Gastrin-Releasing Peptide Gene Expression in Small Cell and large Cell Undifferentiated Lung Carcinomas MARY E. SUNDAY, MD, PHD, NOAH CHOI, MD, ELIOT R. SPINDEL, MD, PHD, WILLIAM W. CHIN, MD, AND EUGENE J. MARK, MD Gastrin-releasing peptide (GRP, mammalian bombesin) is present in the neuroendocrine cells of human fetal lung and in small cell lung carcinomas (SCLCs), where it may act as a growth factor. Considering the potential importance of GRP as a tumor marker, we have conducted a retrospective immunohistochemical analysis of 176 lung tumors for markers of GRP gene expression, as well as several other markers of neuroendocrine cell differentiation: chromogranin A, neuron-specific enolase, and calcitonin. The majority of carcinoids contained mature GRP, in contrast to only a minority of SCLCs and large cell lung carcinomas (LCLCs). However, a majority of SCLCs and LCLCs contained proGRP immunoreactivity. In situ hybridization did not add any information beyond what was obtained using proGRP antisera. In spite of sharing these neuroendocrine cell markers, SCLCs are associated with a graver prognosis than LCLCs. No prognostic significance was associated with immunostaining for GRP or several other markers of neuroendocrine cell differentiation. HUM PATHOL 22:10301039. Copyright i!Zl1991 by W.B. Saunders Company
Gastrin-releasing peptide (GRP), the mammalian homologue of amphibian bombesin, is present at high levels in human fetal lung neuroendocrine cells, where it may act as a growth and differentiation factor for developing pulmonary tissues.‘“’ Although the normal adult lung has low to undetectable levels of GRP, elevated levels of GRP immunoreactivity have been demonstrated in bronchial lavage fluid from asymptomatic cigarette smokers’ and in lungs from patients with asbestosis, bronchiectasis, and chronic obstructive pulmonary disease. ’ ,5-7In many of these chronic diseases, the source of GRP may be pulmonary neuroendocrine cell (PNEC) hyperplasia or pulmonary carcinoid-type tumorlets, which may occur later in the disease course, possibly as a regenerative response to hypoxia and/or pulmonary epithelial injury.“*’ It is generally recognized that most pulmonary carcinoids (well-differentiated neuroendocrine tumors) exFrom the Department ()I’ Pathology, Harvard Medical School. Boston, MA; the Department of Radiation Medicine, Massachusetts General Hospital. Boston. M.4; the Departments of Medicine and Pathology, Brigham 8c Women’s Hospital, Boston. MA; and the Department of Pathology, Massachusetts General Hospital, Boston, MA. Accepted for publication December l-1, 199tl. Supported by National Institutes of’ Health grant no. R21)-38_LOl0 1, Council on Tobacco Research grant no. 2-I 1 1 Rl, and American Cancer Society Junior Faculty Research Award no. JFRA-21-L Kq words: bombesin, immunohistr)chemistr)i, in situ hybridization, lung tumors. prognosis. Address correspondence and reprint requests to Mary E. Sunday, MD, PhD, Brigham & Women’s Hospital, Department of Pathology, 7.5 Francis St. Boston, MA 02 115. Copyright C:, 1991 by W.B. Saunders Compaq 0046-8177/91/2210-0011$5.00/O
1030
press a variety of neuroendocrine cell markers, including GRP.‘.” Small cell carcinomas of the lung (SCLCs), being poorly differentiated, synthesize neuropeptides in a smaller percentage of cases.‘“-‘5 Those SCLC tumors exhibiting a more well-differentiated phenotype, containing hormones in dense-core neurosecretory granules and able to respond to growth factors, have been termed “classic” lines.‘” In contrast, the more undifferentiated “variant” SCLC lines do not make or respond to hormones, but have been associated with amplified N- or c-myc oncogenes and may lead to death even more rapidly than the “classic” SCLC lines. The classic SCLC tumors are thus reported to have a more indolent course than these “variant” SCLC lines. These data suggest that the neuroendocrine phenotype of SCLC might be correlated with the biologic behavior of these lung tumors. It has been recently reported that a percentage of large cell undifferentiated carcinomas of the lung (LCLCs) may also have neuroendocrine features.“-” Thus, we considered it important to analyze the prevalence of neuroendocrine markers, especially GRP, in LCLC as well as in subtypes of SCLCs, carcinoids, and other lung tumors. These observations could then be combined with clinical survival data to determine whether there might be a correlation between biologic behavior of malignant tumors and neuroendocrine phenotype.
METHODS Study Population and Tissues Over 380 ases of lung neoplastns were initially selected randomly frotn cases diagnosed and treated at the Massachusetts General Hospital (MGH) between 1974 and 1986, as
retrieved from the MGH Tumor Registry files and the suqical patholow computer database files. To interpret survival data comparing different tumor groups. we selected for cases with local disease, that is, limited to the lungs and mediastinal lymph nodes, with the exception of 29 cases of XIX in addition to 65 SCLC cases with limited disease. Many other cases had to be randomly and blindly discarded due to one or more of the following reasons: (1) missing blocks; (2) insufficient tissue present in existing blocks; (3) fixation in Bouins (paraformaldehyde-picric acid) or decalcification in acid, both of which were incompatible with our immunohistochemical and in situ hybridization analyses; (4) lung tumors representing metastases from primaries elsewhere (eg. colon, breast); and (5) completely necrotic tumor present in available blocks. All cases used for this study were reviewed blindly by two of us (E.J.M. and M.E.S.) on both the original glass slides and
GRP IN LUNG TUMORS (Sunday et al:
*
m
CTEP-2
*
Antibodies
CTEP-I
)
CTEP-3
FIGURE 1. Gastrin-releasing peptide and CTEP antibodies. Rabbit polyclonal antisera were raised against keyhole limpet hemocyonin conjugates of the peptides, which are schematically depicted OS bars adjacent to their corresponding fulllength form of proGRP (all of which encode GRP, but differ in the most distcll region of the proGRP CTEPs due to altered splicing, including a frame shift mutation between exons 2 and 3). Thus, CTEP-1 antibodies recognize a 27 amino acid synthetic peptide unique to GRP mRNA form 1 (representing two of three GRP mRNAs) and form 2 (a negligible amount of GRP mRNAs). Similarly, CTEP-3 antibodies recognize a 17 amino acid synthetic peptide unique to GRP mRNA form 3.14 Antibodies to mature GRP and to mature bombesin recognize these peptides only after cleavage from the prohormone and amidation. Thus, antibodies to bombesin/GRP will not detect DroGRP. on 11.esh Ilem;~tos~litl-eosin-stainrd recuts of the tumor. blocks II& li)r our ;~al\ses Di;~gnoses were made according to the
c,ritr.ria 01 tl~c CVorld Halth OrganiLarion (WI-IO) classificaticm. Patient dat;l, inc-luding age, sex. stage. treatnit’nt, and survival. ~\.ere obtainc~l from tile M(GH Tumor Registrv and fi-ant iiiedi~:il I wuda. TABLE
1.
Patient
.x ti 1 10 -lJ iti 5X 71
75
Rabbit heteroaitisera were raiaetl ill New %r;tland White rabbits using the following peptide colljugatc:s (Fig 1): amphibiarl bomb&n (Cambridge Research (10 Inc , (;anlbridge, LIE;) coqjugated to bovine thyroglobulin; “(3 EP-I ,” ;I 27amino acid c;lrt,osl-rerminal extt.nsion peptide of
Gastrin-releasing peptide (GRP), the mammalian homologue of amphibian bombesin, is present at high levels in human fetal lung neuroendocrine cells, where it may act as a growth and differentiation factor for developing pulmonary tissues.‘“’ Although the normal adult lung has low to undetectable levels of GRP, elevated levels of GRP immunoreactivity have been demonstrated in bronchial lavage fluid from asymptomatic cigarette smokers’ and in lungs from patients with asbestosis, bronchiectasis, and chronic obstructive pulmonary disease. ’ ,5-7In many of these chronic diseases, the source of GRP may be pulmonary neuroendocrine cell (PNEC) hyperplasia or pulmonary carcinoid-type tumorlets, which may occur later in the disease course, possibly as a regenerative response to hypoxia and/or pulmonary epithelial injury.“*’ It is generally recognized that most pulmonary carcinoids (well-differentiated neuroendocrine tumors) exFrom the Department ()I’ Pathology, Harvard Medical School. Boston, MA; the Department of Radiation Medicine, Massachusetts General Hospital. Boston. M.4; the Departments of Medicine and Pathology, Brigham 8c Women’s Hospital, Boston. MA; and the Department of Pathology, Massachusetts General Hospital, Boston, MA. Accepted for publication December l-1, 199tl. Supported by National Institutes of’ Health grant no. R21)-38_LOl0 1, Council on Tobacco Research grant no. 2-I 1 1 Rl, and American Cancer Society Junior Faculty Research Award no. JFRA-21-L Kq words: bombesin, immunohistr)chemistr)i, in situ hybridization, lung tumors. prognosis. Address correspondence and reprint requests to Mary E. Sunday, MD, PhD, Brigham & Women’s Hospital, Department of Pathology, 7.5 Francis St. Boston, MA 02 115. Copyright C:, 1991 by W.B. Saunders Compaq 0046-8177/91/2210-0011$5.00/O
1030
press a variety of neuroendocrine cell markers, including GRP.‘.” Small cell carcinomas of the lung (SCLCs), being poorly differentiated, synthesize neuropeptides in a smaller percentage of cases.‘“-‘5 Those SCLC tumors exhibiting a more well-differentiated phenotype, containing hormones in dense-core neurosecretory granules and able to respond to growth factors, have been termed “classic” lines.‘” In contrast, the more undifferentiated “variant” SCLC lines do not make or respond to hormones, but have been associated with amplified N- or c-myc oncogenes and may lead to death even more rapidly than the “classic” SCLC lines. The classic SCLC tumors are thus reported to have a more indolent course than these “variant” SCLC lines. These data suggest that the neuroendocrine phenotype of SCLC might be correlated with the biologic behavior of these lung tumors. It has been recently reported that a percentage of large cell undifferentiated carcinomas of the lung (LCLCs) may also have neuroendocrine features.“-” Thus, we considered it important to analyze the prevalence of neuroendocrine markers, especially GRP, in LCLC as well as in subtypes of SCLCs, carcinoids, and other lung tumors. These observations could then be combined with clinical survival data to determine whether there might be a correlation between biologic behavior of malignant tumors and neuroendocrine phenotype.
METHODS Study Population and Tissues Over 380 ases of lung neoplastns were initially selected randomly frotn cases diagnosed and treated at the Massachusetts General Hospital (MGH) between 1974 and 1986, as
retrieved from the MGH Tumor Registry files and the suqical patholow computer database files. To interpret survival data comparing different tumor groups. we selected for cases with local disease, that is, limited to the lungs and mediastinal lymph nodes, with the exception of 29 cases of XIX in addition to 65 SCLC cases with limited disease. Many other cases had to be randomly and blindly discarded due to one or more of the following reasons: (1) missing blocks; (2) insufficient tissue present in existing blocks; (3) fixation in Bouins (paraformaldehyde-picric acid) or decalcification in acid, both of which were incompatible with our immunohistochemical and in situ hybridization analyses; (4) lung tumors representing metastases from primaries elsewhere (eg. colon, breast); and (5) completely necrotic tumor present in available blocks. All cases used for this study were reviewed blindly by two of us (E.J.M. and M.E.S.) on both the original glass slides and
GRP IN LUNG TUMORS (Sunday et al:
*
m
CTEP-2
*
Antibodies
CTEP-I
)
CTEP-3
FIGURE 1. Gastrin-releasing peptide and CTEP antibodies. Rabbit polyclonal antisera were raised against keyhole limpet hemocyonin conjugates of the peptides, which are schematically depicted OS bars adjacent to their corresponding fulllength form of proGRP (all of which encode GRP, but differ in the most distcll region of the proGRP CTEPs due to altered splicing, including a frame shift mutation between exons 2 and 3). Thus, CTEP-1 antibodies recognize a 27 amino acid synthetic peptide unique to GRP mRNA form 1 (representing two of three GRP mRNAs) and form 2 (a negligible amount of GRP mRNAs). Similarly, CTEP-3 antibodies recognize a 17 amino acid synthetic peptide unique to GRP mRNA form 3.14 Antibodies to mature GRP and to mature bombesin recognize these peptides only after cleavage from the prohormone and amidation. Thus, antibodies to bombesin/GRP will not detect DroGRP. on 11.esh Ilem;~tos~litl-eosin-stainrd recuts of the tumor. blocks II& li)r our ;~al\ses Di;~gnoses were made according to the
c,ritr.ria 01 tl~c CVorld Halth OrganiLarion (WI-IO) classificaticm. Patient dat;l, inc-luding age, sex. stage. treatnit’nt, and survival. ~\.ere obtainc~l from tile M(GH Tumor Registrv and fi-ant iiiedi~:il I wuda. TABLE
1.
Patient
.x ti 1 10 -lJ iti 5X 71
75
Rabbit heteroaitisera were raiaetl ill New %r;tland White rabbits using the following peptide colljugatc:s (Fig 1): amphibiarl bomb&n (Cambridge Research (10 Inc , (;anlbridge, LIE;) coqjugated to bovine thyroglobulin; “(3 EP-I ,” ;I 27amino acid c;lrt,osl-rerminal extt.nsion peptide of
