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Thoracic Imaging |
1 From the Departments of Clinical Radiology (S.D., D.W., H.L., N.R., W.H.), Thoracic and Cardiovascular Surgery (M.S.), and Hematology/Oncology and Respiratory Medicine (M.T.), University of Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. Received February 21, 2001; revision requested March 30; revision received June 13; accepted August 8. Supported by grants from the German Cancer Society (Deutsche Krebshilfe: 70-2021-Di 2 and 70-2501-Di 3) and the Medical Faculty of Münster University (IMF Di-1-4-I/97-22). Address correspondence to S.D. (e-mail: diestef@uni-muenster.de).
| ABSTRACT |
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MATERIALS AND METHODS: Eight hundred seventeen asymptomatic volunteers (age range, 4078 years; median age, 53 years; median tobacco consumption, 45 pack-years) underwent spiral low-dose CT of the chest without contrast material enhancement. We regarded all noncalcified pulmonary nodules greater than 10 mm in diameter as potentially malignant and recommended histologic examination or follow-up after 3, 6, 12, and 24 months to exclude growth. For noncalcified pulmonary nodules of 10 mm or smaller, repeat low-dose CT was recommended to exclude growth.
RESULTS: In 43% (350 of 817) of individuals, 858 noncalcified pulmonary nodules were found. Thirty-two nodules in 29 subjects were larger than 10 mm. Biopsy of 15 lesions revealed lung cancer in 12 lesions in 11 subjects (prevalence for all ages, 1.3% [11 of 817 subjects]; >50 years of age, 2.1% [11 of 519 subjects]; >60 years of age, 3.9% [eight of 206 subjects]), with a high proportion of early tumor stages (seven tumors, stage I; two, stage II; and three, stage III); three lesions were benign. In 17 nodules larger than 10 mm, follow-up with low-dose CT for a minimum of 24 months did not demonstrate growth.
CONCLUSION: Lung cancer screening with low-dose CT demonstrated a prevalence of asymptomatic cancers in 1.3% of a smoking population, including a high proportion of early tumor stages and a 20% (three of 15) rate of invasive procedures for benign lesions.
© RSNA, 2002
Index terms: Cancer screening, 60.12111, 60.12115, 60.12119 Computed tomography (CT), radiation exposure Lung neoplasms, CT, 60.12111, 60.12115
| INTRODUCTION |
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Lung cancer most commonly manifests as a noncalcified pulmonary nodule. Computed tomography (CT) is superior to chest radiography for detecting pulmonary nodules, particularly with spiral technology (1417). Typical chest CT protocols are, however, associated with relatively high radiation exposure to the patient (1821), which causes concern about induction of malignant disease (22,23), particularly in a screening setting. Because dose reduction at CT does not substantially decrease sensitivity for small pulmonary nodules (2426), low-radiation-dose CT should depict more tumors than does chest radiography, potentially improving the early detection and prognosis of lung cancer. Many small pulmonary nodules are, however, due to benign lesions (eg, granulomas, hamartomas, intrapulmonary lymph nodes), and differentiation from malignant nodules may require biopsy (27,28). We are currently involved in an ongoing study to assess the prevalence and incidence of pulmonary nodules at low-dose CT in a population at high risk for lung cancer and to test a simple algorithm for further work-up of detected nodules on the basis of their size and attenuation. The purpose of this article was to present the prevalence screening data from a nonrandomized screening trial by using low-dose CT and a simple algorithm based on the size and attenuation of detected nodules to guide their diagnostic work-up.
| MATERIALS AND METHODS |
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Study Population
Eight hundred seventeen asymptomatic volunteers were enrolled in the study from November 1995 to July 1999. The sample size was based on the assumption that the prevalence of lung cancer would be approximately 1% (79) and on experience from the initial phase of the study (29), which suggested 30% prevalence of pulmonary nodules in smokers older than 40 years. Subjects were recruited by means of announcements in the appropriate clinics of the hospital (ie, respiratory medicine, thoracic and cardiovascular surgery, and peripheral vascular surgery), and in the local media (ie, newspapers, radio, and television), including a phone number to call to arrange low-dose CT examinations. For study participation, minimum age of 40 years, minimum tobacco consumption of 20 pack-years, and ability to hold ones breath for approximately 25 seconds at low-dose CT were recommended. The minimum age of 40 years was chosen on the basis of the inclusion criteria of the screening studies in which chest radiography was performed (minimum age, 45 years) (79) and of a Japanese CT screening study (30). This method of recruitment was thought to be appropriate for a feasibility study, even if the study population did not precisely reflect the smoking population in Germany at risk of developing lung cancer.
Before undergoing low-dose CT, each individual underwent a standardized interview based on a questionnaire in which personal data (eg, name, sex, date of birth, and address), smoking history, and other risk factors for developing lung cancer (eg, asbestos exposure) were recorded. Exclusion criteria were a recent (<6 weeks) history of febrile respiratory tract disease or known pulmonary metastases. To avoid inclusion of subjects with pulmonary abnormalities, we pointed out that low-dose CT was appropriate in only symptom-free individuals with no previous tumors and asked about previous tumors, as well as other previous disease, in the questionnaire. We also pointed out that individuals with symptoms such as hemoptysis and chest pain should not undergo low-dose CT in our trial. However, we were not able to check the previous medical records of individuals recruited through the media. Written informed consent was obtained to perform low-dose CT of the chest and additional examinations according to the study protocol if an abnormality was detected.
Low-Dose CT Examination and Analysis
Spiral low-dose CT without contrast material enhancement (Tomoscan SR 7000; Philips, Eindhoven, the Netherlands) was performed at end inspiration in one breath hold. Examination parameters were 120 kVp, 50 mAs, 5-mm collimation, 10-mm-per-rotation table feed, and a 5-mm reconstruction interval. The lowest possible dose setting available on our CT scanner at the time of study initiation was 50 mAs with a pitch of 2. The average effective dose of this protocol (0.6 mSv in men and 1.1 mSv in women) approximates three (in men) and five (in women) chest radiographs, respectively, in two views (31). Additional limited thin-section low-dose CT was performed within 2 weeks to more precisely characterize pulmonary nodules by using 120 kVp, 50 mAs, 1-mm collimation, 2-mm-per-rotation table feed, and a 1-mm reconstruction interval. Limited thin-section low-dose CT covered a maximum of 50 mm in the craniocaudal axis (breath hold of 25 seconds). The maximum effective dose of this examination was less than that of chest radiography in two views.
Analysis was performed by one of two radiologists (S.D., D.W.). In all studies, assessment was performed first by using laser film (Ektascan 2180; Kodak, Stuttgart, Germany) (17 x 14 inches, 4 x 5 images, high-resolution reconstruction algorithm) and subsequently by using a workstation (Easy Vision; Philips, Eindhoven, the Netherlands) and a standard reconstruction algorithm at lung (window width, 1,500 HU; window level, -600 HU) and mediastinal (window width, 400 HU; window level, 40 HU) windows. Assessment at the workstation included image-by-image analysis and separate cine-mode analysis on a 19-inch monitor. The triple assessment (hard copy, monitor single frame, and monitor cine mode) was performed to obtain the optimum sensitivity for nodules. Findings were recorded on a standardized form.
A scan was judged as normal, as demonstrating pulmonary nodules, or as demonstrating other diseases. Any focal lesion surrounded by aerated lung was regarded as a pulmonary nodule if the ratio of maximum and minimum diameter was less than 2. In addition, all other lesions were regarded as nodules if there was the least suspicion of a space-occupying lesion, for example, a focal swelling within a linear structure. Thin linear densities contacting the pleura were not regarded as nodules.
If nodules were detected, their sizes were measured on the monitor by using electronic calipers in 1-mm increments with a 400% zoom. We assessed the largest diameter in the transverse plane, the largest diameter perpendicular to this diameter, and the maximum craniocaudal extension by counting the number of consecutive images showing the nodule at thin-section low-dose CT (collimation, 1 mm; increment, 1 mm). We paid particular attention to correct classification of the maximum diameter into one of three size categories (
5 mm, 610 mm, or >10 mm). Nodule attenuation was recorded as noncalcified (soft-tissue attenuation) or as partially or homogeneously calcified by using an arbitrary threshold of 100 HU to differentiate between calcification and soft-tissue attenuation. For this purpose, we used an attenuation window with a width of 1 HU and a center of 100 HU at standard reconstruction algorithm, which depicts all pixels with attenuation of 100 HU or greater.
The location of nodules was recorded with regard to segment, as well as central or peripheral (within 2 cm of costal pleura) location. We also assessed the bronchi for endobronchial lesions. The criteria for reporting (eg, defining pulmonary nodule, differentiating pleural and intrapulmonary lesions, technique of measuring maximum and minimum diameter, recording of location) had been defined before study initiation and practiced by the two readers together. We did not perform double reading, but cases of unclear size (
10 mm or >10 mm) or possible growth were resolved with consensus.
Diagnostic Algorithms
Low-dose CT scans showing no noncalcified pulmonary nodules were regarded as negative results, and subjects were invited to undergo repeat low-dose CT after 12 months. If solitary or multiple nodules were detected, recommendations for further procedures were guided by their attenuation: Homogeneously calcified nodules were regarded as benign and were not followed up with low-dose CT (negative test result).
Nodules of 10 mm or less appearing at least partially noncalcified at baseline low-dose CT were regarded as indeterminate findings, and additional spiral thin-section low-dose CT was recommended for further characterization and exact size measurement. In cases of multiple lesions, we suggested only thin-section low-dose CT if all nodules could be included in two spiral thin-section scans obtained in one breath hold each, due to limitations of the computing power of our CT software at the time of study initiation, which allowed reconstruction of a maximum of only 100 images at a time. Otherwise, repeat low-dose CT of the whole lung after 3 months was recommended. However, we obtained thin-section low-dose CT scans of every nodule larger than 10 mm.
If thin-section low-dose CT revealed homogeneous calcification, the nodule was regarded as benign, and no further follow-up of the lesion was suggested. If it confirmed a noncalcified nodule, further procedures were recommended according to nodule size measured at thin-section low-dose CT: In nodules 10 mm or less, follow-up 3 months after the initial examination with repeat limited thin-section low-dose CT was performed to detect fast growth. If growth was demonstrated at 3 months, biopsy was performed. If we were not absolutely certain that there was no growth at 3 months, another limited thin-section low-dose CT examination was performed 6 months after the initial examination. If no growth was demonstrated at 3 and 6 months, repeat low-dose CT of the whole lung was performed at 12 and 24 months after the initial examination, and findings were compared with those of initial low-dose CT. Only if this comparison showed questionable growth was thin-section CT repeated. All measurements at all studies were made at the same window settings (1,500 HU, -600 HU).
Absence of growth for a minimum of 24 months was regarded as evidence of a benign lesion (negative examination result). Growth was defined as an increase of the nodules diameter in at least one dimensioncraniocaudal, ventrodorsal, or mediolateral (positive test result).
In individuals with multiple noncalcified nodules, we followed up every individual nodule, since we felt that even in the presence of multiple benign nodules in a heavy smoker, one lesion could represent cancer. Therefore, we did not classify multiple nodules as diffuse disease.
Nodules larger than 10 mm were considered potentially malignant; however, it was decided individually whether the lesion was more likely to be benign or malignant on the basis of its morphology (eg, shape, good or poor definition, or smooth or irregular margination) and associated findings (eg, calcified hilar or mediastinal lymph nodes).
The decision was made with consensus of the two radiologists (S.D., D.W.), as well as the chest surgeon (M.S.) and chest physician (M.T.) on our team, as would have been done for other cases in our clinical practice. For example, if a pulmonary lesion was located in the apex, with a broad base against the pleura (Fig 1), we would consider this most likely a pleural scar and follow it up with only CT. If a lesion was round and ill-defined with spiculated borders (Fig 2), it was considered very suspicious for cancer, and bronchoscopic, percutaneous, or thoracoscopic biopsy was performed according to standard medical care. If the probability of malignancy was considered low (indeterminate test result), follow-up with repeat thin-section low-dose CT at 3, 6, 12, and 24 months after initial examination was performed as in smaller nodules. When growth was documented, biopsy was performed. This approach, similar to that in our clinical practice, was chosen to avoid biopsy in lesions considered most likely benign, despite the fact that there are no reliable imaging features, to our knowledge, that allow differentiation between benign and malignant nodules. The diagnostic algorithm is summarized in Figure 3.
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We subsequently present the prevalence-screen data from our screening trial based on the initial low-dose CT examination and the first thin-section low-dose CT examination or the 3-month follow-up examination.
| RESULTS |
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Nodule Size and Attenuation
A total of 1,001 nodules was reported at the baseline and first thin-section low-dose studies, of which 858 (85.8%) were noncalcified and 143 (14.3%) showed homogeneous calcification (Table 2). Of the 350 individuals with noncalcified nodules, 196 had noncalcified nodules with a maximum diameter of 5 mm or less; 125 had nodules of 610 mm; and 29 had nodules larger than 10 mm. The 858 noncalcified lesions included 624 lesions 5 mm or smaller, 202 lesions of 610 mm, and 32 lesions larger than 10 mm. The proportion of nodule size categories was similar in different age groups, although this was not tested statistically because of the small numbers of nodules larger than 5 mm and, particularly, larger than 10 mm (Table 3).
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In 18 of the 32 nodules, it was decided to not perform immediate biopsy, since the morphologic features at thin-section low-dose CT suggested benign lesions: Nine lesions were regarded as most likely representing pleuropulmonary scars because of their location in the lung apex, with broad contact with the chest wall, with no evidence of infiltration (Fig 1).
Two lesions were considered to represent areas of inflammatory disease because of tree-in-bud phenomena. Two well-defined lesions with popcornlike calcification were regarded as potentially representing chondrohamartoma (Fig 4). Two branching soft-tissue-attenuation lesions were believed to most likely represent small mucus plugs. One well-defined soft-tissue-attenuation nodule in the presence of multiple calcified nodules in a subject with a history of tuberculosis was regarded as most likely representing a noncalcified granuloma (subjects 29, 1115). One well-defined 11-mm lesion was seen retrospectively on a chest radiograph obtained 18 months previously, with no apparent growth (subject 1). In all of these cases, further follow-up was performed, which demonstrated no change over a minimum of 24 months in 13 lesions, size decrease in two, and resolution in another two lesions. One nodule that had been regarded as a scar at initial low-dose CT exhibited slow growth after 24 months (Fig 5). Biopsy was performed on this lesion, revealing stage I adenocarcinoma (subject 10) (Table 4).
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In the 12 lung cancers, tumor stage was IA in six cases and IB in one case, IIA in one case and IIB in one case, IIIA in two cases, and IIIB in one case; no case was considered stage IV (subjects 18b27) (Table 5). Of the 12 carcinomas, nine were found in eight subjects aged 60 years or older (eight of 206 subjects; prevalence, 3.9%), and all 12 were found in 11 subjects older than 50 years (11 of 519 subjects; prevalence, 2.1%). No tumor was detected in subjects aged 50 years or younger. The prevalence of cancer in the total population was 1.3% (11 of 817 subjects).
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In a subject with a 12-mm left-upper-lobe nodule at low-dose CT that was subsequently confirmed as adenocarcinoma, a second 21-mm lesion was shown in the right hilum at staging CT with intravenously administered contrast material. Bronchoscopic findings in this region were negative, and percutaneous biopsy was not indicated because of adjacent pulmonary vessels. Thoracoscopy was not feasible because of adhesions; therefore, exploratory thoracotomy was performed, and histologic examination demonstrated lymphadenopathy from pneumoconiosis of coal workers (subject 18a). We did not detect any purely endobronchial lesions.
Of the 11 subjects with lung cancer, six are alive at the time of this writing, with no evidence of recurrent tumor (follow-up of 240 months; mean, 27 months); one is alive 18 months after diagnosis of local recurrence 30 months after initial diagnosis; and four have died, three of lung cancer (survival, 69 months) and one of an unknown cause (sudden unexpected death after 6 months).
| DISCUSSION |
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In our study, pulmonary nodules were detected with low-dose CT in 50% of individuals screened. The proportion of individuals with noncalcified nodules (43%) in the current study was much higher than the 23% reported in the Early Lung Cancer Action Project (ELCAP) study by Henschke et al (35). This may be due to the higher sensitivity of the CT protocol used in the current study for small pulmonary nodules (5- vs 10-mm collimation in the ELCAP study) but may also reflect differences between the screened populations. It can be assumed that a large proportion of these small lesions represent benign nodules in which invasive procedures are not required.
Consequently, we used an algorithm similar to one suggested by Henschke et al (36) based on the size and attenuation of pulmonary nodules, which included biopsy of soft-tissue-attenuating nodules larger than 10 mm or of smaller lesions with documented growth, unless low-dose CT features strongly suggested a benign nodule. In contrast with the algorithm suggested by Henschke et al (36), we chose to not obtain standard-dose thin-section CT scans routinely at 3, 6, 12, and 24 months but to perform low-dose thin-section CT at 3 and 6 months and 5-mm-collimation low-dose CT routinely at 12 and 24 months. Additional thin-section low-dose CT was only performed at 12 and 24 months if growth was suspected with 5-mm collimation.
This algorithm led to diagnosis of 12 malignant nodules. One individual had bilateral malignant nodules. Therefore, the prevalence of subjects with lung cancer detected with this approach was 1.3% (11 of 817) in the total study population, 2.1% (11 of 519) in the subjects older than 50 years, and 3.9% (eight of 206) in the subjects older than 60 years. For comparison, in the ELCAP study (35), in which a similar approach was used in a population of smokers older than 60 years, the prevalence of malignancy was 1.3% (13 of 1,000) with baseline low-dose CT and baseline thin-section low-dose CT and increased to 2.7% with follow-up thin-section low-dose CT; prevalence with chest radiography in that study was 0.7% (seven of 1,000). In a Japanese study in which smokers and nonsmokers older than 40 years were screened with low-dose CT (37), the prevalence of lung cancer was 0.40%. In studies in which chest radiography was performed in men older than 40 years (10) and older than 45 years (79), the prevalence was 0.3%0.8% (710). The data available, including data from comparable Japanese studies (30,38) suggest that the detection rate for lung cancer is, in fact, markedly higher with low-dose CT than with chest radiography.
The fact that fewer cancers were diagnosed with low-dose CT in the current study than in the ELCAP study (35) is explained by the lower average age in the subjects of the current study (minimum age, 40 years vs 60 years in the ELCAP study). In fact, in the current study subjects older than 60 years, the prevalence was higher than in the subjects of the ELCAP study (3.9% vs 2.7%, respectively), suggesting that future screening trials should focus on older age groups.
In the current study, 75% (nine of 12) of detected cancers represented tumor stages I and II (50%, stage IA; 8%, stage IB; 17%, stage II), and 92% (11 of 12) of tumors were resectable at diagnosis, similar to 89% (24 of 27) stage I and II cancers and 96% (26 of 27) resectable tumors in the ELCAP study (35), also by using low-dose CT. In contrast, only 51% (30 of 59) of the prevalence cases in the Mayo Lung Project, in which chest radiography was performed, were resectable (8). These figures suggest that low-dose CT is particularly superior to chest radiography for detecting early-stage tumors.
Although the figures for lung cancer in the current study are small, and the maximum follow-up was only 40 months, survival in these subjects is favorable, as compared with that in other populations with lung cancer (3). At the time of writing, 55% of subjects in the current study are alive with no evidence of recurrence or metastases, with a maximum follow-up of 40 months (mean follow-up, 27 months).
In our study, only three invasive procedures were performed for benign lesions, as compared with 12 invasive procedures for lung cancer. Similarly, in the ELCAP study (35), only one biopsy was recommended, which revealed a benign lesion. These data suggest an acceptable ratio of biopsy procedures for benign nodules and malignant nodules if procedures are guided by the size and attenuation of lesions.
The potential benefit of low-dose CT (ie, early detection of lung cancer) must be weighed against its risks (eg, induction of malignancy by using ionizing radiation). Estimations by the International Commission on Radiological Protection predict that examination of 100,000 individuals with one low-dose CT examination each by using the CT parameters applied in the current study (effective dose: 0.6 mSv in men, 1.1 mSv in women) will induce three (men) to six (women) additional cancers within the next 1520 years (23). Obviously, the likelihood increases with every additional thin-section low-dose CT or annual repeat low-dose CT examination. These figures must be balanced against the detection rate of potentially curable cancer (eg, 1,300 cases in 100,000 low-dose CT examinations if the prevalence at low-dose CT is 1.3%).
Because low-dose CT can be performed in approximately 30 seconds with no injection of intravenous contrast medium, it is less costly than other CT examinations. However, because of subject preparation, image transfer, and, particularly, image analysis, the time required for an individual screening examination is much longer. Therefore, we estimate the cost of a single screening low-dose CT examination to be higher than that of chest radiography and lower than that of contrast-enhanced standard-dose chest CT. Currently, the cost at our institution is probably $100$200, as compared with $30$50, as reported by Japanese authors (37,39). However, general recommendations for low-dose CT screening would result in immense cost (40).
Study Limitations
The algorithm used in the current study certainly does not depict all cancers: First, it is possible that some of the nodules 10 mm or smaller in which no histologic findings were obtained were malignant. Although follow-up should lead to diagnosis of these cancers by demonstrating growth, it may allow tumor spread in the interval between the baseline and follow-up study. In addition, detection of cancer at follow-up relies on compliance of the individual with study recommendations.
In a recent study, Yankelevitz et al (41) demonstrated that by using 1-mm collimation, a pitch of 1, a 0.5-mm reconstruction interval, and a 9.6-cm field of view, growth of 4- to 11-mm nodules could be detected at repeat thin-section CT after only 30 days. If confirmed by the findings of other studies, this approach offers the possibility of decreasing the number of follow-up examinations for indeterminate nodules to only one after 30 days.
Also, nodules larger than 10 mm currently followed up with low-dose CT because of low-dose CT features strongly suggestive of benign lesions may actually include lung cancer. In this relatively small group of nodules, other classifications such as contrast-enhanced CT (42) or positron emission tomography (43) could be used. Both techniques, however, do not provide 100% sensitivity for malignancy. We did not use a course of antibiotics between initial and repeat examinations. However, we found that some of the lesions still regressed spontaneously. We believe that this should be no surprise, since small asymptomatic inflammatory pulmonary nodules should heal even without antibiotics.
Because CT scans in the current study were assessed by one radiologist only, we cannot assess interrater reliability in the detection and classification of pulmonary lesions.
The current study was designed to assess the feasibility of detection and classification of pulmonary nodules with low-dose CT by using diagnostic algorithms as previously described. Its one-armed noncomparative design is not adequate to analyze the effect to which mortality from lung cancer can be reduced with low-dose CT screening. In fact, although low-dose CT appears to be much more sensitive for small pulmonary nodules representing lung cancer, this does not automatically translate into reduction of lung cancer mortality. Different biases such as lead time, length time, and overdiagnosis suggest that, despite detection of a high proportion of small early-stage resectable lung cancers at low-dose CT, mortality from lung cancer may be unchanged (4446). Furthermore, it has been suggested that prognosis is not as clearly related to tumor size as previously believed (6) and that, therefore, detection of stage IA tumors at the sizes shown by prevalence CT screening (median, 15 mm in the present series) (Table 5) may not necessarily lead to improved survival. Also, even in small tumors, lymph node and distant metastases may occur (35,46).
Future Perspectives
In the subjects in our study, we will follow up all soft-tissue-attenuation nodules with low-dose CT to detect growth and obtain information on the proportion of enlarging nodules 10 mm or smaller that represent malignancy.
Annual follow-up low-dose CT in all individuals with a normal baseline low-dose CT examination will allow assessment of the incidence of pulmonary nodules; further procedures in new lesions will be guided by the study algorithms. Analysis of the stage of interval cancers is hoped to provide information on useful intervals between screening studies.
In the future, multisection spiral CT will enable examination of the entire lung by using thin collimation in one breath hold, facilitating simultaneous detection and characterization of nodules. Innovative equipment with solid-state detectors and dose modulation will allow further decrease in patient dose. Modern modalities of CT data presentation or computer-aided diagnosis show promise for increasing sensitivity and decreasing reporting time (47,48).
In conclusion, in the current study, low-dose spiral CT was feasible for depicting small lung cancers by using a simple algorithm based on the size and attenuation of detected nodules to guide invasive procedures. Lung cancer was histologically confirmed in 12 nodules, 75% of which represented stage I or II tumors. Only three (20%) of 15 invasive procedures were performed for benign lesions.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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Author contributions: Guarantor of integrity of entire study, S.D.; study concepts, S.D., H.L., N.R.; study design, S.D., N.R.; literature research, S.D., D.W., H.L.; clinical studies, S.D., D.W., M.S., M.T.; data acquisition and analysis/interpretation, S.D., D.W., M.S., M.T.; manuscript preparation, S.D.; manuscript definition of intellectual content, S.D., D.W., W.H.; manuscript editing, S.D.; manuscript revision/review, S.D., D.W., H.L., N.R., W.H.; manuscript final version approval, all authors.
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