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Adenocarcinoma of the lung: its development and malignant progression
zmg cancer, 9 (1993) 99-108 @ 1993 Elxvier
99
Scicntif~ Publishers Ireland Ltd. All rights reserved 0169-5OOu93iW6.00
LUNG 00124
Adenocarcinoma of the lung: its development and malignant progression Yukio Shimosato,
Masayuki
Noguchi
and Yoshihiro
Matsuno
Clinical Laboratory and Pathology Divisions, National Cancer Center Hospitaland Research Institute, Tokyo, Japan
Key words: Adenocarcinoma; Human lung; Development; Malignant progression; clear DNA content @loidy); myc gene; p53 gene; Immunohistochemistry
Nu-
Introduction Adenocarcinoma of the lung is the most common lung cancer in Japan and is said to be increasing in some other countries. Etiologically it is less strongly associated with cigarette smoking than squamous cell carcinoma and small cell carcinoma. Histologically and cytologically, it is divided into many subtypes, and biologically it is a heterogeneous group of tumors ranging from very slow growing and low grade malignant tumors to fast growing highly malignant tumors. This paper will focus on the development and malignant progression of adenocarcinoma of the lung from the morphological, biological, molecular and genetic points of view.
The majority of adenocarcinomas of the lung develop in the peripheral airway, that is, the bronchioloalveolar region. Most peripheral adenocarcinomas contain a central or subpleural anthracotic and fibrotic focus, which is often associated with a pleural indentation. This characteristic feature is the basis for the concept of scar cancer, which has been believed to be common in the lung for many years. Although we do not deny the existence of such scar cancers in the lung, we believe that most adenocarcinomas encountered in clinical practice develop either de novo or through the stage of atypical adenomatous hyperplasia or adenoma not associated with scar [16].
Correspondence to: Y. Shimosato, M.D., Clinical Laboratory Division, National Cancer Center Hospital, 5-I-1, Tsukiji, Chuoku Tokyo 104, Japan.
Fig. I. Histology of atypical adenomatous hyperplasia (or adenoma). Slightly to moderately atypical cells with increased nuclear cytoplasmic ratio replace the lining of alveoli, whose septae are slightly thickened. Cytoplasm of some cells are finely vesicular, suggesting type II alveolar epithelial cell type. Within this lesion is a small focus of well differentiated adenocarcinoma.
Rebuttal of the scar cancer concept Although carcinoma arising in diffuse pulmonary fibrosis may be categorized as an example of scar cancer, we believe that in most adenocarcinomas seen in routine practice the central fibrosis or fibrotic focus with anthracosis forms not before, but after, the development of carcinoma [ 171. This occurs as the result of collapse of alveoli secondary to degeneration and death of tumor cells which had lined the alveoli, as a result of impaired blood flow. This idea is supported by the fact that in early stage adenocarcinoma a fibrotic focus is usually absent and also by the absence of collagenization and fibroblastic proliferation in areas with dense aggregates of elastic fibers, which constituted alveolar septae. Convergence of bronchi and pulmonary vessels toward the tumor, which is often associated with pleural indentation, is observed frequently on chest X-ray films and provides evidence supporting the morphological events described above [ 181. Subsequently, fibroblastic cell proliferation and collagenization follow. It is also easy to understand the presence of central fibrosis in distant metastatic foci. There are several other reasons to support this explanation of the process of central fibrosis [17]. De novo carcinogenesis or adenoma-carcinoma sequence If the occurrence of scar cancer is uncommon in the lung, how do adenocarcinomas develop? The first concept to consider is de novo development, but this is very difficult to
Fig. 2. Immunostaining for PCNA of well differentiated adenocarcinoma associated with atypical adenomatous hyperplasia (or adenoma). In the periphery of the lesion with slight nuclear atypia, PCNA positive cells are rare (left), but in the central portion with increased nuclear atypia, PCNA positive nuclei are scattered (right).
prove. We have not infrequently encountered papillary or bronchioloalveolar lesions, which suggest the development of adenocarcinoma in a focus of atypical adenomatous hyperplasia or adenoma [7,11]. Miller and her associates have also suggested the adenomacarcinoma sequence in the lung [9,10]. Such adenomatous lesions are often less than 1 cm in diameter, single or multiple and are composed of atypical cuboidal cells with increased nuclear cytoplasmic ratio (but not atypical enough to call carcinoma) replacing the lining of alveoli. Scanty cytoplasm may contain small vesicles or needle like clefts. They resemble either Clara cells or type II alveolar epithelial cells. Mitotic figures are hardly seen (Fig. 1). Within such a lesion, an area with increased cellularity and cell atypia sufficient enough to call well differentiated adenocarcinoma may be seen. Such atypical adenomatous lesions are not infrequently seen at the periphery of incontrovertible papillary adenocarcinoma, suggesting a late stage of the adenoma-carcinoma sequence. So far we have investigated 5 cases of solitary atypical adenomatous lesions and 9 cases of adenocarcinoma associated with atypical adenomatous lesions [ 111. Analysis of the nuclear DNA content of atypical adenomatous lesions reveals either diploidy, polyploidy or aneuploidy suggesting clonal growth, with or without development of subclones with more abnormal DNA content. Positively stained cells for proliferating cell nuclear antigen (PCNA) are few in number in atypical adenomatous lesions, but increase in areas with increased cell atypia and density (Fig. 2). CEA is generally negative in the former and positive in the latter. From such findings, we now believe such lesions to be neoplastic, either adenoma or extremely well-differentiated adenocarcinoma with
Fig. 3. Histology of adenocarcinoma showing malignant progression, An atypical adenomatous lesion (or adenoma) is seen in the periphery of the tumor (left), features of well differentiated papillary adenocarcinoma are seen in the intermediate zone of the tumor (center), and solid nests consisting of more atypical cells are present in the center of the tumor (right).
bronchioloalveolar carcinoma.
growth pattern.
If the latter is the case, it may be called de novo
Malignant progression Similar to the process of development of adenocarcinoma in adenoma, histologically and cytologically more atypical or more malignant appearing areas develop within carcinoma (Fig. 3). Such morphological progression can be confirmed not only in hematoxylin and eosin stained sections but also by other methods such as immunohistochemistry for CEA (Fig. 4) and markers for proliferative activity, cytofluorometry or flow cytometry, and probably by analysis of genetic changes. Nuclear DNA content and occurrence of aneuploid stem line Mean nuclear DNA content has been shown to increase in less differentiated adenocarcinoma (Fig. 5), and is significantly greater in stage 1 adenocarcinomas with recurrence within 5 years after surgery than those without recurrence [ 1,2]. Within a primary tumor, the mean nuclear DNA content of tumor cells in areas with increased amount of stroma or with apparent invasive growth is definitely greater than that in areas with thin stroma made up of alveolar septae [12]. Comparison of mean nuclear DNA contents of
Fig. 4. CEA immunostaining showing malignant progression in a single tumor. No positively stained cells are present in the atypical adenomatous lesion (adenoma) (left). Moderate cytoplasmic stain is noted in areas with features of well differentiated adenocarcinoma (center), and marked cytoplasmic staining for CEA in the poorly differentiated area (right).
primary tumors and metastatic foci in lymph nodes, brain and liver disclosed that hematogenous metastases were frequently composed of tumor cells with greater mean nuclear DNA content showing more abnormal histogram patterns than those in the primary tumor; whereas the mean nuclear DNA contents in lymph node metastases, particularly metastases to the hilar lymph nodes, were not greater than the primary tumor, although cells with greater DNA contents were noted in the mediastinal node metastases. Furthermore, analysis of the histogram patterns of hematogenous metastases suggested that a small focus with greater DNA content, which had been undetected, developed in some primary tumors, and was the source of metastases [13]. However, histogram patterns in other cases suggested the appearance of abnormal cell lines with increased DNA contents in foci of hematogenous metastases. From these observations, nuclear DNA content is considered to be an excellent parameter of malignancy at least in adenocarcinoma of the lung. Growth properties The growth of tumors can be evaluated by the frequency of mitotic figures, but to obtain mitotic indices by counting the mitotic figures is not only time consuming but also inaccurate. Greater accuracy is obtained by counting the number of DNA synthesizing (S phase) cells by labeling ‘H-thymidine followed by autoradiography, or labeling the cells with bromodeoxyuridine followed by immunohistochemistry. With this method, we demon-
Fig. 5. Histology, mean nuclear DNA contents and frequency histograms of nuclear DNA content in well differentiated and moderately differentiated adenocarcinoma. Well differentiated papillary adenocarcinoma with slight nuclear atypia reveals a diploid pattern with a mean nuclear DNA content of 2.26C (left). Moderately differentiated adenocarcinoma with moderate nuclear atypia shows diploid and aneuploid stem lines with a mean nuclear DNA content of 3.75C (right) (from ref. I), reproduced with permission.
strated that the labeling index was found to be around 19% and 11% in small cell carcinoma and squamous cell carcinoma, respectively. In adenocarcinoma the labeling index ranged widely from less than 1% to 19%, two thirds of the cases being 3% or less, indicating that adenocarcinoma is a very heterogeneous group of tumors with respect to their growth properties [19J. More recently, proliferating cells have been identified by immunohistochemistry for proliferating cell nuclear antigen PCNA and proliferation associated nuclear antigen G-67 [3,8]. With this technique, adenocarcinoma areas with more atypical cells were found to show an increased number of positive cells, that is, increased growth properties, compared to areas of the tumor with less atypical cells (Fig. 2). The bromodeoxyuridine-labeling index of brain metastases of adenocarcinoma of the lung was higher than 5% [4], indicating that adenocarcinoma with a labeling index of 3% or less may have no ability to form metastases in the brain. Abnormalities in oncogenes and antioncogenes Point mutation in the c-Ki-ras gene is seen in 15 to 30% of lung adenocarcinomas [6,15]. This appears to be associated with cell type, but not with malignant progression [6]. Amplification of myc family genes has not been seen in the primary adenocarcinoma of the lung (O/18), but is frequently seen in brain metastases (13/l 7) (Nakajima et al., unpublished
Fig. 6. ~53 immunostaining of well differentiated papillary adenocarcinoma. Only a few tumor cell nuclei are positively stained in the periphery of the tumor with slight nuclear atypia (left), but most tumor cell nuclei are positively stained near the center of the tumor with increased nuclear atypia (right).
data) (Table 1). Allelic loss of ~53 gene also appeared more frequently in brain metastases than in primary tumors in non-small cell carcinomas (Nishikawa et al., unpublished data). These findings may indicate malignant progression due to genetic abnormalities. Nuclear staining due to accumulation of the p53 gene product is said to be closely associated with point mutation of the gene [14]. Positive immunostaining was statistically significantly associated with lymph node and distant organ metastases, and with the stage of the disease in adenocarcinoma, although such an association was not seen in squamous cell carcinoma and small cell carcinoma [5]. Positive nuclear staining was seen more frequently in nuclei showing increased atypia. In a single tumor composed of areas with negative staining and areas with positive staining, the former often consisted of cells with slight nuclear atypia and the latter with increased nuclear atypia (Fig. 6). In addition to nuclear atypia, cell type may also be important, since both of two cases of goblet cell type bronchioloalveolar carcinoma of the lung examined so far showed entirely negative immunostaining. From the above, the following conclusions can be drawn. 1. Papillary adenocarcinoma and bronchioloalveolar carcinoma develop in some instances in type II pneumocyte type or Clara cell type adenoma (or extremely well differentiated, low grade malignant adenocarcinoma). 2. Malignant progression is not infrequently seen in papillary adenocarcinoma and bronchioloalveolar carcinoma. Parameters of malignant progression include histology, cytology, immunostaining of CEA, mean nuclear DNA content and occurrence of aneuploid stem line, proliferative
106 TABLE 1 Amplification of myc family oncogenes in human lung cancer (No. of tumor with amplification/No. examined) Histologic type
Primary tumor
Brain metastasis
Adenocarcinoma Squamous cell carcinoma Adenosquamous carcinoma Large cell carcinoma Small cell carcinoma
O/l8 2112 o/2 O/4 3112
13117 616 l/l 4111
of tumor
activity (frequency of S phase cells or PCNA positive cells), and myc oncogene and ~53 suppressor gene abnormalities.
References 1 Asamura H, Nakajima T, Mukai K, Noguchi M, Shimosato Y. DNA cytofluorometric and nuclear morphometric analyses of lung adenocarcinoma. Cancer 1989;64:1657-1664. 2 Asamura H, Nakajima T, Mukai K, Shimosato Y. Nuclear DNA content by cytofluorometry of stage 1 adenocarcinoma of the lung in relation to postoperative recurrence. Chest 1989;96:312-318. 3 Brown DC, Gatter KG. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 1990;17:489503. 4 Cho KG, Hoshino T, Pitts LH, Nomura K, Shimosato Y. Proliferative potential of brain metastases. Cancer 1988;62:512-515. 5 Hiyoshi H, Matsumo Y, Noguchi M, Kato H, Shimosato Y, Hirohashi S. Clinical significance of ~53 tumor suppressor gene abnormalities in lung cancer. Lung Cancer 1991;7(suppl):38. 6 Kobayashi T, Tsuda H, Noguchi M et al. Association of Point mutation in c-Ki-ras oncogene in lung adenocarcinoma with particular reference to cytologic subtypes. Cancer 1990;66:289-294. 7 Kodama T, Biyajima S, Watanabe S, Shimosato Y. Morphometric study of adenocarcinomas and hyperplastic epithelial lesions in the peripheral lung. Am J Clin Pathol 1986;85:146-I 5 I. 8 Matsuno Y, Hirohashi S, Furuya S et al. Heterogeneity of proliferative activity in nodule-in-nodule lesions of small hepatocellular carcinoma. Jpn J Cancer Res 1990;81:1137-I 140. 9 Miller RR. Bronchioloalveolar cell adenomas. Am J Surg Path01 1990;14:904-912. 10 Miller RR, Nelems B, Evans KG, Muller NL. Ostrow DN. Glandular neoolasia of the lunn. _ A nronosed . . analogy to colonic tumors. Cancer 1988;61:1009-1014. 11 Nakayama H, Noguchi M, Tsuchiya R, Kodama T, Shimosato Y. Clonal growth of atypical adenomatous hyperplasia of the lung: cytofluorometric analysis of nuclear DNA content. Modern Pathol 1990;3:314320. 12 Nomori H, Hirohashi S, Noguchi M, Matsuno Y, Shimosato Y. Tumor cell heterogeneity and subpopulations with metastatic ability in differentiated adenocarcinoma of the lung. Histologic and cytofluorometric DNA analyses. Chest 199 1;99:934-940. 13 Nomori H, Nakajima T, Noguchi M, Iga R, Shimosato Y. Cytofluorometric analysis of metastases from lung adenocarcinoma with special reference to the difference between hematogenous and lymphatic metastases. Cancer 1991;67:2941-2947. 14 Rodrigues NR, Rowan A, Smith MEF et al. ~53 mutations in colorectal cancer. Proc Natl Acad Sci USA 1990;87:7555-7559. 15 Rosenhuis S, Slebos RJC, Boot AJM et al. Incidence and possible clinical significance of K-rus oncogene activation in adenocarcinoma of the human lung. Cancer Res 1988;48:5738-5741. 16 Shimosato Y. Pulmonary neoplasms. In: S.S. Sternberg, ed., Diagnostic surgical pathology. New York: Raven Press, 1989, 785-827.
107 17 Shimosato Y, Hashimoto T, Kodama T et al. Prognostic implications of fibrotic focus (scar) in small peripheral lung cancer. Am J Surg Pathol 1989;4:365-373. 18 Suzuki A. Growth characteristics of peripheral type adenocarcinoma of the lung in terms of roentgenologic findings. In: Y. Shimosato, M.R. Melamed, P Nettesheim, eds., Morphogenesis of lung cancer. Boca Raton, Florida: CRC Press, 1982, 91-110. 19 Yoshida K, Morinaga S, Shimosato Y, Hayata Y. A cell kinetic study of pulmonary adenocarcinoma by an immunoperoxidase procedure after bromodeoxyuridine labeling. Cancer 1989;64:2284-2291.
99
Scicntif~ Publishers Ireland Ltd. All rights reserved 0169-5OOu93iW6.00
LUNG 00124
Adenocarcinoma of the lung: its development and malignant progression Yukio Shimosato,
Masayuki
Noguchi
and Yoshihiro
Matsuno
Clinical Laboratory and Pathology Divisions, National Cancer Center Hospitaland Research Institute, Tokyo, Japan
Key words: Adenocarcinoma; Human lung; Development; Malignant progression; clear DNA content @loidy); myc gene; p53 gene; Immunohistochemistry
Nu-
Introduction Adenocarcinoma of the lung is the most common lung cancer in Japan and is said to be increasing in some other countries. Etiologically it is less strongly associated with cigarette smoking than squamous cell carcinoma and small cell carcinoma. Histologically and cytologically, it is divided into many subtypes, and biologically it is a heterogeneous group of tumors ranging from very slow growing and low grade malignant tumors to fast growing highly malignant tumors. This paper will focus on the development and malignant progression of adenocarcinoma of the lung from the morphological, biological, molecular and genetic points of view.
The majority of adenocarcinomas of the lung develop in the peripheral airway, that is, the bronchioloalveolar region. Most peripheral adenocarcinomas contain a central or subpleural anthracotic and fibrotic focus, which is often associated with a pleural indentation. This characteristic feature is the basis for the concept of scar cancer, which has been believed to be common in the lung for many years. Although we do not deny the existence of such scar cancers in the lung, we believe that most adenocarcinomas encountered in clinical practice develop either de novo or through the stage of atypical adenomatous hyperplasia or adenoma not associated with scar [16].
Correspondence to: Y. Shimosato, M.D., Clinical Laboratory Division, National Cancer Center Hospital, 5-I-1, Tsukiji, Chuoku Tokyo 104, Japan.
Fig. I. Histology of atypical adenomatous hyperplasia (or adenoma). Slightly to moderately atypical cells with increased nuclear cytoplasmic ratio replace the lining of alveoli, whose septae are slightly thickened. Cytoplasm of some cells are finely vesicular, suggesting type II alveolar epithelial cell type. Within this lesion is a small focus of well differentiated adenocarcinoma.
Rebuttal of the scar cancer concept Although carcinoma arising in diffuse pulmonary fibrosis may be categorized as an example of scar cancer, we believe that in most adenocarcinomas seen in routine practice the central fibrosis or fibrotic focus with anthracosis forms not before, but after, the development of carcinoma [ 171. This occurs as the result of collapse of alveoli secondary to degeneration and death of tumor cells which had lined the alveoli, as a result of impaired blood flow. This idea is supported by the fact that in early stage adenocarcinoma a fibrotic focus is usually absent and also by the absence of collagenization and fibroblastic proliferation in areas with dense aggregates of elastic fibers, which constituted alveolar septae. Convergence of bronchi and pulmonary vessels toward the tumor, which is often associated with pleural indentation, is observed frequently on chest X-ray films and provides evidence supporting the morphological events described above [ 181. Subsequently, fibroblastic cell proliferation and collagenization follow. It is also easy to understand the presence of central fibrosis in distant metastatic foci. There are several other reasons to support this explanation of the process of central fibrosis [17]. De novo carcinogenesis or adenoma-carcinoma sequence If the occurrence of scar cancer is uncommon in the lung, how do adenocarcinomas develop? The first concept to consider is de novo development, but this is very difficult to
Fig. 2. Immunostaining for PCNA of well differentiated adenocarcinoma associated with atypical adenomatous hyperplasia (or adenoma). In the periphery of the lesion with slight nuclear atypia, PCNA positive cells are rare (left), but in the central portion with increased nuclear atypia, PCNA positive nuclei are scattered (right).
prove. We have not infrequently encountered papillary or bronchioloalveolar lesions, which suggest the development of adenocarcinoma in a focus of atypical adenomatous hyperplasia or adenoma [7,11]. Miller and her associates have also suggested the adenomacarcinoma sequence in the lung [9,10]. Such adenomatous lesions are often less than 1 cm in diameter, single or multiple and are composed of atypical cuboidal cells with increased nuclear cytoplasmic ratio (but not atypical enough to call carcinoma) replacing the lining of alveoli. Scanty cytoplasm may contain small vesicles or needle like clefts. They resemble either Clara cells or type II alveolar epithelial cells. Mitotic figures are hardly seen (Fig. 1). Within such a lesion, an area with increased cellularity and cell atypia sufficient enough to call well differentiated adenocarcinoma may be seen. Such atypical adenomatous lesions are not infrequently seen at the periphery of incontrovertible papillary adenocarcinoma, suggesting a late stage of the adenoma-carcinoma sequence. So far we have investigated 5 cases of solitary atypical adenomatous lesions and 9 cases of adenocarcinoma associated with atypical adenomatous lesions [ 111. Analysis of the nuclear DNA content of atypical adenomatous lesions reveals either diploidy, polyploidy or aneuploidy suggesting clonal growth, with or without development of subclones with more abnormal DNA content. Positively stained cells for proliferating cell nuclear antigen (PCNA) are few in number in atypical adenomatous lesions, but increase in areas with increased cell atypia and density (Fig. 2). CEA is generally negative in the former and positive in the latter. From such findings, we now believe such lesions to be neoplastic, either adenoma or extremely well-differentiated adenocarcinoma with
Fig. 3. Histology of adenocarcinoma showing malignant progression, An atypical adenomatous lesion (or adenoma) is seen in the periphery of the tumor (left), features of well differentiated papillary adenocarcinoma are seen in the intermediate zone of the tumor (center), and solid nests consisting of more atypical cells are present in the center of the tumor (right).
bronchioloalveolar carcinoma.
growth pattern.
If the latter is the case, it may be called de novo
Malignant progression Similar to the process of development of adenocarcinoma in adenoma, histologically and cytologically more atypical or more malignant appearing areas develop within carcinoma (Fig. 3). Such morphological progression can be confirmed not only in hematoxylin and eosin stained sections but also by other methods such as immunohistochemistry for CEA (Fig. 4) and markers for proliferative activity, cytofluorometry or flow cytometry, and probably by analysis of genetic changes. Nuclear DNA content and occurrence of aneuploid stem line Mean nuclear DNA content has been shown to increase in less differentiated adenocarcinoma (Fig. 5), and is significantly greater in stage 1 adenocarcinomas with recurrence within 5 years after surgery than those without recurrence [ 1,2]. Within a primary tumor, the mean nuclear DNA content of tumor cells in areas with increased amount of stroma or with apparent invasive growth is definitely greater than that in areas with thin stroma made up of alveolar septae [12]. Comparison of mean nuclear DNA contents of
Fig. 4. CEA immunostaining showing malignant progression in a single tumor. No positively stained cells are present in the atypical adenomatous lesion (adenoma) (left). Moderate cytoplasmic stain is noted in areas with features of well differentiated adenocarcinoma (center), and marked cytoplasmic staining for CEA in the poorly differentiated area (right).
primary tumors and metastatic foci in lymph nodes, brain and liver disclosed that hematogenous metastases were frequently composed of tumor cells with greater mean nuclear DNA content showing more abnormal histogram patterns than those in the primary tumor; whereas the mean nuclear DNA contents in lymph node metastases, particularly metastases to the hilar lymph nodes, were not greater than the primary tumor, although cells with greater DNA contents were noted in the mediastinal node metastases. Furthermore, analysis of the histogram patterns of hematogenous metastases suggested that a small focus with greater DNA content, which had been undetected, developed in some primary tumors, and was the source of metastases [13]. However, histogram patterns in other cases suggested the appearance of abnormal cell lines with increased DNA contents in foci of hematogenous metastases. From these observations, nuclear DNA content is considered to be an excellent parameter of malignancy at least in adenocarcinoma of the lung. Growth properties The growth of tumors can be evaluated by the frequency of mitotic figures, but to obtain mitotic indices by counting the mitotic figures is not only time consuming but also inaccurate. Greater accuracy is obtained by counting the number of DNA synthesizing (S phase) cells by labeling ‘H-thymidine followed by autoradiography, or labeling the cells with bromodeoxyuridine followed by immunohistochemistry. With this method, we demon-
Fig. 5. Histology, mean nuclear DNA contents and frequency histograms of nuclear DNA content in well differentiated and moderately differentiated adenocarcinoma. Well differentiated papillary adenocarcinoma with slight nuclear atypia reveals a diploid pattern with a mean nuclear DNA content of 2.26C (left). Moderately differentiated adenocarcinoma with moderate nuclear atypia shows diploid and aneuploid stem lines with a mean nuclear DNA content of 3.75C (right) (from ref. I), reproduced with permission.
strated that the labeling index was found to be around 19% and 11% in small cell carcinoma and squamous cell carcinoma, respectively. In adenocarcinoma the labeling index ranged widely from less than 1% to 19%, two thirds of the cases being 3% or less, indicating that adenocarcinoma is a very heterogeneous group of tumors with respect to their growth properties [19J. More recently, proliferating cells have been identified by immunohistochemistry for proliferating cell nuclear antigen PCNA and proliferation associated nuclear antigen G-67 [3,8]. With this technique, adenocarcinoma areas with more atypical cells were found to show an increased number of positive cells, that is, increased growth properties, compared to areas of the tumor with less atypical cells (Fig. 2). The bromodeoxyuridine-labeling index of brain metastases of adenocarcinoma of the lung was higher than 5% [4], indicating that adenocarcinoma with a labeling index of 3% or less may have no ability to form metastases in the brain. Abnormalities in oncogenes and antioncogenes Point mutation in the c-Ki-ras gene is seen in 15 to 30% of lung adenocarcinomas [6,15]. This appears to be associated with cell type, but not with malignant progression [6]. Amplification of myc family genes has not been seen in the primary adenocarcinoma of the lung (O/18), but is frequently seen in brain metastases (13/l 7) (Nakajima et al., unpublished
Fig. 6. ~53 immunostaining of well differentiated papillary adenocarcinoma. Only a few tumor cell nuclei are positively stained in the periphery of the tumor with slight nuclear atypia (left), but most tumor cell nuclei are positively stained near the center of the tumor with increased nuclear atypia (right).
data) (Table 1). Allelic loss of ~53 gene also appeared more frequently in brain metastases than in primary tumors in non-small cell carcinomas (Nishikawa et al., unpublished data). These findings may indicate malignant progression due to genetic abnormalities. Nuclear staining due to accumulation of the p53 gene product is said to be closely associated with point mutation of the gene [14]. Positive immunostaining was statistically significantly associated with lymph node and distant organ metastases, and with the stage of the disease in adenocarcinoma, although such an association was not seen in squamous cell carcinoma and small cell carcinoma [5]. Positive nuclear staining was seen more frequently in nuclei showing increased atypia. In a single tumor composed of areas with negative staining and areas with positive staining, the former often consisted of cells with slight nuclear atypia and the latter with increased nuclear atypia (Fig. 6). In addition to nuclear atypia, cell type may also be important, since both of two cases of goblet cell type bronchioloalveolar carcinoma of the lung examined so far showed entirely negative immunostaining. From the above, the following conclusions can be drawn. 1. Papillary adenocarcinoma and bronchioloalveolar carcinoma develop in some instances in type II pneumocyte type or Clara cell type adenoma (or extremely well differentiated, low grade malignant adenocarcinoma). 2. Malignant progression is not infrequently seen in papillary adenocarcinoma and bronchioloalveolar carcinoma. Parameters of malignant progression include histology, cytology, immunostaining of CEA, mean nuclear DNA content and occurrence of aneuploid stem line, proliferative
106 TABLE 1 Amplification of myc family oncogenes in human lung cancer (No. of tumor with amplification/No. examined) Histologic type
Primary tumor
Brain metastasis
Adenocarcinoma Squamous cell carcinoma Adenosquamous carcinoma Large cell carcinoma Small cell carcinoma
O/l8 2112 o/2 O/4 3112
13117 616 l/l 4111
of tumor
activity (frequency of S phase cells or PCNA positive cells), and myc oncogene and ~53 suppressor gene abnormalities.
References 1 Asamura H, Nakajima T, Mukai K, Noguchi M, Shimosato Y. DNA cytofluorometric and nuclear morphometric analyses of lung adenocarcinoma. Cancer 1989;64:1657-1664. 2 Asamura H, Nakajima T, Mukai K, Shimosato Y. Nuclear DNA content by cytofluorometry of stage 1 adenocarcinoma of the lung in relation to postoperative recurrence. Chest 1989;96:312-318. 3 Brown DC, Gatter KG. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 1990;17:489503. 4 Cho KG, Hoshino T, Pitts LH, Nomura K, Shimosato Y. Proliferative potential of brain metastases. Cancer 1988;62:512-515. 5 Hiyoshi H, Matsumo Y, Noguchi M, Kato H, Shimosato Y, Hirohashi S. Clinical significance of ~53 tumor suppressor gene abnormalities in lung cancer. Lung Cancer 1991;7(suppl):38. 6 Kobayashi T, Tsuda H, Noguchi M et al. Association of Point mutation in c-Ki-ras oncogene in lung adenocarcinoma with particular reference to cytologic subtypes. Cancer 1990;66:289-294. 7 Kodama T, Biyajima S, Watanabe S, Shimosato Y. Morphometric study of adenocarcinomas and hyperplastic epithelial lesions in the peripheral lung. Am J Clin Pathol 1986;85:146-I 5 I. 8 Matsuno Y, Hirohashi S, Furuya S et al. Heterogeneity of proliferative activity in nodule-in-nodule lesions of small hepatocellular carcinoma. Jpn J Cancer Res 1990;81:1137-I 140. 9 Miller RR. Bronchioloalveolar cell adenomas. Am J Surg Path01 1990;14:904-912. 10 Miller RR, Nelems B, Evans KG, Muller NL. Ostrow DN. Glandular neoolasia of the lunn. _ A nronosed . . analogy to colonic tumors. Cancer 1988;61:1009-1014. 11 Nakayama H, Noguchi M, Tsuchiya R, Kodama T, Shimosato Y. Clonal growth of atypical adenomatous hyperplasia of the lung: cytofluorometric analysis of nuclear DNA content. Modern Pathol 1990;3:314320. 12 Nomori H, Hirohashi S, Noguchi M, Matsuno Y, Shimosato Y. Tumor cell heterogeneity and subpopulations with metastatic ability in differentiated adenocarcinoma of the lung. Histologic and cytofluorometric DNA analyses. Chest 199 1;99:934-940. 13 Nomori H, Nakajima T, Noguchi M, Iga R, Shimosato Y. Cytofluorometric analysis of metastases from lung adenocarcinoma with special reference to the difference between hematogenous and lymphatic metastases. Cancer 1991;67:2941-2947. 14 Rodrigues NR, Rowan A, Smith MEF et al. ~53 mutations in colorectal cancer. Proc Natl Acad Sci USA 1990;87:7555-7559. 15 Rosenhuis S, Slebos RJC, Boot AJM et al. Incidence and possible clinical significance of K-rus oncogene activation in adenocarcinoma of the human lung. Cancer Res 1988;48:5738-5741. 16 Shimosato Y. Pulmonary neoplasms. In: S.S. Sternberg, ed., Diagnostic surgical pathology. New York: Raven Press, 1989, 785-827.
107 17 Shimosato Y, Hashimoto T, Kodama T et al. Prognostic implications of fibrotic focus (scar) in small peripheral lung cancer. Am J Surg Pathol 1989;4:365-373. 18 Suzuki A. Growth characteristics of peripheral type adenocarcinoma of the lung in terms of roentgenologic findings. In: Y. Shimosato, M.R. Melamed, P Nettesheim, eds., Morphogenesis of lung cancer. Boca Raton, Florida: CRC Press, 1982, 91-110. 19 Yoshida K, Morinaga S, Shimosato Y, Hayata Y. A cell kinetic study of pulmonary adenocarcinoma by an immunoperoxidase procedure after bromodeoxyuridine labeling. Cancer 1989;64:2284-2291.