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Prognostic significance of tumor deoxyribonucleic acid content in surgically resected small-cell carcinoma of lung
General Thoracic Surgery
Prognostic significance of tumor deoxyribonucleic acid content in surgically resected small-cell carcinoma of lung The prognostic role of deoxyribonucleic acid flow cytometry was investigated in 53 cases of surgically resected small-cell lung cancer. Deoxyribonucleic acid aneuploidy was detected in 26 patients (49.1 %), the remaining tumors being either diploid or tetraploid. Patients with aneuploid tumors had a significantly reduced 2-year survival (38.5 % ) when compared with patients with diploid or tetraploid tumors (70.3 %; p < 0.05). This finding was independent of tumor stage on multiple logistic regression analysis. Diploid or tetraploid deoxyribonucleic acid content was associated with a particularly good 2-year survival (85 % ) in NO or Nl disease. Tumor deoxyribonucleic acid ploidy should be taken into account in planning of management and assessment of prognosis in small-cell lung cancer. (J 'fHORAC CARDInVASe SURG 1992;103:1214-7)
Francis A. Carey, MRCPath,a U. Sai Prasad, FRCS,b William S. Walker, FRCS,b Evan W. J. Cameron, FRCS,b David Lamb, FRCPath,a and Colin C. Bird, FRCPath,a Edinburgh, United Kingdom
Small-cell carcinoma of the lung (SCCL) is the most lethal form of lung cancer. It differs from the other main histologic types of bronchial carcinoma in its biology' and also in having a more aggressive natural history.i Although combination chemotherapy has been the cornerstone of treatment in this disease, an increasing number of studies have shown considerable merit in surgical resection, particularly for patients with limited-stage disease. 3-7 Flow cytometric estimation of tumor deoxyribonucleic From the Departments of Pathology"and Thoracic Surgery.?University of Edinburgh, Edinburgh, United Kingdom. Partially supported by the Cancer Research Campaign (U.K.). Receivedfor publicationOct. 16, 1990. Accepted for publication March IS, 1991. Address for reprints: F. A. Carey, MRCPath, BCh, Department of Pathology, University Medical School, Teviot Place, Edinburgh EH8 9AG, United Kingdom. 12/1/30410
12 I 4
acid (DNA) content has been shown to be a useful indicator of prognosis in many common neoplasms, including surgically treated non-small-celllung cancer."!' In the case of SCCL, which is routinely surgically resected at our center whenever possible, the most consistent prognostic indicator has been tumor stage or TNM status'? We present an evaluation of the application of DNA flow cytometry in this tumor and an assessment of its prognostic use in conjunction with clinicopathologic staging. Methods Formalin-fixed, paraffin-embedded pathologic material from 55 patients with a diagnosis of SCCL after examination of lobectomy or pneumonectomy specimens in the years 19811987, inclusive, was retrieved from the files of the Department of Pathology, University of Edinburgh. Material received before 1981 was excluded because the tissue had been postfixed in Bouin's solution and was found to be unsuitable for DNA analysis. Hematoxylin and eosin-stained sections were reviewed for each tumor, and in two patients the diagnosis was revised to "atypical carcinoid tumor." These patients were excluded from further study. All tumor blocks from the remaining patients
Volume 103 Number 6 June 1992
Tumor DNA content in small-cell carcinoma of lung
Table I. Effect of DNA content on 2-year survival
Go/G.
according to node status
Limited stage (NO/NO Advanced stage (N2) All patients
Diploid/ tetraploid
Aneuploid
17/20* (85%) 2/7 (29%) 19/27 (70%)
9/22 (41%) 1/4 (25%) 10/26 (38%)
I2I5
The difference in survival between ploidysubgroupsis significant (p < 0.05 multiplelogistic regression). 'Survivors out of total in each category.
were included for DNA analysis to avoid possible errors arising in the sampling of these potentially heterogeneous neoplasms. 12 Tissue blocks for DNA analysis had 50 ~m sections cut and plated onto glass slides. Portions of nonnecrotic tumor tissue were selected with a scalpel blade from these slides to obtain the best possible sample for estimation of the true tumor DNA content, avoiding errors caused by masking of tumor aneuploidy by normal (diploid) lung material present in most tissue blocks.'? The tumor tissue was dewaxed in xylene and rehydrated in alcohol according to the protocol of Hedley and coworkers. 14 The sample was mechanically minced and digested in 0.5% trypsin in 0.9% sodium chloride adjusted to pH 1.5 with hydrochloric acid. The nuclear suspension obtained was incubated overnight in propidium iodide and filtered through nylon wool before being analyzed on an EPICS CS flow cytometer (Coulter Electronics, Inc., Hialheah, Fla). At least 10,000 nuclei were analyzed for each sample. The resulting DNA histograms were classified as diploid, tetraploid, or aneuploid (Figs. I and 2). A coefficient of variation was measured on the diploid peak in each patient as a quality control assessment. Only histograms with a coefficient of variation of less than 5.0 were accepted. For aneuploid samples the DNA index was defined as the ratio of the channel number of the aneuploid peak to that of the diploid peak. A tetraploid histogram was defined by a DNA index in the range of 1.9 to 2.1. Data on clinical outcome were obtained from case notes in the department of thoracic surgery and have been reported as part of a previous study," TNM staging was performed by correlation of operative and histologic findings. All patients had preoperative posteroanterior and lateral chest radiographs, bronchoscopy, barium swallow, and diaphragmatic screening. Mediastinoscopy was not routinely performed. Computed tomographic scans and radioisotope and ultrasound examinations for extra thoracic metastatic disease were performed only when indicated by clinical or biochemical abnormality. Since the diagnosis of SCCL is usually followed rapidly by death, less than 20% of patients being alive 2 years after diagnosis in most series, survival at 2 years after operation was taken as a suitable end point in assessing the value of prognostic indicators. Patients with early-stage (NO N 1) cancer were treated by surgery alone, whereas those with more advanced disease (N2, stage III) received postoperative chemotherapy. The standard regimen consisted of cyclophosphamide (I gm/rn-), doxorubicin (40 mg/m''), and vincristine (I mg/m'') given at 3week intervals for three cycles. The relative prognostic significance of TNM staging and DNA content was estimated on univariate and multivariate analysis (multiple logistic regression test).
Fig. 1. DNA histogram from a diploid tumor. A single GO/G 1 and a smaller G2/M peak are identified.
Fig. 2. DNA histogram from an aneuploid tumor. A second (aneuploid) GO/GI peak is obvious (arrow).
Results Full clinical data were available in 53 patients. On pathologic staging 11 patients had mediastinal lymph node involvement (N2). The remaining patients had NO (no lymph node involvement) or N I (ipsilateral hilar node metastasis) disease. All patients were clinically free of distant metastases (MO). The cumulative 2-year survival was, irrespective of node status, 54.7% (29/53 patients), for limited-stage (NO/NI) disease, 61.9% (26/42), and for advanced disease (N2), 27.3% (3/11). DNA analysis was performed on 250 samples (mean 4.5 per patient). A tumor was characterized as aneuploid or tetraploid if anyone of the samples showed the characteristic abnormality. DNA heterogeneity (variation in ploidy between different samples within a tumor) was noted in 18 patients (40%). Aneuploidy was detected in 26 patients (49.1 %), 15 patients had tetraploid histograms (28.3%), and all samples were diploid in 12 tumors (22.5%). Since, generally, tetraploid tumors tend to behave in a similar fashion to those with diploid DNA content, 15 these patients were considered as a group when looking at prognostic data. At 2 years 19 of 27 (70.3%) patients with diploid or tetraploid tumors but only 10 of
The Journal-of Thoracic and Cardiovascular Surgery
1 2 1 6 Carey et al.
26 (38.5%) patients with aneuploid tumors were still alive. Table I shows that diploid or tetraploid tumors tended to do better at any stage of disease than did those with an aneuploid DNA content. On univariate analysis, limited-stage disease, diploid or tetraploid DNA content, and homogeneity of ploidy status were all significantly associated with increased survival at 2 years (p < 0.05 in each case). When the relationship of each of these prognostic indicators to each other was tested on multivariate analysis, ploidy homogeneity dropped out as an independent variable, leaving tumor stage and flow cytometric DNA content as independent prognostic variables with comparable predictive powers.
Discussion In studies of survival in SCCL many clinical, biochemical, and pathologic parameters have been investigated for prognostic significance. In general good performance status at diagnosis and limited stage of disease have been consistently associated with a more favorable outcome. 16, 17 Serologic markers such as carcinoembryonic antigen'" and neuron-specific enolase'? have been extensively investigated, but the clinical application of these assays is as yet uncertain. Careful histopathologic review of all patients is of particular importance in studies reporting survival figures for SCCL to exclude morphologically similar lesions, particularly atypical carcinoid tumor (well-differentiated neuroendocrine carcinomaj.P 21 This task is relatively easier on surgically resected than on biopsy material, two such patients being identified and excluded from the series reported here. The extremely low cure rates reported in large series of patients treated by cytotoxic chemotherapy or radiotherapy, or both, have led an increasing number of workers to question the prevailing wisdom that SCCL is not amenable to surgical intervention.Y' We have previously reported a relatively good prognosis in patients undergoing resection for SCCL7 and have noted that TNM stage is a good predictor of outcome in these patients. We have now described, in DNA ploidy, the first prognostic indicator that is independent of tumor stage while being an equally good predictor of clinical outcome. It is particularly interesting to note that patients with early-stage tumors (NO/NO with a diploid or tetraploid DNA content have an 85% survival at 2 years (see Table I). Although it may be that patients who have limited disease at initial presentation are both biologically and clinically the exception rather than the rule, we believe that careful clinicopathologic staging of newly diagnosed SCCL is of importance in identifying patients for whom surgical
treatment may offer a relatively good hope of cure. Indeed, it is our belief that all patients with operable SCCL should be offered resection. The technique of DNA flow cytometry that is becoming increasingly available in clinical laboratories is reliable, reproducible, and applicable to both fresh and fixed paraffin-embedded tumor material," There is also some evidence that, in the case of SCCL, DNA content analysis may make a contribution in selecting patients who might benefit from postoperative adjuvant chemotherapy. Abe and coworkers.F using a microspectrophotometer to measure nuclear DNA content in cells obtained by bronchial brushing, found that patients with SCCL showing aneuploid DNA histograms were significantly more responsive to chemotherapy than those having a more normal DNA content. In the future, judicious application of clinical, TNM, and ploidy data may allow for more rational treatment options in this common and highly malignant tumor. We thank R. A. Elton, PhD, for statistical advice. REFERENCES 1. Weynants P, Humblet Y, Canon JL, Symann M. Biology of small cell lung cancer: an overview. Eur Respir J 1990; 3:699-714. 2. Souhami RL, Law K. Longevity in small cell lung cancer: a report to the lung cancer subcommittee of the United Kingdom coordinating committee for cancer research. Br J Cancer 1990;61:584-9. 3. Shore DF, Paneth M. Survival after resection of small cell carcinoma of the bronchus. Thorax 1980;35:819-22. 4. Meyer JA. Surgical treatment of lung carcinoma: indications for surgical treatment in small cell carcinoma of the lung. Surg Clin North Am 1987;67:1103-5. 5. Karrer K, Shields TW, Denck H, Hrabar B, Vogt-Moykopf I, Salzer GM. The importance of surgical and multimodality treatment for small cell bronchial carcinoma. J THORAC CARDIOVASC SURG 1989;97:168-76. 6. Ishida T, Nisino T, Oka T, et al. Surgical treatment of patients with small cell carcinoma of the lung: a histochemical and immunohistochemical study. J Surg Oncol 1989; 40:188-93. 7. Prasad US, Naylor AR, Walker WS, Lamb D, Cameron EWJ, Walbaum PRo Long term survival after pulmonary resection for small cell carcinoma of the lung. Thorax 1989;44:784-7. 8. Hedley DW. Flow cytometry using paraffin-embedded tissue: five years on. Cytometry 1989;10:229-41. 9. Zimmerman PV, Hawson GAT, Bint MH, Parsons PG. Ploidy as a prognostic determinant in surgically treated lung cancer. Lancet 1987;2:530-3. 10. Volm M, Drings P, Mattern J, et al. Prognostic significance of DNA patterns and resistance-predictive tests in nonsmall cell lung carcinoma. Cancer 1985;59:1396-403.
Volume 103 Number 6 June 1992
Tumor DNA content in small-cell carcinoma of lung
II. Dazzi H, Thatcher N, Hasleton PS, Swindell R. DNA analysis by flow cytometry in non-small cell lung cancer: relationship to epidermal growth factor receptor, histology, tumour stage and survival. Respir Med 1990;84:217-23. 12. Carey FA, Lamb D, Bird CC. Intratumoral heterogeneity of DNA content in lung cancer. Cancer 1990;65:2266-9. 13. Carey FA, Lamb D, Bird Cc. Importance of sampling method in DNA analysis of lung cancer. J Clin Pathol 1990;43:820-3. 14. Hedley DW, Friedlander ML, Taylor IW, etal. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem 1983;31:1333-5. 15. Quirke P, Dixon MF, Clayden AD, et al. Prognostic significance of DNA aneuploidy and cell proliferation in rectal adenocarcinoma. J PathoI1987;151:285-91. 16. Niiranen A. Long-term survival in small cell carcinoma of the lung. Eur J Cancer Clin OncoI1988;24:749-52. 17. Rawson NS, Peto J. An overview of prognostic factors in small cell lung cancer. A report from the subcommittee for the management of lung cancer of the United Kingdom coordinating committee on cancer research. Br J Cancer 1990;61:597-604.
12 1 7
18. Bepler G, Ostholt M, Neumann K, et al. Carcinoembryonic antigen as differentiation marker for small cell lung cancer in vitro and its clinical relevance. Anticancer Res 1989;9:1525-30. 19. Nou E, Steinholtz L, Bergh J, Nilsson K, Pahlman S. Neuron-specific enolase as a follow-up marker in small cell bronchial carcinoma: a prospective study in an unse1ected series. Cancer 1990;65:1380-5. 20. Warren WH, Menoli VA, Jordan AG, Gould VE. Reevaluation of pulmonary neoplasms resected as small cell carcinomas: significance of distinguishing between well-differentiated and small cell neuroendocrine carcinomas. Cancer 1990;65:I003-10. 21. Lamb D. Lung cancer and its classification. In: Anthony PP, MacSween RNM, eds. Recent advances in histopathology 13, Edinburgh: Churchill Livingstone, 1987;45-59. 22. Abe S, Tsuneta Y, Makirnura S, Itabashi K, Nagai T, Kawakami Y. Nuclear DNA content as an indicator of chemosensitivity in small cell carcinoma of the lung. Anal Quant CytoI1987;9:425-8.
Prognostic significance of tumor deoxyribonucleic acid content in surgically resected small-cell carcinoma of lung The prognostic role of deoxyribonucleic acid flow cytometry was investigated in 53 cases of surgically resected small-cell lung cancer. Deoxyribonucleic acid aneuploidy was detected in 26 patients (49.1 %), the remaining tumors being either diploid or tetraploid. Patients with aneuploid tumors had a significantly reduced 2-year survival (38.5 % ) when compared with patients with diploid or tetraploid tumors (70.3 %; p < 0.05). This finding was independent of tumor stage on multiple logistic regression analysis. Diploid or tetraploid deoxyribonucleic acid content was associated with a particularly good 2-year survival (85 % ) in NO or Nl disease. Tumor deoxyribonucleic acid ploidy should be taken into account in planning of management and assessment of prognosis in small-cell lung cancer. (J 'fHORAC CARDInVASe SURG 1992;103:1214-7)
Francis A. Carey, MRCPath,a U. Sai Prasad, FRCS,b William S. Walker, FRCS,b Evan W. J. Cameron, FRCS,b David Lamb, FRCPath,a and Colin C. Bird, FRCPath,a Edinburgh, United Kingdom
Small-cell carcinoma of the lung (SCCL) is the most lethal form of lung cancer. It differs from the other main histologic types of bronchial carcinoma in its biology' and also in having a more aggressive natural history.i Although combination chemotherapy has been the cornerstone of treatment in this disease, an increasing number of studies have shown considerable merit in surgical resection, particularly for patients with limited-stage disease. 3-7 Flow cytometric estimation of tumor deoxyribonucleic From the Departments of Pathology"and Thoracic Surgery.?University of Edinburgh, Edinburgh, United Kingdom. Partially supported by the Cancer Research Campaign (U.K.). Receivedfor publicationOct. 16, 1990. Accepted for publication March IS, 1991. Address for reprints: F. A. Carey, MRCPath, BCh, Department of Pathology, University Medical School, Teviot Place, Edinburgh EH8 9AG, United Kingdom. 12/1/30410
12 I 4
acid (DNA) content has been shown to be a useful indicator of prognosis in many common neoplasms, including surgically treated non-small-celllung cancer."!' In the case of SCCL, which is routinely surgically resected at our center whenever possible, the most consistent prognostic indicator has been tumor stage or TNM status'? We present an evaluation of the application of DNA flow cytometry in this tumor and an assessment of its prognostic use in conjunction with clinicopathologic staging. Methods Formalin-fixed, paraffin-embedded pathologic material from 55 patients with a diagnosis of SCCL after examination of lobectomy or pneumonectomy specimens in the years 19811987, inclusive, was retrieved from the files of the Department of Pathology, University of Edinburgh. Material received before 1981 was excluded because the tissue had been postfixed in Bouin's solution and was found to be unsuitable for DNA analysis. Hematoxylin and eosin-stained sections were reviewed for each tumor, and in two patients the diagnosis was revised to "atypical carcinoid tumor." These patients were excluded from further study. All tumor blocks from the remaining patients
Volume 103 Number 6 June 1992
Tumor DNA content in small-cell carcinoma of lung
Table I. Effect of DNA content on 2-year survival
Go/G.
according to node status
Limited stage (NO/NO Advanced stage (N2) All patients
Diploid/ tetraploid
Aneuploid
17/20* (85%) 2/7 (29%) 19/27 (70%)
9/22 (41%) 1/4 (25%) 10/26 (38%)
I2I5
The difference in survival between ploidysubgroupsis significant (p < 0.05 multiplelogistic regression). 'Survivors out of total in each category.
were included for DNA analysis to avoid possible errors arising in the sampling of these potentially heterogeneous neoplasms. 12 Tissue blocks for DNA analysis had 50 ~m sections cut and plated onto glass slides. Portions of nonnecrotic tumor tissue were selected with a scalpel blade from these slides to obtain the best possible sample for estimation of the true tumor DNA content, avoiding errors caused by masking of tumor aneuploidy by normal (diploid) lung material present in most tissue blocks.'? The tumor tissue was dewaxed in xylene and rehydrated in alcohol according to the protocol of Hedley and coworkers. 14 The sample was mechanically minced and digested in 0.5% trypsin in 0.9% sodium chloride adjusted to pH 1.5 with hydrochloric acid. The nuclear suspension obtained was incubated overnight in propidium iodide and filtered through nylon wool before being analyzed on an EPICS CS flow cytometer (Coulter Electronics, Inc., Hialheah, Fla). At least 10,000 nuclei were analyzed for each sample. The resulting DNA histograms were classified as diploid, tetraploid, or aneuploid (Figs. I and 2). A coefficient of variation was measured on the diploid peak in each patient as a quality control assessment. Only histograms with a coefficient of variation of less than 5.0 were accepted. For aneuploid samples the DNA index was defined as the ratio of the channel number of the aneuploid peak to that of the diploid peak. A tetraploid histogram was defined by a DNA index in the range of 1.9 to 2.1. Data on clinical outcome were obtained from case notes in the department of thoracic surgery and have been reported as part of a previous study," TNM staging was performed by correlation of operative and histologic findings. All patients had preoperative posteroanterior and lateral chest radiographs, bronchoscopy, barium swallow, and diaphragmatic screening. Mediastinoscopy was not routinely performed. Computed tomographic scans and radioisotope and ultrasound examinations for extra thoracic metastatic disease were performed only when indicated by clinical or biochemical abnormality. Since the diagnosis of SCCL is usually followed rapidly by death, less than 20% of patients being alive 2 years after diagnosis in most series, survival at 2 years after operation was taken as a suitable end point in assessing the value of prognostic indicators. Patients with early-stage (NO N 1) cancer were treated by surgery alone, whereas those with more advanced disease (N2, stage III) received postoperative chemotherapy. The standard regimen consisted of cyclophosphamide (I gm/rn-), doxorubicin (40 mg/m''), and vincristine (I mg/m'') given at 3week intervals for three cycles. The relative prognostic significance of TNM staging and DNA content was estimated on univariate and multivariate analysis (multiple logistic regression test).
Fig. 1. DNA histogram from a diploid tumor. A single GO/G 1 and a smaller G2/M peak are identified.
Fig. 2. DNA histogram from an aneuploid tumor. A second (aneuploid) GO/GI peak is obvious (arrow).
Results Full clinical data were available in 53 patients. On pathologic staging 11 patients had mediastinal lymph node involvement (N2). The remaining patients had NO (no lymph node involvement) or N I (ipsilateral hilar node metastasis) disease. All patients were clinically free of distant metastases (MO). The cumulative 2-year survival was, irrespective of node status, 54.7% (29/53 patients), for limited-stage (NO/NI) disease, 61.9% (26/42), and for advanced disease (N2), 27.3% (3/11). DNA analysis was performed on 250 samples (mean 4.5 per patient). A tumor was characterized as aneuploid or tetraploid if anyone of the samples showed the characteristic abnormality. DNA heterogeneity (variation in ploidy between different samples within a tumor) was noted in 18 patients (40%). Aneuploidy was detected in 26 patients (49.1 %), 15 patients had tetraploid histograms (28.3%), and all samples were diploid in 12 tumors (22.5%). Since, generally, tetraploid tumors tend to behave in a similar fashion to those with diploid DNA content, 15 these patients were considered as a group when looking at prognostic data. At 2 years 19 of 27 (70.3%) patients with diploid or tetraploid tumors but only 10 of
The Journal-of Thoracic and Cardiovascular Surgery
1 2 1 6 Carey et al.
26 (38.5%) patients with aneuploid tumors were still alive. Table I shows that diploid or tetraploid tumors tended to do better at any stage of disease than did those with an aneuploid DNA content. On univariate analysis, limited-stage disease, diploid or tetraploid DNA content, and homogeneity of ploidy status were all significantly associated with increased survival at 2 years (p < 0.05 in each case). When the relationship of each of these prognostic indicators to each other was tested on multivariate analysis, ploidy homogeneity dropped out as an independent variable, leaving tumor stage and flow cytometric DNA content as independent prognostic variables with comparable predictive powers.
Discussion In studies of survival in SCCL many clinical, biochemical, and pathologic parameters have been investigated for prognostic significance. In general good performance status at diagnosis and limited stage of disease have been consistently associated with a more favorable outcome. 16, 17 Serologic markers such as carcinoembryonic antigen'" and neuron-specific enolase'? have been extensively investigated, but the clinical application of these assays is as yet uncertain. Careful histopathologic review of all patients is of particular importance in studies reporting survival figures for SCCL to exclude morphologically similar lesions, particularly atypical carcinoid tumor (well-differentiated neuroendocrine carcinomaj.P 21 This task is relatively easier on surgically resected than on biopsy material, two such patients being identified and excluded from the series reported here. The extremely low cure rates reported in large series of patients treated by cytotoxic chemotherapy or radiotherapy, or both, have led an increasing number of workers to question the prevailing wisdom that SCCL is not amenable to surgical intervention.Y' We have previously reported a relatively good prognosis in patients undergoing resection for SCCL7 and have noted that TNM stage is a good predictor of outcome in these patients. We have now described, in DNA ploidy, the first prognostic indicator that is independent of tumor stage while being an equally good predictor of clinical outcome. It is particularly interesting to note that patients with early-stage tumors (NO/NO with a diploid or tetraploid DNA content have an 85% survival at 2 years (see Table I). Although it may be that patients who have limited disease at initial presentation are both biologically and clinically the exception rather than the rule, we believe that careful clinicopathologic staging of newly diagnosed SCCL is of importance in identifying patients for whom surgical
treatment may offer a relatively good hope of cure. Indeed, it is our belief that all patients with operable SCCL should be offered resection. The technique of DNA flow cytometry that is becoming increasingly available in clinical laboratories is reliable, reproducible, and applicable to both fresh and fixed paraffin-embedded tumor material," There is also some evidence that, in the case of SCCL, DNA content analysis may make a contribution in selecting patients who might benefit from postoperative adjuvant chemotherapy. Abe and coworkers.F using a microspectrophotometer to measure nuclear DNA content in cells obtained by bronchial brushing, found that patients with SCCL showing aneuploid DNA histograms were significantly more responsive to chemotherapy than those having a more normal DNA content. In the future, judicious application of clinical, TNM, and ploidy data may allow for more rational treatment options in this common and highly malignant tumor. We thank R. A. Elton, PhD, for statistical advice. REFERENCES 1. Weynants P, Humblet Y, Canon JL, Symann M. Biology of small cell lung cancer: an overview. Eur Respir J 1990; 3:699-714. 2. Souhami RL, Law K. Longevity in small cell lung cancer: a report to the lung cancer subcommittee of the United Kingdom coordinating committee for cancer research. Br J Cancer 1990;61:584-9. 3. Shore DF, Paneth M. Survival after resection of small cell carcinoma of the bronchus. Thorax 1980;35:819-22. 4. Meyer JA. Surgical treatment of lung carcinoma: indications for surgical treatment in small cell carcinoma of the lung. Surg Clin North Am 1987;67:1103-5. 5. Karrer K, Shields TW, Denck H, Hrabar B, Vogt-Moykopf I, Salzer GM. The importance of surgical and multimodality treatment for small cell bronchial carcinoma. J THORAC CARDIOVASC SURG 1989;97:168-76. 6. Ishida T, Nisino T, Oka T, et al. Surgical treatment of patients with small cell carcinoma of the lung: a histochemical and immunohistochemical study. J Surg Oncol 1989; 40:188-93. 7. Prasad US, Naylor AR, Walker WS, Lamb D, Cameron EWJ, Walbaum PRo Long term survival after pulmonary resection for small cell carcinoma of the lung. Thorax 1989;44:784-7. 8. Hedley DW. Flow cytometry using paraffin-embedded tissue: five years on. Cytometry 1989;10:229-41. 9. Zimmerman PV, Hawson GAT, Bint MH, Parsons PG. Ploidy as a prognostic determinant in surgically treated lung cancer. Lancet 1987;2:530-3. 10. Volm M, Drings P, Mattern J, et al. Prognostic significance of DNA patterns and resistance-predictive tests in nonsmall cell lung carcinoma. Cancer 1985;59:1396-403.
Volume 103 Number 6 June 1992
Tumor DNA content in small-cell carcinoma of lung
II. Dazzi H, Thatcher N, Hasleton PS, Swindell R. DNA analysis by flow cytometry in non-small cell lung cancer: relationship to epidermal growth factor receptor, histology, tumour stage and survival. Respir Med 1990;84:217-23. 12. Carey FA, Lamb D, Bird CC. Intratumoral heterogeneity of DNA content in lung cancer. Cancer 1990;65:2266-9. 13. Carey FA, Lamb D, Bird Cc. Importance of sampling method in DNA analysis of lung cancer. J Clin Pathol 1990;43:820-3. 14. Hedley DW, Friedlander ML, Taylor IW, etal. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem 1983;31:1333-5. 15. Quirke P, Dixon MF, Clayden AD, et al. Prognostic significance of DNA aneuploidy and cell proliferation in rectal adenocarcinoma. J PathoI1987;151:285-91. 16. Niiranen A. Long-term survival in small cell carcinoma of the lung. Eur J Cancer Clin OncoI1988;24:749-52. 17. Rawson NS, Peto J. An overview of prognostic factors in small cell lung cancer. A report from the subcommittee for the management of lung cancer of the United Kingdom coordinating committee on cancer research. Br J Cancer 1990;61:597-604.
12 1 7
18. Bepler G, Ostholt M, Neumann K, et al. Carcinoembryonic antigen as differentiation marker for small cell lung cancer in vitro and its clinical relevance. Anticancer Res 1989;9:1525-30. 19. Nou E, Steinholtz L, Bergh J, Nilsson K, Pahlman S. Neuron-specific enolase as a follow-up marker in small cell bronchial carcinoma: a prospective study in an unse1ected series. Cancer 1990;65:1380-5. 20. Warren WH, Menoli VA, Jordan AG, Gould VE. Reevaluation of pulmonary neoplasms resected as small cell carcinomas: significance of distinguishing between well-differentiated and small cell neuroendocrine carcinomas. Cancer 1990;65:I003-10. 21. Lamb D. Lung cancer and its classification. In: Anthony PP, MacSween RNM, eds. Recent advances in histopathology 13, Edinburgh: Churchill Livingstone, 1987;45-59. 22. Abe S, Tsuneta Y, Makirnura S, Itabashi K, Nagai T, Kawakami Y. Nuclear DNA content as an indicator of chemosensitivity in small cell carcinoma of the lung. Anal Quant CytoI1987;9:425-8.