall motion abnormality (WMA) was detected at baseline, infusion was started at 5 μg/kg/min, after which the dose of dobutamine was increased to 10, 20, 30 and 40 μg/kg/min. Starting dose of dobutamine was 10 μg/kg/min when no WMA was detected at baseline. Imaging started 6 min after each dose increase and required 3 min per dose increase.
Termination criteria for dobutamine stress CMR were the development of new wall motion abnormalities (NWMA) or worsening WMA, a fall of systolic blood pressure of more than 40 mmHg, marked hypertension above 240/120 mmHg, severe chest pain, ventricular arrhythmias and intolerable side effects. NWMA are indicative of myocardial ischemia.
On peak-dose dobutamine a bolus injection of 0.1 mmol/kg gadolinium-DTPA (Omniscan®) was given and a perfusion sequence was started: TrueFisp: TR, 150.5 or 163.1 ms; TE 1.03 ms; TI 100/103 ms; α 45/50°; FOV 300 × 300; slice-thickness 6 mm; matrix 76 × 128; iPAT 2. The same three slices as the cine and tagging series were acquired.
During the examination a radiologist present in the MR suite to monitor the condition of the patient and to visually evaluate the images. When NWMA’s with a corresponding perfusion deficit were seen, a coronary angiography (CAG) was performed within 3 weeks. Patients with NWMA’s and a normal first pass perfusion on peak stress entered follow-up.
Image analysis
Wall motion was scored on six segments of the basal plane, six on the midventricular plane and four on the apical plane. Segmental wall motion was qualitatively graded as 1 = normal or hyperkinesis; 2 = hypokinesis; 3 = akinesis; and, 4 = dyskinesis. Myocardial ischemia was defined as a new (induced) or worsening WMA in at least two segments at consecutive planes of the left ventricle during infusion of dobutamine. Analysis was performed using both cine and tagging images as described in detail previously [4].
Wall Motion Score Index (WMSI) was derived as the mean score of all segments of all short-axis images. WMSI data from the combined analysis of the cine and tagging images were determined from baseline and peak stress images.
The first pass perfusion images on peak-dose dobutamine were visually analyzed by an experienced radiologist and cardiologist in a consensus reading after the wall motion analysis by the same physicians. A perfusion abnormality, corresponding to the coronary artery distribution areas, in at least two segments at consecutive short-axis slices or one segment of the most apical slice of the left ventricle was defined as myocardial ischemia.
Follow-up
Follow-up data were obtained in September 2006. The present status of the patient was determined by review of the hospital records or contacting the patient’s general physician. The date of the last review was used to calculate follow-up time.
Evaluated end points were non-fatal myocardial infarction (angina of >30 min duration and either 2 mm ST segment elevation in two consecutive ECG leads or a rise in creatine kinase level and its myoglobine fraction two times the upper limit of normal), cardiac death (death in the presence of acute myocardial infarction, significant cardiac arrhythmias or refractory congestive heart failure) and coronary revascularization.
Results
Patient population
From the 124 consecutively included patients, in nine patients the examination could not be completed due to: intolerable side effects (nausea, vomiting) in four patients, claustrophobia in three patients. Two patients were excluded due to insufficient image quality, one of whom had an irregular rhythm with triggering difficulties, and the other patient was unable to sustain breath holds.
Therefore, 115 patients all with good image quality were analysed (93%) with a mean age 61 ± 11 years, 20 women (30%). Demographic and hemodynamic data are listed in Table 1.
Table 1Demographic and hemodynamic dataVariableMean or %Age, years61 ± 11Female, %29.6Previous myocardial infarction, %38.0Revascularization, %31.0Rest wall motion abnormalities (RWMA), %40.9Body weight, kg78 ± 12Resting diastolic blood pressure, mmHg87 ± 11Peak diastolic blood pressure, mmHg78 ± 12Resting systolic blood pressure, mmHg152 ± 26Peak systolic blood pressure, mmHg151 ± 31Resting heart rate, bpm79 ± 15Peak heart rate, bpm119 ± 21Rate-pressure producta at rest12,030 ± 3,545Rate-pressure product at peak stress17,935 ± 4,807Wall Motion Score Index (WMSI) at baseline1.18 ± 0,32Wall Motion Score Index (WMSI) at peak dose1.21 ± 0.34Values are expressed as mean ± SD or percentageaRate-pressure product = (heart rate) × (systolic blood pressure)RWMA = Rest Wall Motion Abnormality; WMSI = Wall Motion Score Index
Dobutamine stress CMR with myocardial perfusion on peak-dose dobutamine
Eighteen of the 115 patients (16%) had NWMA of whom 14 (78%) showed perfusion deficits on peak-dose dobutamine in the corresponding segments. Four patients (22%) with NWMA did not have a perfusion deficit. In these four patients, NWMA were attributable to a LBBB as could be confirmed with an independent (stress) ECG. Two of these were inducible LBBB, not known prior to the examination. CAG was positive for the corresponding segments in the 14 patients (100%) with NWMA and a corresponding perfusion deficit.
Ninety-seven patients (84%) had no NWMA. The perfusion images on peak-dose dobutamine showed absence of perfusion deficits in 67 of these 97 patients (69%) and perfusion deficts in 30 (31%). Of these patients 29 (97%) were attributable to a known previous myocardial infarction in the patients history combined with the presence of RWMA. One patient (3%) had a small perfusion deficit inferior which could not be assigned to a known previous event in the patients history. Figure 1 illustrates the course and outcome of the study. In Fig. 2 the additional value of cine or grid tagging images combined with stress first-pass perfusion images is illustrated.
Fig. 1Flow chart illustrating course of the study and outcome NWMA = New Wall Motion Abnormality; LBBB = Left Bundle Branch Block; CAG = Coronary AngiogramFig. 2Short axis views at peak-dose dobutamine. Cine image illustrating a NWMA inferior. (A) Perfusion abnormality in the corresponding segment. (B) Dyskinetic septal wall in another patient. (C) No perfusion abnormalities in the corresponding segment. (D) Dyskinetic septal wall in C was due to a LBBB, this differentiation could be made by a perfusion sequence on peak-dose dobutamine. Arrows indicate the wall motion abnormality or perfusion abnormality NWMA = New Wall Motion Abnormality; LBBB = Left Bundle Branch Block
Follow-up results (median 13.5 months, range 6–20 months) were obtained from all patients with NWMA’s and a normal first-pass perfusion. None of these patients had cardiac events or revascularizations at follow-up.
Discussion
This study demonstrates that adding first-pass myocardial perfusion imaging during peak-dose dobutamine has direct clinical relevance for the interpretation of dobutamine stress CMR examinations, by increasing the interpretation of possible wall motion abnormalities (NWMA) of the left ventricle. The addition of first pass perfusion relies on the conceptual use of the ischemic cascade. Ischemic wall motion abnormalities are preceded by perfusion abnormalities, therefore visualized NWMA due to myocardial ischemia should also show perfusion abnormalities. A normal perfusion study is used to identify wall motion abnormalities not due to myocardial ischemia, and an abnormal corresponding perfusion deficit is used to confirm NWMA, indicative for myocardial ischemia.
This is the first study to assess the additional value of a first pass myocardial perfusion imaging sequence on peak-dose dobutamine during a dobutamine stress CMR. Previous studies have reported a broad range of sensitivity and specificity for dobutamine stress CMR in the detection of myocardial ischemia [4]. In this protocol we have chosen for a prolonged infusion time of dobutamine from 3 to 6 min without atropine and the use of the target heart rate rule, as described before [4, 12–17].
The use of the target heart rate rule from a physical exercise based concept can not be generalized to a pharmacological stress setting and has been questioned in several reports [12–14, 18]. It has also been shown that the target heart rate rule can not be extrapolated to a pharmacological stress examination [19]. In a recent overview of published dobutamine stress CMR examinations by Strach et al. [20], the approach we used [4] showed to provide the highest diagnostic accuracy for significant coronary artery disease defined by a >50% luminal stenosis on a coronary angiogram. Specificity, although already high, was lowered, according to our opinion, in part due to LBBB. This could be overcome with a perfusion sequence, taking into account the high negative predictive value of normal myocardial perfusion imaging. The additional value of myocardial perfusion MRI could be used for this purpose and may add significant diagnostic information.
In this study we added a perfusion sequence on peak-dose dobutamine in all patients. The results show a good agreement between the absence of NWMA and myocardial perfusion. The main purpose of this study was to assess the additional value of first-pass perfusion imaging in the presence of NWMA. Our recommendation for future clinical use is to add a first pass perfusion sequence on indication, namely if there is doubt on whether a NWMA is due to myocardial ischemia. In this way, the specificity of dobutamine stress CMR can be even further increased. By combining dobutamine stress CMR with myocardial perfusion, one still has the opportunity to examine for viability, which seems even superior to scar quantification [21].
Absolute specificity values cannot be given with this study, because a CAG was not performed in case of a negative dobutamine stress examination. The outcome of the dobutamine stress CMR and myocardial perfusion on peak-dose dobutamine was used as a direct arbiter for subsequent clinical follow-up. In this respect follow-up was considered the reference standard. None of the patients with NWMA’s and a normal myocardial perfusion had an adverse outcome at follow-up. Therefore, it is reasonable to state that adding first pass myocardial perfusion imaging on peak-dose dobutamine increases the specificity of dobutamine stress CMR in this patient group.
Our data could have been influenced by a referral bias, but in light of previous results, we found it not justifiable to perform a CAG in case of a negative examination [12]. Furthermore, the high negative predictive value of dobutamine stress CMR examination without perfusion imaging has been proven before [12, 22]. Mahrholdt et al. [23]. examined 139 patients with a LBBB. All 139 patients had fixed perfusion defects with SPECT, 19 could not be confirmed with CAG. Rest wall motion analysis and myocardial perfusion (between 5 and 30 min after contrast administration) was performed on these 19 patients using MRI. All 19 patients showed septal wall motion abnormalities, but none showed subendocardial or transmural contrast enhancement. This also indicates that contrast enhanced imaging can help differentiate between WMA on the basis of coronary artery disease or a LBBB.
Long term follow-up will provide information about the case in which a small perfusion abnormality was seen inferior without NWMA. Whether this abnormality is a “true” abnormality or an artefact is unclear. This patient did not have any adverse cardiac event at 14 months follow-up.
Another limitation of our study is the fact that images were analysed semi-quantitatively. Quantitative wall motion analysis could possibly provide additional information, but due to the time-consuming nature this is not yet feasible in clinical practice. Furthermore, no decisions can be made during the examination, which we believe is crucial, since overstressing may lead to serious complications [13].
Visual analysis of wall motion and perfusion images was performed in a consensus reading by an experienced radiologist and cardiologist. This can be regarded as a limitation since the observers are not blinded for the previous wall motion images. However, this does represent routine clinical practice and is fundamental to the underlying concept of using first-pass perfusion imaging in the presence of NWMA. In recent studies the inter-observer agreement of dobutamine stress CMR was investigated and good agreement was found [24, 25].
In a clinical post-infarct setting, delayed contrast enhancement imaging may provide valuable diagnostic information. To our opinion, this would in our study not have provided additional information regarding the fact whether a NWMA was caused by myocardial ischemia or a non-ischemic cause, and guide the necessity for an invasive coronary angiogram. Cine and grid tagging images were acquired at rest to look for rest wall motion abnormalities. New wall motion abnormalities in this setting would represent myocardial ischemia (whether or not in the presence of a previous myocardial infarction). Delayed contrast enhancement imaging was therefore not performed in this protocol.
In this protocol we only acquired stress first-pass perfusion images. Perfusion imaging at rest may be very useful in stress-rest perfusion imaging studies, but the main focus in this protocol is on a normal perfusion in case of NWMA. A rest perfusion exam would in this context not provide additional information (the rest perfusion exam will also be normal). In this way the protocol is not unnecessarily prolonged and a second bolus injection of gadolinium-DTPA can be omitted.
Conclusion
Good agreement exists between the absence of NWMA and a normal myocardial perfusion. Furthermore, a perfusion sequence on peak-dose dobutamine can help decide whether a NWMA is caused by ischemia or is due to a LBBB. First pass myocardial perfusion during peak-dose dobutamine can be used as an additional tool to reduce the number of false-positive NWMA’s, to improve the detection of myocardial ischemia.


Tree thinking cannot taken for granted: challenges for teaching phylogenetics

Tree thinking is an integral part of modern evolutionary biology, and a necessary precondition for phylogenetics and comparative analyses. Tree thinking has during the 20th century largely replaced group thinking, developmental thinking and anthropocentricism in biology. Unfortunately, however, this does not imply that tree thinking can be taken for granted. The findings reported here indicate that tree thinking is very much an acquired ability which needs extensive training. I tested a sample of undergraduate and graduate students of biology by means of questionnaires. Not a single student was able to correctly interpret a simple tree drawing. Several other findings demonstrate that tree thinking is virtually absent in students unless they are explicitly taught how to read evolutionary trees. Possible causes and implications of this mental bias are discussed. It seems that biological textbooks can be an important source of confusion for students. While group and developmental thinking have disappeared from most textual representations of evolution, they have survived in the evolutionary tree drawings of many textbooks. It is quite common for students to encounter anthropocentric trees and even trees containing stem groups and paraphyla. While these biases originate from the unconscious philosophical assumptions made by authors, the findings suggest that presenting unbiased evolutionary trees in biological publications is not merely a philosophical virtue but has also clear practical implications.
Introduction
Modern evolutionary biology relies on population thinking and tree thinking (O’Hara 1997). Both ways of thinking have replaced earlier philosophies that turned out to be incompatible with the empirical basis (Hull 1965; Simberloff 1980; Sober 1980). The transition from essentialism to population thinking has been described by Ernst Mayr (1988, 1996; but see Levit and Meister 2006; Winsor 2003). This transition followed directly from the Darwinian revolution (Darwin 1859): it is organisms with unique trait combinations that make up populations, that constitute biodiversity, and that are the basis of evolutionary change. The earlier paradigm, which viewed organisms as blueprints of unchangeable essences, was incompatible with these observations.
Equally important to modern biology is tree thinking. Each and every species occupies one specific place in the tree of life. Consequently all species have shared certain parts of their history with all other living and extinct species. The degree to which this history has been shared, allows us to predict the extent and way in which species differ from and resemble each other. A description of biodiversity is impossible or at least incomplete without taking this knowledge into account. It is necessary both for the idiographic activity of reconstructing evolutionary relationships between species, and for the nomothetic activity of testing hypotheses about biological laws of nature (Ghiselin 1997; Hull 1999). Phylogenetic reconstruction (Felsenstein 2004) and the comparative method (Martins and Hansen 1996) are thus entirely unthinkable without tree thinking.
Tree thinking became widely accepted somewhat later than population thinking, as it can be said to have originated with phylogenetic systematics (Hennig 1950). However, this does not mean that tree thinking has superseded population thinking (O’Hara 1997). Both perspectives are compatible, and both are necessary in biology today. As Robert O’Hara (1997) has shown, what has been replaced by tree thinking, are the earlier paradigms of group thinking and developmental thinking in evolutionary biology. Group thinking had equated systematics with classification, i.e. it perceived taxonomic groups as units that could be defined rather than entities that had to be discovered. Group thinking thus represented essentialism in systematics, because it failed to understand that taxa are evolving entities and thus, ontologically speaking, individuals (Ghiselin 1974, 1997; Hull 1976). Developmental thinking, on the other hand, had assumed that organic evolution was a teleological process (Ghiselin 2002; Hull 1974; Mayr 1988). It was thus based upon the ancient idea that change in nature has an ultimate goal. This is incompatible with the irregular and divergent nature of evolution.
One might presume that the replacement of group and developmental thinking by tree thinking is of merely historical interest—a subject to be dealt with by historians of science, just as the replacement of essentialism by population thinking. To the contrary, I here argue that the transition of both group thinking and developmental thinking to tree thinking is still happening, and that it deserves more attention by those who teach phylogenetics and who communicate phylogenies. The transition is still happening in the sense that students of biology have to accomplish it in the course of their “academic ontogenies”: tree thinking is an ability that has to be acquired and cannot be taken for granted by teachers. Sadly, many text books indirectly and implicitly promote developmental thinking. This further aggravates the problem. Given the importance (even necessity) of tree thinking in order to fully appreciate the meaning of evolutionary results, this problem deserves attention.
Materials and methods
I devised a short questionnaire which was handed out during the first lesson in several classes of a university course in systematics and evolution (at Universitetet i Tromsø, Norway). The course was intended for students who had studied biology for several terms, but this was not a formal requirement. The students who followed normal progression had been taught evolutionary principles and processes in earlier courses. The same questionnaire was used in a similar course at a regional university college (Høgskolen i Bodø, Norway). The students were asked to answer the questions, using the time they needed, and were assured anonymity. They did not have access to any books or notes.
The questions (and correct answers) were:“Given the following evolutionary tree [reproduced in Fig. 1], where letters symbolise species: which species is (are) most closely related to species B?”1(The correct answer was “C, D, E and F”.)“How many classes of vertebrates and angiosperms are there? Fill in the scientifically correct answer:...“(There is no scientifically correct answer to this question.)“What are the differences between species and genera, or between orders and classes? Give one or more keywords that describe these differences. (a) Differences between species and genera:...(b) Differences between orders and classes:...“(The correct answer was that species differ from genera [and orders and classes], for instance by being populations within which gene flow can occur. The difference between orders and classes is merely one of definition, none of nature.)“What does the ‘tree of life’ look like? Please draw the evolutionary tree which describes the relationships between groups of organisms. Include as many groups as you are able to recall”.Fig. 1Example phylogeny, where letters symbolise species. Not a single student (n = 31) was able to give the correct answer to the question “which species is/are most closely related to species B”
Results
Twenty one university students returned questionnaires that were at least partly answered. On average they had previously taken biology courses for 1.6 years of full time study load (median 1.5, range 0.3–4.0 years). Four were graduate students, the remainder were undergraduates. A further ten students of the regional university college returned the questionnaire.
Not a single correct answer was given to question 1. In most cases, species A (cf. Fig. 1) was the whole answer or part of the answer (17 of 20 university students, and 8 of 9 college students).
Of 20 answers to question 2, 18 consisted of numbers. One student entered strokes, and 1 left the answers blank.
In response to question 3, 5 of 18 respondents answered that species were fundamentally different from genera, while there was no such difference for orders and classes. The remainder answered that there was no important differences at all (n = 8) or that there were important differences between both species and genera, and between orders and classes (n = 5).
As an answer to question 4, 20 university students produced a drawing, however, 7 of them did not enter any names, and 1 entered one name only. The remainder provided between 4 and 23 taxon names. See Fig. 2 for an example.
Fig. 2Phylogenetic tree drawn by a university student. The question posed was “What does the ‘tree of life’ look like? Please draw the evolutionary tree which describes the relationships between groups of organisms. Include as many groups as you remember”. Note the prominent position of mammals and the unresolved “side branches”. Taxon names provided by the student were translated verbatim into English, the drawing itself is unchanged
Seven of 13 students depicted 1 to 9 extant taxa as stem groups in their drawings. A total of 14% of all names provided were drawn as stem taxa. 11 of 13 students drew 1 to 11 branches which they named with paraphyletic groups. A total of 31% of all names provided referred to paraphyla.
As regards the placement of our own species, or the taxon containing Homo sapiens, the placement of our taxon seemed arbitrary in four drawings, and in two cases no names provided referred to taxa that contained our species. Our taxon was placed at top of the drawing in five cases, and at the outermost position in two cases. This means that 7 of 13 respondents gave a special position to our taxon. Counting the number of nodes at which the path to H. sapiens leads to the uttermost or uppermost position, a continuous “human utterness score” can be calculated (Sandvik 2008). Combined for the eleven drawings to which it was applicable, the average score was 0.90 (43 of 48 nodes), which is highly significantly biased in favour of man (P < 10−8).
The resolution of the tree also favoured our species. I calculated scores of the relative attention the tree drawings devoted to three species, viz. H. sapiens (Mammalia), Vanessa atalanta (Lepidoptera) and Veronica fruticans (Lamiales), in the following manner: a score of 1 was assigned if the species was named, of 2 if at least the “order” was named, of 3 if at least the “class” was named, of 4 if at least the “phylum” was named, of 5 if a higher taxon was named, and of 6 if no named branch contained the species. The average attention score was 3.6 ± 1.5 (SD) for man, 4.8 ± 1.2 for the butterfly, and 4.6 ± 1.3 for the flower. A one-tailed Wilcoxon two-sample tests showed that man received more attention than the other two species (W = 155.5, P < 10−5). If using the “human attention score” defined elsewhere (Sandvik 2008), a continuous measure that expresses the degree to which tree resolution favours our own species, the average for the 13 tree drawings was 0.60, which is highly significantly biased in favour of man (P < 10−4).
Discussion
O’Hara (1997:327) remarked that “beginning students in biology should be taught how to read trees [...] just as beginning students in geography need to be taught how to read maps”. My findings drastically support this opinion. None of the undergraduate or graduate students in my (admittedly small) sample was able to correctly interpret a simple cladogram (Fig. 1). The cladogram was certainly not the first evolutionary tree that the students had met, since most had studied biology for several terms, including introductory courses in evolutionary biology.
It does not require many sentences to explain how a cladogram should be read. The concept of evolutionary relatedness is not hard to grasp once it is explained. If it is not, however, erroneous conceptions seem to be able to survive for several years during prospective biologists’ educational careers. At the end of my course all university students were able to give correct interpretations of cladograms. Matters differed for the students of the regional university college. These students were asked which of the small cladograms in Fig. 3b–i were compatible with the large cladogram (Fig. 3a). This latter question was posed after the meaning of cladograms had been explained and after a considerable amount of time had been spent practicing the reading of cladograms. Still, only one answer out of fourteen correctly identified all of the three compatible cladograms. The remainder failed to identify at least one compatible cladogram and/or indicated at least one incompatible tree as compatible. This sobering result might be related to the fact that many of the college students simply needed some biology courses in order to study, e.g. fishery. They might thus not have been genuinely interested in biology per se, so that these findings would need to be replicated with university students. Still, it is independent evidence that reading cladograms is very much an acquired ability rather than one that can be presupposed.
Fig. 3Nine phylogenetic trees over Mollusca with different topologies. a was used as a reference phylogeny. Students were asked which of the eight small trees were compatible with the reference tree. The eight topologies b–i differ from a in either the phylogenetic relationships displayed (making them incompatible with a), resolution of some of the branches, placement of taxa along the left–right axis, or a combination of those (only c, g and h are compatible with a. In the tree distributed to students, all names were given as unabbreviated Norwegian vernacular names)
In their answers, the students proved to pay more attention to how the taxon names at the tree tips were ordered along the left–right axis, than to the topology of the cladogram. Given that the information of cladograms is conveyed in the branching order of taxa, while the left–right ordering is arbitrary, this wrong focus necessarily leads to wrong conclusions.
The main problem is presumably that students simply have not been taught how to read cladograms. Teachers may often assume that cladograms are self-explicatory graphic devices to illustrate phylogenetic relationships, when in fact their interpretation is not trivial. As Halstead (1978:760) put it: “Cladograms are difficult enough for experts in the field to comprehend fully”. Ironically, what he was advocating as the obvious alternative was phylograms—which were meant to not only express phylogenetic relationships, but, in addition, phenotypic similarity, distribution through geologic time and species number. As such, phylograms are not only harder to interpret for untrained readers than cladograms. They may also convey incorrect information on any one of the aspects because they represent a two-dimensional compromise of several multidimensional measures.
Regrettably, the presentation of systematics in text books has been lagging behind the cutting edge of systematic research by several decades. It is thus only quite recently that most biological text books have replaced phylograms with cladograms. A less appreciated fact is that even displaying cladograms does not guarantee that the information provided is unbiased, as many cladograms are drawn in an anthropocentric fashion (Sandvik 2008). It might thus well be that one source of confusion for students is the ambiguity of evolutionary tree drawings in text books. Drawings of evolutionary trees cannot be clearer than their authors’ thoughts about evolutionary processes. And the latter have long been rather muddled, as can be exemplified by the occurrence of “stem groups” in many text book trees (e.g. Villee et al. 1984: Fig. 19.6; Willmer 1990: Fig. 14.2, which has even been reprinted by a number of other text books). However, stem groups have never existed in nature. A taxon above the species level cannot possibly give rise to other taxa—nor to anything else (Ghiselin 1997). One might say, therefore, that stem groups are just another way of expressing (or, worse still, trying to hide) ignorance. That some taxonomists have chosen to even give scientific names to their ignorance (e.g. “Procoelomata”; Bergström 1989), does not really make the matter more transparent for biologists under education. No wonder, then, that stem groups occurred in 54% of the student drawings.
One problem is thus that many tree drawings which students meet during their studies are biased and thereby distort the evolutionary understanding of their readers. Another is that this distorted understanding even impinges on the interpretation of correctly drawn phylogenetic trees. In other words, both problems re-enforce each other. I have demonstrated elsewhere that even cladograms in phylogenetic text books are biased in an anthropocentric way (Sandvik 2008). In terms of ordering of taxa and differential resolution of branches, otherwise correct cladograms may distort the understanding of evolution. These findings make the evidence presented here even more alarming: If taxa are ordered in an anthropocentric manner in most cladograms, and if students rely more on ordering than topology, this affects the interpretation of relationships. The taxon containing our own species is most often placed in the rightmost position in cladograms (Sandvik 2008). This can be illustrated with Fig. 4, which displays the phylogeny of Osteognathostomata in an anthropocentric manner. The topology of the cladogram is the same as in Fig. 1. Accordingly, many untrained student of biology can be expected to conclude that coelacanths are more closely related to ray-finned fishes than to mammals.
Fig. 4Cladogram depicting the phylogeny of bony vertebrates (Osteognathostomata). The cladogram is anthropocentric in that both the resolution of its branches and the ordering of taxon names are biased in favour of mammals. Note that the tree topology is identical to Fig. 1. See text for further details
The students’ inability to answer question 1 (Fig. 1) might be argued to have other reasons. An obvious alternative explanation would be that the error was caused by a simple misunderstanding of the word “relationship”, and that it is sufficient to tell students that relationship, as used in evolutionary biology, is a technical term meaning “the relative recency of common ancestry” (Mayr 1974). However, the question to the students was posed in Norwegian, were the word “slektskap” only has the latter meaning. In other words, while knowledge of the precise meaning of “relationship” is necessary (especially in English), it is not sufficient for students to understand cladograms.
In passing I would like to mention a speculation on the reason why phylogenetic systematics and cladistic methodology was rather quickly accepted in Germany (Ax 1977; Remane 1956; Schlee 1969), but provoked intense debates in English-language journals (verifiable with almost any issue of Systematic Zoology from the 1970s). I suspect that part of the problem was semantic. The German word for “relationship” is “Verwandtschaft”, but while the English word has all kind of abstract and symbolic connotations, including overall similarity, the German term is reserved for true, genealogical bonds (as is the Norwegian “slekt”, see footnote 1). The statement that for instance the lungfish is more closely “verwandt” to the cow than to the salmon is quite uncontroversial in German. On the other hand, the statement that the lungfish is more closely related to the cow than to the salmon, was able to create a heated discussion—which was only peripherally concerned with the actual phylogeny of the groups concerned (Gardiner et al. 1979; Halstead 1978; Halstead et al. 1979).
A final observation concerns the importance attributed to Linnean categories (i.e. labels such as “family”, “order”, etc.) by students. It is well-established that Linnean categories above the species level do not carry information, that they are not comparable across taxa, and that it is entirely arbitrary to which taxa they are assigned in the first place (de Queiroz and Gauthier 1992; Donoghue 2001; Ereshefsky 1994, 2001, 2002). Many taxonomists have abandoned categories altogether, simply referring to taxa by their names. However, few students are aware of the arbitrariness of Linnean categories. In many undergraduate text books, the enumeration (!) of animal “phyla” or insect “orders” seems to be more important than the discussion of interrelationships between the taxa concerned (e.g. Barnes et al. 1998). This leaves students with the impression that categories must express something. Unless told otherwise, they tend to make up their own explanations. As my results indicate, only a tiny proportion of students seems to question the existence and reality of Linnean categories. Of course, blank answers to question 2 do not necessarily indicate the deliberate rejection of categories—the two students may also have wished to give a number, but were unable to “recall the correct one”. On the other hand, question 2 was clearly a leading (not to say, loaded) question, which may have biased the respondents into thinking that a number was the only acceptable answer. Still, findings from question 3 show that at least 27% of the students perceive the distinction between “orders” and “classes” to be a reflection of real differences between natural levels of organisation.
The findings presented here re-enforce earlier reports that reading cladograms is an ability that has to be practiced (O’Hara 1992, 1997). Even though the transition from developmental thinking to tree thinking is more or less completed in the science of systematics at the collective level, it has to be accomplished anew by every generation of biology students on the individual level. This must not be forgotten when teaching phylogenetics or writing or illustrating text books. Other topics that need to be addressed explicitly are the artificiality of Linnean categories, the non-existence of stem taxa, and the arbitrariness of paraphyla. It does not seem to be sufficient to “get the facts right” in teaching and text books. Students tend to fall back to group and developmental thinking unless explicitly told otherwise.

group thinking
evolutionary tree
stem group
essentialism
cladogram
biological education


Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray

Metastatic colorectal carcinomas (CRC) resistant to chemotherapy may benefit from targeting monoclonal therapy cetuximab when they express the epidermal growth factor receptor (EGFR). Because of its clinical implications, we studied EGFR expression by immunohistochemistry on tissue sections of primary CRC (n=32) and their related metastases (n=53). A tissue microarray (TMA) was generated from the same paraffin blocks to determine whether this technique could be used for EGFR screening in CRC. On tissue sections, 84% of the primary CRC and 94% of the metastases were EGFR-positive. When matched, they showed a concordant EGFR-positive status in 78% of the cases. Moreover, staining intensity and extent of EGFR-positive cells in the primary CRC correlated with those observed in the synchronous metastases. On TMA, 65% of the primary CRC, 66% of the metastases, and 43% of the matched primary CRC metastases were EGFR-positive. There was no concordant EGFR status between the primary and the metastatic sites. A strong discrepancy of EGFR status was noted between TMA and tissue sections. In conclusion, EGFR expression measured in tissue sections from primary CRC and their related metastases was found to be similar and frequent, but it was significantly underestimated by the TMA technique.
Introduction
Epidermal growth factor receptor (EGFR), also called HER1, is a member of the transmembrane tyrosine kinase receptor family. In normal and malignant cells, the activation of EGFR receptor cascades has multiple consequences, such as cell growth, differentiation, and proliferation [10]. The EGFR signaling pathway may also promote malignant transformation, angiogenesis, and metastatic dissemination [10]. To block the activation of this receptor, targeted therapies have been developed, representing a new and promising strategy for cancer management [15]. Thus, antibodies directed against EGFR have recently been integrated into the treatment of metastatic colorectal carcinomas (CRC) resistant to chemotherapy [3, 23]. Such a strategy requires an EGFR immunohistochemical assessment by the pathologist to ensure that the targeted receptor is present in the tumor. Cancer patients included in these protocols are not always treated in the same institute for their primary disease and for their metastatic disease. Moreover, metastases are not always available for histology. It is thus possible that EGFR expression be evaluated in only one of the tumor locations (i.e., primary or metastatic). However, few and controversial data are available concerning the EGFR status in its primary site and the related metastatic sites [6, 11, 13, 24].
The first goal of our study was to analyze whether EFGR expression in colorectal primary tumors and their metastatic sites was similar or not. To answer this question, formalin-fixed and paraffin-embedded primary CRC from 32 patients and their related synchronous metastases (n=45) were examined using a standardized EGFR immunohistochemical procedure. Additionally, metachronous metastases (n=8) from four out of 32 patients were also processed for EGFR immunohistochemistry.
The second goal of this study was to evaluate EGFR expression on a tissue microarray (TMA) generated from the same paraffin blocks and to compare these results with those obtained by using the whole tissue sections. This comparison was carried out to determine whether the increasingly used TMA technology is a reliable tool for high-throughput EGFR screening in CRC [17, 26].
Materials and methods
Patient characteristics
Clinical and histopathological characteristics of the patients are reported in Table 1. Thirty-two patients, having undergone surgical resection of both the primary tumor and the corresponding metastatic sites in our institution between 2000 and 2004, were selected from a pathological database of colorectal cancer cases. There were 15 men (47%) and 17 women (53%). Median age at surgery was 60 years (ranging from 45 to 81 years). Twenty-one patients (66%) had colon cancer and 11 (34%) had rectal cancer. The most common histological type was adenocarcinoma (91%), the remaining three cases (9%) being mucinous carcinomas. According to the latest TNM classification [28], all the patients had stage-IV disease because of synchronous metastases at the time of diagnosis. Among them, four patients received neoadjuvant radiotherapy (n=2) or radiochemotherapy (n=2) for rectal carcinomas and six others received neoadjuvant chemotherapy before surgical resection of their metastases. Eighty-five paraffin-embedded tumor samples from CRC (n=32) and liver, pulmonary, and ovarian metastases (n=51, 1, and 1, respectively) were available. Synchronous metastases (n=45) were unique in 23 patients and multiple (n=22) in nine patients, consisting of two, three, or four locations in the liver (Table 1). Metachronous metastases (n=8) from four patients were also collected and assessed for EGFR immunohistochemistry. They consisted of five multiple liver metastases for one patient, one single liver metastasis for two patients, and one pulmonary metastasis for one patient. Synchronous and metachronous metastases were obtained by metastasectomy. All resected samples were received fresh, then immediately fixed in 10% pH neutral formalin for 48 h and embedded in paraffin before processing.
Table 1Clinical data and EGFR status among primary CRC and their related metastasesClinical dataEGFR statusa on tissue sectionsCase no.SexAge (years)pTNMLocationNeoadjuvant treatmentPrimary siteSynchronous metastases (location number)Metachronous metastases (location number)1234123451M60T3N1M1C+++++2M60T3N2M1RRCT++++3M66T2N0M1R++++4M49T3N2M1RCT++++++++5M70T3N1M1C+++6F74T3N1M1RRCT+++7F59T3N2M1RCT+++−8M71T3N2M1C+++9F55T3N2M1R−+−10F61T4N1M1CCT+++11F68T3N1M1CCT+++12M75T3N1M1R++13M72T3N1M1C++14M54T3N0M1C++15F55T4N1M1C++16F57T4N2M1C++17F52T3N1M1R++18M53T3N0M1C++19M62T4N2M1C++20F77T3N2M1C++21F49T3N1M1C++22F51T3N2M1C++23M62T3N2M1RRT++24F65T3N1M1CCT++25M56T4N1M1CCT++26F47T3N1M1RRT++27F59T3N1M1C+−28F45T3N2M1C+−29F81T3N1M1R−+30M58T2N0M1C−+31M69T3N1M1C−+32F68T3N2M1C−+Liver metastases in all cases except for a metachronous pulmonary metastasis and a synchronous ovarian metastasis in patients 7 and 15, respectivelyC colon, R rectum, RCT radio-chemotherapy, CT chemotherapy before surgical resection of metastasis, RT radiotherapyaPositive if >1% of tumor cells expressed EGFR
EGFR immunohistochemistry
EGFR immunohistochemistry was carried out on freshly sectioned tissue slides which has been shown to be critical for optimal EGFR immunoreactivity [1]. Immunohistochemistry of the tumors was performed by using the Dako autostainer and the EGFR pharmDx™ kit (K1494, Dako, Glostrup, Denmark), according to the manufacturer’s instructions. Briefly, 4-μm-thick sections were mounted on silanized slides and allowed to dry overnight at 37°C. After deparaffinization and rehydratation, slides were incubated with 3% hydrogen peroxide for 5 min. After washing with the supplied buffer, tissue sections were covered for 5 min with a proteinase K solution. The slides were then incubated for 30 min with the primary mouse anti-EGFR monoclonal antibody (clone 2-18C9), which binds to a formalin-resistant epitope near the ligand-binding site on the extracellular domain of EGFR. After two rinses in buffer, the slides were incubated with a horseradish peroxidase-labeled polymer coupled to secondary antibodies for 30 min. Tissue staining was visualized with diaminobenzidine as substrate-chromogen solution. Slides were counterstained with hematoxylin, dehydrated, and mounted. Negative control sections were processed without the primary antibody but with an irrelevant murine IgG1 supplied with the kit. Negative and positive control cell slides (CAMA-1 and HT29 cell lines, respectively), provided with the EGFR pharmDx™ kit, were also used to ensure that each assay run was performed appropriately and according to protocol specifications. Furthermore, perineurium and normal mucosae on primary CRC and hepatocytes surrounding the metastases were considered as positive internal controls on tumor slides.
Tissue microarray
Tissue blocks appearing to have enough material upon gross inspection were initially selected and hematoxylin–eosin-stained sections were evaluated by two observers (F. Bibeau and F. Boissière-Michot) for the presence of carcinoma. The areas to be used for the construction of the TMAs were marked on the slide and the donor block. Particular attention was made to select invasive cells near the tumor front. The tissues corresponding to selected areas were sampled using a manual arraying instrument (Manual Tissue Arrayer 1, Beecher Instruments, Sun Prairie, WI, USA). Two TMAs were constructed using 0.6-mm tissue cores. The sampling consisted of three malignant cores from different areas of the tumor from a single case of invasive colorectal cancer, placed at specified coordinates. When possible, normal mucosa was also sampled as internal control. After the arraying was completed, TMA blocks were sectioned at a thickness of 4 μm. One section was stained with hematoxylin–eosin, and EGFR immunostaining using the immunohistochemical system kit EGFR pharmDx™ was performed on the adjacent section. Among the 32 cases sampled, 31 primary CRC, 29 metastatic sites, and 28 matched sites were assessable with this technology.
EGFR immunohistochemical staining scoring system
EGFR assessment was realized according to the EGFR pharmDx™ scoring guidelines. EGFR expression was evaluated on sections including the deepest region of tumor invasion as this region was shown to contain the greatest density of EGFR-positive cells [6]. Results were reported as positive when a complete or incomplete circumferential membrane staining was observed in at least 1% of the tumor cells. Staining was defined as immunostaining of tumor cell membranes above background level and scored as follows: 1+=weak, 2+=moderate, and 3+=strong. The absence of membrane staining or cytoplasmic staining was reported as negative. The percentage of stained cells was assessed as follows: 1–10, 10–50, and >50%. Slides were scored by two pathologists (F. Bibeau and J.C. Sabourin), who were blinded to the patients’ characteristics. In the case of disagreement (three out of 91 specimens), the EGFR status was determined by consensus after simultaneous dual reexamination.
In addition to these standardized criteria, a semiquantitative analysis was performed taking into account both the staining intensity and the percentage of positive tumor cells.
Statistical analysis
The cut-off value for negative/positive expression of EGFR was 1% of tumor cells displaying a membranous staining, as specified by the EGFR pharmDx™ scoring guidelines. Data were summarized by frequency and percentage for categorical variables and by means, standard deviations, median, and range for continuous variables. For TMA analysis, the percentage of positive cells was calculated from the mean of triple cores.
The correlation between the percentage of positive cells observed on primary tumors and matched metastases was evaluated with the Spearman correlation coefficient. Associations between categorical variables were examined using McNemar’s chi2 test. A P value of less than 0.05 was considered statistically significant. Due to the small number of patients with metachronous metastases, only the correlation between the matched primary CRC and the synchronous metastatic sites was made.
Results
EGFR expression of primary CRC and their related metastases on tissue sections
EGFR reactivity was not homogeneous throughout the tumors. EGFR immunostaining was mainly observed in the deepest region of the primary CRC or at the periphery of metastases, especially in the liver. Of note, EGFR immunoreactivity was often greater in isolated tumor cells and in small clusters of tumor cells.
Eighty-four percent of primary CRC (27 out of 32) and 94% of the synchronous metastases (30 out of 32) displayed EGFR reactivity in more than 1% of the tumor cells (Table 1). Overall, all patients displayed an EGFR-positive status considering EGFR-expression in the primary tumor and/or the metastatic sites. EGFR expression was positive at both sites in most of the cases (25 out of 32; 78%) and rarely in only one site, i.e., primitive (two out of 32; 6%) or metastastic (five out of 32; 16%). However, the discordant cases (i.e., positive primary sites with negative metastasis and conversely) were not statistically significant (P=0.453).
Nine (28%), 10 (31%), and eight (25%) of the primary CRC and seven (22%), 14 (44%), and nine (28%) of the synchronous metastases displayed a weak, moderate, and strong positivity, respectively. According to the extent of EGFR immunoreactivity, five (16%) primary CRC had no reactivity, 12 (37.5%) had <10% reactive cells, eight (25%) had >10–50% positive cells, and seven (22%) had >50% labeled cells. In the synchronous metastases, two cases (6%) were negative, 13 (41%) had <10% reactive cells, 11 (34%) had >10–50% positive cells, and six (19%) had >50% labeled cells.
Interestingly, the percentage of EGFR-positive tumor cells in the primary site was significantly correlated with that observed in the synchronous metastases (r2=0.616, P=0.0002). This correlation was also demonstrated when combining EGFR scoring (labeling intensity) with the percentage of EGFR-positive tumor cells (r2=0.559, P=0.0009). One example of the pattern of EGFR labeling in both sites is shown in Fig. 1. EGFR expression was positive among the multiple synchronous liver metastases in eight out of nine patients (i.e., 21 out of 22 samples, Table 1). The related primary site of the discordant case (two synchronous metastases: one was EGFR-positive, the other was EGFR-negative) displayed an EGFR-negative status (case number 9, Table 1).
Fig. 1Example of EGFR expression in tissue sections in a primary colorectal carcinoma (a) and the matched metastasis (b). Strong membranous staining of numerous cells in both sites (immunoperoxidase; bar 50 μm)
Regarding the analysis of EGFR expression in metachronous metastases, we observed an analogous EGFR-positive status between all the available liver metastases (i.e., five metastases for one patient, one metastasis for two patients) and their related primary sites and synchronous metastases (case numbers 4, 10, and 11, Table 1). The only metachronous pulmonary metastasis available for analysis did not show any EGFR immunoreactivity, whereas both the corresponding hepatic synchronous metastases and the primary site expressed EGFR (case number 7, Table 1).
EGFR expression of primary CRC and their related metastases on TMA
On the first TMA block, the EGFR status was found to be positive in 18 out of the 31 (58%) assessable primary tumors and 18 out of 29 metastases (62%) (Table 2). Only nine out of 28 (32%) assessable matched tumors were positive for both the primary and the metastatic sites, whereas four out of 28 (14%) were negative. Fifteen out of 28 (54%) cases were discordant (i.e., seven positive primary and negative metastases, and eight, conversely).
Table 2Comparison of EGFR expression in primary CRC and their synchronous metastatic sites on whole tissue sections and TMA Primary siteMetastasesMatched sitesTissue sections27/32 (84%)30/32 (94%)25/32 (78%)First TMA18/31 (58%)18/29 (62%)9/28 (32%)Second TMA14/31 (45%)16/29 (55%)8/28 (29%)Both TMAs20/31 (65%)19/29 (66%)12/28 (43%)Data are expressed as the number of positive cases/total number of  specimens
On the second TMA block, 14 out of 31 (45%) primary tumors and 16 out of 29 (55%) metastases were EGFR-positive (Table 2). Eight out of 28 (29%) assessable matched tumors were positive for both sites, whereas nine (32%) were negative. Eleven out of 28 cases (39%) were discordant (i.e., four positive primary sites and negative metastases, and seven, conversely).
When combining the two TMAs (six tissue cores), the number of positive cases increased, to reach 20 out of 31 of the primary tumors (65%) and 19 out of 29 of the metastases (66%) (Table 2). Twelve out of 28 (43%) assessable matched tumors were positive for both sites, whereas three (11%) were negative. Nevertheless, 13 cases (46%) remained discordant (i.e., six positive primary and negative metastases, and seven, conversely). Moreover, using the TMA technology and whatever the number of cores (three or six), we failed to demonstrate any correlation of EGFR expression between primitive sites and matched metastases.
Comparison of EGFR expression between tissue sections and TMA
The EGFR status on the triple core-tissue array was significantly underestimated at both sites when compared with tissue sections (P=0.011 for both primitive and metastatic sites for the first TMA; P=0.0005 and P=0.0023 for primitive and metastatic sites, respectively, for the second TMA). When combining both TMAs (six tissue cores), although the number of positive cases increased, we still observed a significant discrepancy between both techniques for primitive sites (P=0.034) and metastases (P=0.0001).
Discussion
EGFR is a 170-kDa transmembrane cell surface receptor encoded by the human HER1 gene, which promotes malignant cell proliferation and cancer progression [15]. Interfering with EGFR cell signaling by targeting strategies represents a novel approach to the treatment of solid tumors [15]. Among them, cetuximab (Erbitux®), a chimeric monoclonal human–mouse antibody, is used for the treatment of EGFR-expressing metastatic CRC that have progressed following chemotherapy [3, 23]. To identify patients eligible for this treatment, an immunohistochemical evaluation of EGFR expression is required. Although numerous studies have demonstrated EGFR expression in 70–97% of CRC [4, 6, 29], only few and controversial data are available on the EGFR status in distant metastases [6, 11, 13, 24]. Consequently, knowledge of EGFR metastastic status could be of potential value for therapeutic decisions, particularly if it differs from the primary tumors.
To answer the question as to whether EGFR expression is similar in different tumor sites, we compared the EGFR status of a set of primary CRC and their corresponding synchronous distant metastases, using a standardized immunohistochemistry test (EGFR pharmDx™, Dako). We also analyzed EGFR expression in metachronous metastases, when available, to study possible changes of EGFR expression during the metastatic process of CRC.
On whole tissue sections, our results demonstrated that EGFR expression was frequent and similar in both the primary and the metastatic sites. In our study, immunoreactivity was observed in 84 and 94% of the primary tumors and the metastases, respectively (Table 1). The 32 matched primary and synchronous metastatic CRC expressed a concordant EGFR status in 78% of the cases, all immunopositive, and the discordant cases were not statistically significant (P=0.453). Our data are in agreement with three recent studies that showed a high frequency and concordance of EGFR expression in matched primary tumors and distant metastatic lesions of CRC [8, 11, 13]. To our knowledge, only one study reported no correlation of EGFR expression on primary CRC and related metastases [24].
In addition to the concordant EGFR status between primary and metastatic locations, our study showed that both the staining intensity and the percentage of EGFR-positive cells in the primary site were significantly correlated with those observed in synchronous metastases (Fig. 1). Our work also emphasized the EGFR-positive status among multiple synchronous and metachronous metastatic sites in most of the patients (Table 1). Furthermore, we noticed an analogous EGFR-positive status between all metachronous metastases but one, their related synchronous metastases, and their primary CRC (Table 1). Such data, to our knowledge, have never been reported in clinical samples and seem to agree with the preclinical results, suggesting that EGFR expression is required for the tumor to acquire metastatic potential [19, 22, 27]. Moreover, in our cohort, all the patients displayed an EGFR-positive status. This would mean that most of the patients are potentially eligible for therapy with EGFR-targeted monoclonal antibodies, given the results of the immunohistochemistry.
The clinical relevance of EGFR immunohistochemical detection is today a matter of debate because EGFR-negative patients might respond to cetuximab and EGFR-positive patients might not [2, 3, 5, 23]. One reason proposed to explain this lack of correlation is a possible difference in the EGFR status between the primary tumor and the metastatic sites [24]. Our results do not support this hypothesis. The lack of response in EGFR-positive patients could more likely be explained by alterations in the critical downstream signals, activated via other receptors or other pathways. It is also plausible that the lack of correlation between EGFR expression and antitumor activity could be due to the potential for cetuximab to induce antibody-dependent cell-mediated cytotoxicity and to the absence of discrimination between the high- and low-affinity receptors with the commonly used antibodies [2, 14, 16]. Moreover, negative immunohistochemical results may be linked to the heterogeneity of EGFR expression and could, in part, represent false negative cases that might respond to EGFR-targeted therapy.
The second goal of our study was to perform the same analysis on TMA generated with the same paraffin blocks used for EGFR assessment on the whole tissue sections. TMA has been employed to study the molecular profile of different cancer types and to test hypotheses regarding colorectal carcinogenesis and prognosis [9, 12, 20]. However, EGFR expression in primary CRC and their related metastases has rarely been studied with this approach and the few published results are controversial [8, 25]. In our work, TMA was carried on multiple (three) cores, selected from the deepest region of tumor invasion. Nevertheless, the results obtained were systematically lower than those observed on the whole tissue sections. Even when combining the two TMAs, EGFR positivity only reached 65 and 66% in primary CRC and their related metastases, respectively (Table 2). In contrast to what we observed on tissue sections, we failed to demonstrate a correlation of EGFR expression between both sites using the TMA technology. The discordant results between the two technologies may be explained by cases containing rare stained cells (i.e., <10%) or small invasive clusters, which may be not selected when TMA is performed even using three or six cores. These small tumor clusters, also called tumor “budding,” belong to a phenomenon which might be involved in metastatic spreading and seem to represent a poor prognostic factor [18, 21, 30]. Such data are of importance because TMA has been recognized as a useful and powerful tool for screening immunohistochemical markers for prognostic purposes and for carcinogenesis studies, as well.
In conclusion, we demonstrated that EGFR expression on whole tissue sections is frequent and similar in primary CRC and their related metastases. Our results also indicated that the EGFR status of the primary is concordant with those of metastases, whatever the number of sites and the time of occurrence, i.e., synchronous or metachronous. This may have clinical implications because patients are not always treated in the same institute for their primary disease and for their metastatic disease. In our experience, TMA does not represent an appropriate technique for EGFR screening in CRC because it significantly underestimates EGFR expression and does not provide data similar to those obtained with whole tissue sections. Undoubtedly, this technology is a reliable tool for the high-throughput assessment of homogeneously distributed proteins [7], but it appears to generate unreliable information with very heterogeneous markers such as EGFR.

epidermal growth factor receptor
colorectal carcinoma
metastases
tissue microarray


Evaluation of the Occupational Doses of Interventional Radiologists

The aim of the present study was to determine whether there is a linear relation between the doses measured above and those measured under the lead apron of the radiologists performing interventional procedures. To monitor radiation exposure the International Commission of Radiological Protection (ICRP) recommends the use of a single dosimeter under the protective apron. To determine the exposure more accurately an additional dosimeter is recommended above the protective apron. The exposure of eight radiologists was monitored with two personal dosimeters during 3 consecutive years. To measure the doses uniformly the two dosimeters were worn in a special holder attached to the lead apron. The two personal dosimeters were replaced every 4 weeks on the same day. The doses above and under the protective aprons of seven radiologists did not differ significantly. A significant lower dose above and under the protective apron was measured for one of the radiologists. During a 4-week period the average dose measured above the lead apron was 3.44 mSv (median, 3.05 mSv), while that under the 0.25-mm lead apron was 0.12 mSv (median, 0.1 mSv). The coefficients of the regression line result in the equation Y = 0.036X − 0.004, with Y as the dose under the lead apron and X as the dose above the lead apron. The statistical analysis of the data established a linear relation between the doses above and those under the lead apron (R2 = 0.59). Before the special holder was introduced it was not possible to derive a relation between the doses above and those under the lead apron, as the doses were measured at varying places above and under the lead apron. There is no evidence that the effective dose can be estimated more accurately when an additional dosimeter is used. The present study revealed a threshold before doses under the lead apron were measured. Due to the threshold it can be concluded that the doses under the lead apron will not be underestimated easily when doses above the lead apron are used to calculate them. This is not the case when the doses above the lead apron are calculated for the doses under the lead apron.
Introduction
Like many other hospitals the Academic Medical Centre (AMC) in Amsterdam uses personal dosimeters to monitor the occupational doses from x-ray procedures to clinical staff. The occupational doses of the majority of the clinical staff do not exceed 1 mSv in a single year. However, for clinical staff involved in fluoroscopically guided interventional procedures, significant doses are measured. As a consequence of the radiation exposure, the risk of fatal cancer for the radiologists performing interventional procedures increases. This stochastic risk can increase to a value of about 0.1% for radiologists performing these procedures during many years [1]. In recent years the number of fluoroscopically guided interventional procedures at the AMC has increased, as has the number of radiologists performing the procedures. Moreover, the discipline to wear personal dosimeters has improved. As a result, the number of radiologists with doses exceeding 20 mSv in a single year has increased. Whenever the dose exceeds 20 mSv in a single year a report has to be sent to the Dutch labor inspection, even when the doses are measured above the lead apron. In the report evidence has to be given that the limit for stochastic effects is not exceeded. In order to prove that the doses of 20 mSv measured above lead aprons do not exceed the stochastic dose limit, it was decided to provide additional personal dosimeters to the radiologists performing interventional procedures at the AMC. These additional personal dosimeters were meant to monitor the occupational doses under the lead apron.
When in 2004 it was decided to provide the radiologists with an additional dosimeter, the AMC had already substantial data of doses monitored on both sides of the lead apron. These data were collected at the Department of Cardiology, where additional dosimeters were introduced in 1998. Until 2004 the data collected at the Department of Cardiology established no relation between the doses measured above and those measured under the lead apron. This was thought to be caused by the fact that the two dosimeters were often transposed, that the dosimeters were not always returned at the same time, and that unmistakable instruction regarding the position of the two personal dosimeters was missing. To rule out the influence of these factors on the dose measured, it was decided to develop a special holder for the two dosimeters at the time the radiologists were provided with additional dosimeters. Moreover, it was decided to replace all personal dosimeters in the study on the same day by one person, to preclude the influence of differences in measuring periods and problems with transposed dosimeters.
For this department the doses measured under the lead apron were considered to be a substitute for the effective dose as recommended by the ICRP [2], while the doses measured above the lead apron were used to estimate the effective dose more accurately.
The aim of the present study was to determine whether a relation exists between the doses measured above and those measured under the lead aprons of radiologists performing interventional procedures and whether two personal dosimeters provide additional information compared to a single dosimeter.
Materials and Methods
From 2004 onward the occupational doses of eight radiologists involved in interventional fluoroscopically guided procedures were monitored by means of two personal dosimeters. The personal dosimeters were used to measure the doses above and under the lead aprons of the radiologists. To receive consistent measurements the two dosimeters were worn in a specially designed holder that was fixed to the lead apron (Figs. 1 and 2). The holder with the two personal dosimeters was worn breast-high. The two dosimeters were replaced every 4 weeks on the same day, while the holder remained fixed to the lead apron during the whole study.
Fig. 1The specially designed holder fixed to the lead apron breast-highFig. 2The specially designed holder with the two personal dosimeters
The doses were measured during 39 periods of 4 weeks in 3 consecutive years.
At the start of the study six of the radiologists were provided with two personal dosimeters, while throughout the study another two radiologists were provided with two dosimeters. Three radiologists left the AMC before the end of the study.
The personal dosimeters were provided by the Nuclear Research and Consultant Group (NRG, Arnhem, the Netherlands). The personal dosimeters were read by the NRG and reported to the AMC. The dosimeters contain LiF:Ti, Mg (TLD100) detectors behind 2 mm of aluminum for the measurements at a depth of 10 mm. In the dose reports the doses were presented by means of the personal dose equivalent at a depth of 10 mm, the Hp(10). The doses were reported in multiples of 0.01 mSv. Doses below 0.005 mSv were reported as <0.01 mSv [3].
At the Department of Radiology the interventional procedures were performed with two Philips Integris Allura machines (Philips Medical Systems, Best, the Netherlands) with the same potential range of 50−133 kVp. During all interventional procedures the radiologists used wrap-around lead aprons (skirts and vests; Scanflex Medical AB, Täby, Sweden) with an overall lead equivalence of 0.25 mm at 100 kV. The wrap-around apron has an overlap due to which the actual thickness of the protective apron in the front is 0.5 mm lead equivalent. The doses were monitored above and under a layer of 0.25 mm lead equivalence.
The doses measured above and under the lead apron were statistically analyzed using SPSS software (release 11.5.1 for Windows). Under the lead apron doses <0.01 mSv were treated as 0 mSv for the statistical analysis. Above the lead apron doses <0.01 mSv were entered as missing values. For the statistical analysis the doses under the lead apron were entered as dependent variables, while the doses above the lead apron and the eight radiologists were independent variables. Statistical significant differences were set at a level of 5% (p = 0.05).
Results
During the study 392 dosimeters were provided to eight radiologists. All dosimeters were returned to the NRG. The doses were read and reported in 13 4-weekly dose reports. In total 196 doses were recorded above the lead apron, ranging from <0.01 to 16.78 mSv. Under the lead apron the doses ranged from <0.01 to 0.83 mSv. Five readings were excluded from the study, as the doses measured above and under the lead apron were reported to be <0.01 mSv. In 27 cases the doses measured under the lead apron were <0.01 mSv. These doses were treated as 0 mSv in the statistical analysis.
In Tables 1 and 2 and in Figs. 3 and 4, the mean dose, median, and quartiles above and under the lead apron are presented for the eight radiologists. A Kolmogorov-Smirnov test showed that the doses of the eight radiologists were normally distributed (p > 0.05). The doses measured above and under the lead apron differed significantly among the eight radiologists (one-way ANOVA, p < 0.05). The Levenes test established that the variance of the doses measured above and under the lead apron of seven radiologists was equal (p > 0.05). The doses of these seven radiologists did not differ significantly (one-way ANOVA, p > 0.05). The average dose of the seven radiologists was 3.85 mSv in 4 weeks above the lead apron and 0.13 mSv in 4 weeks under the lead apron. The average dose of radiologist 5 was 1.79 mSv in 4 weeks above the lead apron and 0.07 mSv under the lead apron. The dose above the lead apron of radiologist 5 differed significantly from the doses of radiologists 6, 7, and 8 (Tamhane post hoc analysis, p < 0.05). Under the lead apron the dose of radiologist 5 differed significantly from the dose of radiologist 7 (Tamhane post hoc analysis, p < 0.05).
Table 1The number of measurements, mean dose, standard deviation, and percentiles (25th, 50th [median], and 75th) of the doses measured above the lead aprons of radiologists 1 to 8Radiologist no.NMean (mSv)SD25th (mSv)50th (mSv)75th (mSv)Minimum (mSv)Maximum (mSv)143.411.222.163.584.481.884.582343.673.211.323.155.260.0316.78392.331.241.382.173.590.284.00493.652.611.763.564.720.359.245a381.791.090.881.702.290.145.406b393.682.372.253.264.180.1311.527b213.661.712.493.084.570.666.948b374.752.313.104.876.131.0513.23Total1913.442.411.743.054.610.0316.78Note. Doses followed by a superscript a differ significantly from doses followed by a superscript b (p < 0.05).Table 2The number of measurements, mean dose, standard deviation, and percentiles (25th, 50th [median], and 75th) of the doses measured under the lead aprons of radiologists 1 to 8Radiologist no.NMean (mSv)SD25th (mSv)50th (mSv)75th (mSv)Minimum (mSv)Maximum (mSv)140.180.090.100.170.270.090.292340.130.150.030.110.170.000.83390.060.060.010.070.110.000.15490.140.090.060.150.210.000.245a380.070.090.000.060.090.000.496390.120.080.060.110.190.000.377a210.200.120.120.180.270.020.508370.130.120.050.120.180.000.66Total1910.120.110.050.10.170.000.83aThese doses differ significantly (p < 0.05). Fig. 3Boxplot of the doses (mSv) measured above the lead apron. The black line in the red box marks the median, the box demarks the 25th and 75th percentiles, and the whiskers mark the minimum and maximum observed values that are not statistical outliers. Extreme values are marked with an asterisk. The extreme values are more than three box lengths from the upper edge of the box. The box length is the interquartile rangeFig. 4Boxplot of the doses (mSv) measured under the lead apron. The black line in the red box marks the median, the box demarks the 25th and 75th percentiles, and the whiskers mark the minimum and maximum observed values that are not statistical outliers. Extreme values are marked by an asterisk. The extreme values are more than three box lengths from the upper edge of the box. The box length is the interquartile range
In Fig. 5 the doses measured under the lead apron are plotted against the doses above the lead apron. The statistical analysis of the data established a linear relation between the doses above and those under the lead apron (ANOVA, p < 0.05, R2 = 0.59). The coefficients of the regression line representing the relation between the doses measured above and those measured under the leaded apron were derived, resulting in the equation Y = 0.036X − 0.004, with Y as the dependent variable (dose under the lead apron) and X as the predictor value (dose above the lead apron). The 95% confidence interval for the coefficients ranged from 0.034 to 0.038. The standard error of the models tested was 0.07. The standard error of the model was lower than the standard deviation of the average dose under the lead apron (0.11). The residuals were normally distributed (p > 0.05) (Fig. 6).
Fig. 5Plot of doses (mSv) measured above the lead apron (X-axis) and under the lead apron (Y-axis). The line represents the equation for the regressionFig. 6Distribution of the residuals (mean = 0, standard deviation = 1)
The linear relation was not influenced by the doses measured for radiologist 5. The equation remained the same. With the equation and the lowest detectable dose under the lead apron (<0.005 mSv), the corresponding dose above the lead apron was calculated: <0.25 mSv. Above this threshold the relation between doses above and those under the lead apron was determined another time. The statistical analysis once again established a linear relation (ANOVA, p < 0.05). The coefficients of the regression line remained the same. Only the strength of the model was lower (R2 = 0.57).
Discussion
The dose measurements above and under the lead apron established that the occupational exposure of the radiologists performing interventional procedures in the AMC is comparable. There was only one exception: radiologist 5. As radiologist 5 performed fewer procedures than his colleagues, his exposure was expected to be lower. It was, however, unknown what reduction in doses was to be expected, as data on the number and the length of procedures were not collected for the study. Despite this, it is likely that the lower exposure of radiologist 5 was caused by a lower workload, as the doses above and under the lead apron decreased proportionally compared to the other radiologists’: 46% and 50%.
Results of doses measured above and under the lead apron of physicians performing interventional procedures have been published by several authors. Moreover, measurements for cardiologists performing interventional procedures were carried out. Renaud et al. [4] reported measurements above and under the lead aprons of in-room personnel during cardiac catheterization. Vano et al. [5] published measurements of cardiologists during a 15-year follow-up. However, differences in the way radiologists and cardiologists perform interventional procedures make it difficult to compare the doses of cardiologists and radiologists. A limited number of clinical studies on doses above and under the lead apron have been carried out among radiologists. Williams et al. [6] reported average monthly doses above the lead aprons of radiologists that ranged from 1.08 to 6.55 mSv. The highest average dose in this study was found for a radiologist who performed all interventional procedures in patients with liver disease. The average dose of the other radiologists in the study was 1.56 mSv. The findings of Williams et al. are comparable to the average dose above the lead apron in the present study, where the doses varied between 1.09 and 3.21 mSv.
Niklason et al. [7] published annual radiation doses above the lead aprons of 28 radiologists. The average annual dose in their study was 48 mSv. The average annual dose in the study by Niklason et al. was higher than the average annual dose in the present study: 45 mSv. However, the average annual dose found by Niklason et al. was lower than the average annual dose when radiologist 5 was excluded. The average annual dose of the seven remaining radiologists was 50 mSv.
In the studies by Williams et al. and Nicklason et al. the doses under the lead apron were also measured. Williams et al. found that the average monthly dose under the lead apron ranged from 0 to 0.48 mSv. As for the results above the lead apron, Williams et al. reported that the highest average dose was caused by the differences in the nature of work of one of the radiologists. The average monthly dose under the lead apron of all other radiologists (the radiologist with the highest dose was excluded) was 0.08 mSv. In the present study the doses under the lead apron varied between 0.06 and 0.2 mSv in a 4-week period. The average dose, 0.12 mSv, was higher than the average dose reported by Williams et al. The differences between the two studies are possibly caused by the differences in thicknesses of the lead aprons between them. Williams et al. reported that the doses were measured under lead aprons of 0.35 and 0.5 mm, while in the present study doses were measured under a layer of 0.25 mm.
Niklason et al. measured doses under lead aprons of 0.25 and 0.5 mm. As for the doses measured above the lead apron, the doses under the lead apron were annual doses. The average annual dose under the lead apron reported by Niklason et al. was 0.88 mSv, while in the present study the average annual dose was 1.6 mSv (median, 1.3 mSv). As for the results reported by Williams et al., the differences in doses were probably caused by the variation in thickness of the lead aprons worn by the radiologists.
In the study reported by Williams et al., the radiologists ensured that the personal dosimeters were worn in all cases. However, the personal dosimeters under the lead aprons were worn not only during interventional procedures but also during other radiology procedures. The authors described that the dedication of the radiologists to wearing additional dosimeters under the lead apron was probably less consistent than for the other dosimeters. In the present study the dedication of the radiologists was not tested, as the dosimeters were simultaneously worn in the special holder. Moreover, the dosimeters in this study were worn exclusively during interventional procedures.
The duration of the measurements in the study by Niklason et al. was approximately 2 months, while in the present study the doses of the majority of the radiologists were measured during 3 consecutive years.
The regression analysis revealed a linear relationship between the doses measured above and those measured under the lead apron. The relation was described by the formula Y = 0.036X − 0.004. The slope of the line (0.036) is a measure of the transmission of the lead aprons. In the present study a transmission of 3.6% was found for lead aprons of 0.25 mm. This transmission was derived breast-high, at the height of the lead collar of radiologists. According to Kharrati et al. [8], the energy of the scattered radiation at the height of the lead collar of radiologists is ∼70 kVp. The transmission fraction in the present study (3.6%) is in accordance with the data on transmission factors of different primary beam energies found by other authors. Murphy et al. [9] found a transmission factor of 3% for lead aprons of 0.3 mm in the primary beam of 80 kVp. Vano et al. [5] found a fraction transmitted through 0.25-mm lead aprons of between 3.3% at 70 kVp and 5.7% at 80 kVp. Christodoulou et al. [10] reported about transmission fraction through lead aprons of 0.25 mm at a tube potential of 70 kVp. The transmission fraction ranged from 4.3% up to 10%.
From the formula Y = 0.036X − 0.004 the intercept of the regression line can be calculated. The intercept with the X-axis lies at 0.11 mSv. This value theoretically represents the threshold of the doses above the lead apron before doses under the lead apron. This means that for the doses above the lead apron of ≤0.11 mSv, only doses below the diction limit are expected. The doses will be reported as <0.01 mSv. As doses up to 0.005 mSv are below the detection limit of the dosimeters and are reported as doses <0.01 mSv, the theoretical threshold is even higher: 0.25 mSv, versus 0.11 mSv for the doses above the lead apron. As a matter of fact, in the present study for all measurements above the lead apron <0.25 mSv, the corresponding doses under the lead apron were reported to be below the detection limit of 0.01 mSv. The lowest dose in this study above the lead apron for which a dose under the lead apron of 0.01 mSv reported was 0.59 mSv.
The minimum thickness recommended by the IAEA [11] for lead aprons of radiologists performing interventional procedures is 0.35 mm. In the present study the doses were measured at 0.25 mm. The doses were measured under one layer of wrap-around lead aprons. Due to the overlap of these aprons, the thickness of the lead apron in the front of the radiologists was 0.5 mm. For practical reasons it was decided to measure the doses under one layer of 0.25 mm despite the fact that the doses measured were higher than the occupational doses to the body of the radiologists. The occupational doses of the radiologists under the lead apron in this study are therefore overestimated.
The present study showed that additional monitoring under the lead apron is not necessary to estimate the occupational exposure more accurately. This result is in agreement with the conclusions of Schultz et al. [12], based on dose calculations with Monte Carlo simulations. A transmission of 3.6% for lead aprons ≥ 0.25 mm does not underestimate the exposure for radiologists.
In the present study the doses under the lead apron were determined almost at the same place as the doses above the lead apron. This was possible as a special holder was used. Before the special holder was introduced it was not possible to derive a relation between the doses above and those under the lead apron.
Conclusion
The aim of the present study was to determine whether a relation exists between the doses measured above the lead apron and those measured under the lead apron of radiologists performing interventional procedures and whether two personal dosimeters provide additional information compared to a single dosimeter. It might be concluded that a linear relation between the measurements above and those under the lead apron is proved in this study. With this, there is no evidence for a more accurate estimation of the effective dose when an additional dosimeter is used.
As the results of this study were derived from measurements by radiologists performing interventional procedures, it is not known whether a factor of 0.036 is applicable for other specialists performing interventional procedures, such as interventional cardiologists and electrophysiologists. For this reason, more investigation is needed to derive correction factors for other physicians performing interventional procedures.

lead apron
effective dose
dosimetry
fluoroscopy
radiation protection