Semi-automated image cytometry (ASC) has become one fundamental arm in screening study programs for early lung cancer (1, 2). In its current version, ASC represents a sensitive tool for the quantitative analysis of nuclear structure and DNA-content of exfoliated airway epithelial cells (1, 3). Together with “low dose CT-scans” (4), auto fluorescence bronchoscopy (5), and endobronchial ultrasound (6) ASC is a critical element of new technologies promising a significant forward-shift in the diagnosis of early stage lung cancer, and prompting reawakened interest in lung cancer screening. Lung cancer still is one of the most prevalent and lethal cancers, accounting for 18% of all cancer deaths (7). In the last 30 years, 5-year survival rate for lung cancer remained less than 15% (8). Prognosis of lung cancer, however, is strongly associated with the stage of cancer at the time of diagnosis, 5 years survival rates range from 5% for patients with stage IV lesions to 80% at stage I (9). Therefore, improving detection rate of early stage lung cancer is essential for improving the prognosis of lung cancer.

Cigarette smoking is the main source of lung cancer worldwide (10). The undisputed effects of smoking on lung cancer can be multiplied by exposure at the working place - most prominently ionizing radiation or asbestos dust (11). Numerous studies confirmed that lung cancer is increased among uranium miners in Germany (12) and other western countries after incorporation of ionizing irradiation from radon progeny.

In the present, case finding study preparing for a prospective screening program for detection of early lung cancer in high-risk individuals with former occupational exposure, ASC and CY investigations of sputa from uranium miners with silicosis were performed. We studied the correlation between ASC and CY with the final diagnosis of lung cancer.


PATIENTS AND METHODS
1517 former male uranium miners with silicosis (71 ±5 years old, median 71 years) from former East German uranium mining industry, being seen for a regular check up in a hospital, specialized on occupational lung disease (BG-Klinik für Berufskrankheiten Falkenstein, Sachsen) were recruited from January 1999 to December 2004. Sputum specimens were collected as spontaneous 3-day pooled samples or after 3% saline aerosol induction. Patients had worked for 12 ±11 years (median 8 years) in uranium mines and retired 40 ±12 years (median 45 years) since the last exposure. 1033 of them smoked 19 ±17 pack/years, at the time of the investigation and the majority of them (853) were ex-smokers for 21 ±14 years. Sputum samples from 100 healthy non-smokers were taken as controls.

Sputum induction

Sputum was either collected and pooled on 3 consecutive days and stored in a refrigerator by patients, as instructed, or induced by 20 min inhalation of 3.0% saline aerosol (with addition of 2.5 µg Salbutamol in 20 ml saline) under supervision of a respiratory therapist. The aerosol, generated by a jet nebulizer (Pari Master, Starnberg, Germany), was inhaled via a mouth piece, and exhaled through the nose. Subjects were instructed to breath normally, swallow saliva, and to expectorate sputum, from the deeper airways only, into a 50 ml vial filled with 25 ml Saccomanno-preservative with addition of 0.1% dithiothreitol, DTT (13).

Staining

As described in detail in previous papers (1, 2), sputum samples in Saccomanno-DTT solution were completely liquefied and could be treated like a blood smear for a monolayer preparation. After centrifugation for 15 min at 500 x g, the supernatant was decanted. Depending on the weight, the cell pellet was resuspended in 0.5-2.5 ml Saccomanno solution in order to get constant cell concentrations. Two drops of the cell suspension (about 0.2 ml) were applied to 6 slides each, smeared like a blood sample, avoiding nuclear artifacts or distortion by using capillary forces only. The slides were air-dried over night, two were stained according to Papanicolaou for CY, two according to a modified Feulgen reaction (14) for ASC, and 2 slides were kept for future reference.

Cytology

Conventional cytology was performed independently from cytometry. The classification was performed according to the following scheme. O: inadequate material, I: normal cell/nuclear structure, II: benign changes (mostly mild, moderate or severe signs of inflammation), III: metaplastic changes, IV: dysplasias (mild, moderate, or severe), and V: some/numerous malignant cells, carcinoma most probable. In case of mild and moderate dysplasia a second sputum sample was requested for confirmation of the results. In cases of high grade alterations - severe dysplasia or higher - immediate clinical investigations (endoscopy, chest radiography, etc.) were recommended.

Automated Sputum Cytometry

As described in detail in previous publications (1, 2), quantitative cytometry was performed by an automated image cytometer (Cyto-Savant®, Oncometrics, Vancouver, CDN). The technology followed the recommendations of the Task Force on Standardization of Quantitative Methods in Diagnostic Pathology (ESAPC) (15), including diffraction error correction. A specially designed trainable binary classifier for exfoliated cells of the lung was developed (1, 16). Beside photometric DNA-measurement, more than 110 nuclear features were calculated, quantifying chromatin distribution for each nucleus.

The automated mode allowed up to 50 slides daily to be screened unattended for a preset period of time (30 min) or for a limited number of nuclei. Up to 2000 cells (1000 normal epithelial cells, 200 suspicious nuclei with a DNA-index greater than 1.25 and smaller 2.5 and up to 100 highly suspicious ones with a DNA-Index greater 2.5 were collected from each slide. Along with the epithelial cells, up to 200 lymphocytes, polymorphonuclear and eosinophil granulocytes, and 100 alveolar macrophages were collected. For each batch of the 2000 cells, digital values of all features were calculated and stored. The DNA-amount of normal epithelial nuclei, referred to as the 2c-value or euploidy value, derived from a representative group of lymphocytes, was 2c ±0.25c and corresponded to a coefficient of variance of 12.5%. Three parameters were calculated from DNA-values of the epithelial nuclei: the percentual rate of 5c- exceeding nuclei (5cER) and the 2c-deviation index (2cDI) and the euploidy index (EI) in a modified version, log(2cDI*(1+5cER*10)+1)*1.683 (17, 18). Both parameters, 2cDI and 5cER, closely correlate to EI. The 5cER is the rate (in %) of aneuploid nuclei with a DNA-amount >5c. These are different from normal separating mitotic nuclei. Nuclei are called euploid if their DNA-amount is in the range 2c ±0.25c. 2cDI is defined as the sum of all squared deviations of the DNA-amount of all epithelial cells (ci) from the mean value (2c) divided by the number of cells (17).

The procedure allows to quantify the whole spectrum of malignancy with the help of a simple score. Following the recommendations of the ESACP (15) for quantitative cytometry, we used the following categories grade 0, inadequate or too few diagnostic cells from the airways (<1.000 cells), grade I, benign or normal nuclear structure, euploidy, EI <0.10, grade II, suspicious samples with EI >0.10, and grade III, highly suspicious or EI >0.25, and/or abnormal nuclei with an DNA-Index >2.5. In cases of grade II or III ASC and low grade cytological classification (mild and moderate dysplasia, metaplasia or normal and inflammatory CY), a second sputum sample was requested for confirmation.

Statistics

Sensitivity and specificity of the cytometrical investigations were tested against the final diagnosis of histologically confirmed lung cancer as the ”gold standard” (19). Similarly we tested the results of cytology against the final diagnosis. The confidence intervals for the 95% range (CI) were taken from Geigy, Scientific Tables. The ROC curve for EI was plotted to determine the suitable threshold, and to estimate the efficiency of ASC relating to the “gold standard” clinically relevant alterations, which were defined as histopathologically confirmed severe dysplasia and/or higher grade lesions. The comparison between the areas under the ROC curves for MG and 5cER were made using Delong’s method (20).


RESULTS
Sputum collection in patients with silicosis

In the former uranium miners, sputum induction resulted in 7.4 ±4.2 ml sputum, median 6 ml, which was significantly (P<0.005) less compared to 3 days pooled sputum 10.6 ±5.0 ml, median 10 ml. The range of sputum volumes was identical in both groups (1-25 ml).

ASC collected and analyzed fewer epithelial cells from induced sputum (1638 ±1030) compared to the pooled spontaneous samples (2163 ±1544, P<0.005). EI, as a parameter of DNA-distribution ,was not significantly different in both groups (0.065 ±0.016 and 0.064 ±0.017). Only 6 induced samples were inadequate for ASC/CY and another 15 spontaneous sputa. Benign or grade I results were obtained in 88.6% and 85.0% cases, respectively. The rates of suspicious or grade II results were 8.4% and 10.3%, respectively, and the rates of highly suspicious or grade III results were 0.5% and 0.9% and were not significantly different in both types of sputum collection. The counts of inflammatory cells and macrophages did not significantly differ in specimens derived from both methods.

Control Group

ASC of both slides from each control subject turned out to be normal in all but two cases, containing inflammatory cells, epithelial cells, and alveolar macrophages. All epithelial nuclei were found in a small DNA-range close to DNA-Index 1.00. Only few nuclei were found separate from the normal distribution, mainly as a small second peak at DNA-Index 2.0. All but 2 samples from smokers were classified as benign (grade I) by ASC. Quantitative evaluation of all samples derived from the control subjects led to a mean value of EI of 0.068 ±0.023. Comparing the sister slides of healthy subjects resulted in a mean difference of EI of 0.002 ±0.003 units, and led to identical grade I classifications.

Cytologically investigated sputum samples from asymptomatic non-smokers were classified as normal (I), mild, moderate or severe inflammatory (II), and in rare cases as inflammatory with cellular metaplasias (III). No indications for neoplastic alterations could be found in chest X-rays and in the final diagnosis.

Patients with Silicosis

Outcome of ASC. Of the 1517 samples, 21 (0.8%) were inadequate for ASC, 1358 (89.5%) benign or grade I, 120 (7.9%) were grade II or suspicious, and 18 (1.2%) grade III or highly suspicious. The mean value of EI for grade III results was 0.389 ±0.244 (median 0.284) and 0.135 ±0.035 (median 0.123) in grade II. The values for grade III and grade II were significantly (P<0.005) higher compared with EI for grade I (0.064 ±0.014, median 0.062) (Fig. 1).

Fig. 1. Frequency distribution of euploidy index (EI) derived from 1517 sputum samples from former uranium miners with silicosis. Cut-off values for grade II results were 0.10 and 0.25 for grade III results.

Correlation between the results from ASC and CY. Among the 1358 normal samples in ASC, the large majority (1313 or 95%) was mild, moderate or severe inflammatory (grade II) or with cellular metaplasias (grade III). Thirty four specimens contained nuclei with mild dysplasia, 6 with moderate und 5 with severe dysplasia (Table 1).

Table 1. Results for radon miners with silicosis (n = 1517).

In 120 patients with grade II ASC, 36 high grade alterations were seen in CY, 18 were classified as I - II and another 27 included metaplasias, 19 mild and 20 moderate dysplasias. Severe dysplasias were described in 22 cases and cell suspicious for tumor in 14 samples. A total amount of 36 from the 120 ASC positive samples of patients in final diagnosis were pathological by CY and considered for further investigation. Within the 18 patients with grade III results, 9 samples were classified by CY as tumor cell positive, 7 as severe dysplasia and 1 as mild and 1 as moderate dysplasia.

The results of the euploidy index in ASC were significantly (P<0.00005) correlated with the cytological alterations. For the 1158 cytological normal or inflammatory slides the mean value for EI was 0.066 ±0.018, and increased for moderate dysplasia by nearly 100%, and by 500% for slides suspicious for tumor cells (Table 2).

Table 2. Correlation between the euploidy index (EI) and the results of cytology.

Final Diagnosis. Of the 1517 occupationally radon progeny exposed patients 23 cases of lung cancer or metastasis from other cancers were found in the final diagnosis, representing the prevalence of 1.51%. In the 1494 of these patients silicosis was confirmed. Lung cancers were classified histologically as non-small cell (NSCLC) lung cancers, 2/3 were squamous epithelial cell carcinomas (SQuCLC), the others adenocarcinomas (Adeno), and one as an advanced pleural mesothelioma.

Fig. 2. Frequency distribution of euploidy index (EI) for high grade sputum cytology (CY) results (severe dysplasia and higher).

Sensitivity and specificity of ASC. At a cut off value of 0.10 for EI (Fig. 2), sensitivity of ASC was 87% (20/23) with a 95% confidence interval of 66.4 to 97.2% at a specificity of 92%, with a 95% confidence interval of 90.3 to 93.4%. Sensitivity of CY was 83% (19/23) with a 95% confidence interval of 61.2 to 95.1% at 98% specificity and a 95% confidence interval of 96.9 to 98.7%. Taking both methods together sensitivity of sputum investigations was 87% at a specificity of 92%.

Fig. 3. Receiver operated curve for ASC at a cut-off level of euploidy index at 0.10 for suspicious or grade II results. At specificity of 80%, sensitivity of ASC for high grade alterations in sputum and lung cancer was 84%.

DISCUSSION
Quantitative semi-automated image cytometry - ASC - of sputa from radon-exposed former uranium miners with silicosis was shown to be sensitive and reliable in detecting nuclear dysplasias and nuclei suspicious for lung cancer in sputum. Compared with the final diagnosis, we found a sensitivity of 87% (20/23) for ASC, which was slightly higher than that for cytology (83%, or 19/23). ASC-specificity (92%) was lower than that for cytology (98%). The results obtained represent a diagnostic efficiency of 89.3% for ASC and suggest the use of ASC as an effective tool for a screening program for lung cancer in high risk groups. Among the 1517 patients, 120 suspicious samples (grade II) and 18 highly suspicious samples for malignancy (grade III) were found by ASC, in 23 samples cells suspicious for tumor were confirmed by CY, and in 29 samples for severe dysplasias. In the final diagnosis, 23 tumors were reported; 20 of them were detected by ASC.

Comparable to the present study, the prevalence of lung cancer in RIDTELC Lung Study on heavy smokers was 1.1% (27/2480), which was within the expected prevalence of 1-2%. Of the 27 cancers found during the study, 2 did not receive ASC screening. Of the remaining 25 cancers, 11 had only positive results by sputum examination, 1 had both cytology and cytometry positive results, and 10 had only cytometry positive results.

All 9 patients with squamous cell cancer had positive DNA cytometric results, demonstrating a 100% sensitivity of image cytometry for this type of lung cancer. Most importantly, 8 out of 9 patients were found at an early stage (0-II), only one was at a later stage (III). So squamous cell carcinoma of lung could be detected at an early stage by DNA image cytometry. For adenocarcinoma of the lung, a high detection rate was evident as well, 8 from 11 patients (72.7%) had positive DNA cytometric results. Nevertheless, only 2 out of 8 patients detected were at early stage (I-II).

If we compare the early detection rate by sputum image cytometry and by conventional sputum cytology, we could find that among 13 early stage cancers (0-II stage), 10 could be detected by cytometry, only 1 could be detected by sputum cytology. So, we could expect that more cancers, especially more early stage cancers, may be detected if primarily screen high-risk population by sputum cytometry than by conventional cytology examination.

Currently, considerable research is being done using genetic and other molecular markers for early lung cancer on sputum samples, bronchial washings and brushings (21). Using microsattelite markers genetic alterations in bronchial lavage cells were detected in 35% (15/43) patients with lung cancer, but also in 23% (11/43) patients without evidence of neoplasias (22). In bronchial brushings from patients with lung cancer loss of heterozygosity (LOH) in at least one chromosomal locus was present in 79% whereas cytology was positive in 59% only (21). Microsattelite alterations were found in 49% (25/51) in tumor, normal bronchial mucosa and cytological specimens from patients with lung cancer (23). A number of mutations have been found in lung cancer such as allelic deletions of tumor suppressor genes or p53 alterations being present not only in lung cancer (24). Other approaches include specific monoclonal antibodies for recognition of human lung cancer antigen on sputum cells (25). These ongoing studies are still in an experimental stage and still must be validated clinically.

After detailed instruction, most of the patients investigated in the present study were able to expectorate sputum spontaneously. Sputum collection of patients with silicosis performed on three consecutive days as well as sputum induction by inhalation of aerosolized 3% saline - with few exceptions - mobilized sufficient diagnostic cells from the lower airways (26) and was well tolerated. Only 21 out of 1517 samples contained inadequate material for adequate ASC and CY, containing salivary cells but too few alveolar macrophages and columnar cells. Even in a healthy, non-COPD population, saline induction gave adequate results in over 80% (27). Compared with the quality of spontaneous sputum, induction with 3% saline mobilized sufficient sputum from the lower airways (13) and should be preferred to spontaneous sputum samples.

Addition of dithiothreitol (DTT) to the Saccomanno preservative (0.1% solution) resulted in a complete liquification of sputum specimen (13). Samples posted to our laboratory could be directly processed. After centrifugation, the pellet is resuspended in Saccomanno’s fixative depending on the size and smeared on glass slides similar to blood smears without shear forces altering the cellular integrity. This procedure results in an even distribution of diagnostic cells on all slides, which can not be obtained with the conventional “pick and smear” or even with the Saccomanno “blending-method”.

Revisiting 60 representative studies on the sensitivity of sputum cytology (28), an average sensitivity of 64.5% with a range of 22.0 to 98.1% was found. The reviewers’ own results amounted to a sensitivity of 40.3% of sputum cytology (28), which was comparable with earlier reports in the literature (29, 30), but not as high as the sensitivity of sputum cytology reported by Böcking and coworkers (31) using a combination of cytology on three separate samples, quantitative cytometry and immunohistochemistry (paraffin-embedded sputum).

Compared with earlier studies for cytological evaluation of sputum samples, bronchial washings or brush biopsies for the diagnosis of broncho-pulmonary tumors, our results obtained with the semi-automated cytometer had a higher sensitivity (1, 2). Several authors describe a dependency of the sensitivity of cytologically investigated sputa and bronchial washings on the tumor localization and size (28). A parallel investigation of endoscopic findings on 142 patients with suspected broncho-pulmonary tumors showed the following growth patterns: 22% mainly submucosal tumor growth, 44% exophytic tumors localized in the visual range, 11% peripheral lesions not visible with bronchoscopy. In our study on bronchial washings, we found a higher positive identification rate in those cases, with bronchoscopically visible tumors (84% for submucosal, and 90% for exophytic tumor growth). Without visible endoscopical changes, ASC could identify suspicious nuclei in 56% of the cases with peripheral lesions (32).

One could speculate that a certain portion of our ASC and CY “false positive” results represent early oncogenetic changes non-detectable by present means. A number of ASC positive results correspond to CY low grade alterations of different degree and cellular metaplasias, which are thought to be largely reversible. With the possibility of identifying these potential preneoplastic lesions more knowledge about the natural history of carcinogenesis may be acquired in the course of time. Inflammatory alterations of nuclear structure may also lead to misinterpretation. An altered decision threshold will have to be tested as well as a careful follow-up of patients without confirmed tumor and suspicious ASC/CY.

Interpretation of ASC classification in its current version depends on euploidy index (EI) as a measure of DNA-Euploidy and is represented by the morphological characteristics of nuclei presented in the gallery of abnormal nuclei in this report. In addition to the previous paper (1), a receiver operated curve (ROC) shows the alterations in sensitivity and specificity, altering the cut-off value for EI. Only a small percentage of low grade alteration were found with an EI <0.10. The development of a sample classifier, giving one integrative score for normal and abnormal samples, based on a couple of nuclear features differentiating normal from pathological nuclei in experimental stage. Features, such as variation-area, variation-intensity, fractal area, variation-radius, or long 45 runs, which may characterize pathologically transformed nuclei (16), are integrated into a sample classifier for sputum. One may speculate that future developments of ASC may allow the identification of histogenetic tumor types (SCLC, SQuCLC, Adeno, heterogeneously differentiated tumor or metastases of non primary lung cancers).

In case of persistent findings of samples classified as suspicious or highly suspicious by ASC and confirmed by CY as high grade alteration, subjects should be recommended radiological investigations, including low dose CT-scans and endoscopy. In cases where no tumor or preneoplasia was detected, sputum investigations should be repeated at 6 monthly intervals until either spontaneous remission or neoplastic changes occur. A follow-up of severe dysplasias, found in sputum samples, demonstrated malignancy in 46% of the cases (33). Saccomanno et al (34), investigating sputum from uranium miners who developed lung cancer over a follow-up over 10 years, detected atypias consistent with severe dysplasias in 60% of the cases in initial investigations. Squamous cell cancer developed in 83% in the course of time.

In summary, based on the results of our case finding study semi-automated sputum cytometry appears to be a sensitive non-invasive method for detection of malignant and possibly premalignant changes in the respiratory tract. Compared to other methods, it provides automated, attendant free and standardized cell collection, and enables interactive interpretation of the results. Patients with persistent suspicious ASC findings should undergo further diagnostic tests including at least conventional cytology. ASC in combination with low-dose CT and additional molecular diagnostic tests may be used in the near future for screening high risk groups of patients, such as occupationally exposed workers or heavy smokers. It was an essential element in the RIDTELC large scale feasibility study on early lung cancer detection in the high risk group of heavy smokers. Importantly, eupoidy index, combining 5cER and 2cDI measurements was a useful parameter for early detection of lung cancer by automated sputum image cytometry, especially for squamous cell lung carcinoma. Automated sputum image cytometry seems a feasible screening tool to detect lung cancer at an early, asymptomatic stage. Its specificity should be further investigated.


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