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The expression of trophoblastic cell markers by lung carcinomas
The Expression of Trophoblastic Cell Markers by Lung Carcinomas LESLIE D, BOUCHER, MD AND KOKICHI YONEDA, MD The authors studied the expression of trophoblastic cell markers in lung carcinomas cells by immunoperoxidase staining using antibodies against three trophoblastic glycoproteins (human chorionic gonadotropin [hCG]; human placental lactogen [hPL]; pregnancy-specific ~l-glycoprotein [SP-1]). One hundred five tissue sections from 44 lung carcinomas of various histological types were examined for positive staining with three antibodies. Hematoxylin-eosin-stalned sectious from the same tissue blocks were examined for the presence of tumor giant cells. The aim was to study the relationship between tumor giant cells and the trophoblastic glycoprotein expression in lung carcinomas. Small cell carcinoma (SCC) did not show any positive reaction with all three markers. Squamous cell carcinoma (SQCC) showed positive staining with hCG in 21% of cases, hPL in 28%, and SP-1 in 64%. Adenocarcinoma showed positive staining with hCG in 60% of cases, hPL in 10%, and SP-1 in 80%. Large cell carcinoma (LCC) showed positive staining with hCG in 93% of cases, hPL in
56%, and SP-1 in 93%. The positive reaction did not appear to be restricted to nor associated with the tumor giant cells. It was concluded that these trophoblastic cell markers are expressed in various types of lung carcinomas and that they are not associated with certain histological types or with tumor giant cells. HUM PATHOL 26:1201-1206. Copyright © 1995 by W.B. Saunders Company Key words: lung carcinomas, human chorionic gonadotropin, human placental lactogen, pregnancy-specific/~rglycoprotein. Abbreviations: hCG, human chorionic gonadotropin; hPL, human placental lactogen; SP-1, pregnancy-specific/$1-glycoprotein; CIS, carcinoma in situ; SCC, small cell carcinoma; SQCC, squamous cell carcinoma; WD, well differentiated; PD, poorly differentiated; BAC, bronchioloalveolar cell carcinoma; PAP, papillary carcinoma; AC, acinar carcinoma; LCC, large cell carcinoma; CLC, clear cell carcinoma; GC, giant cell carcinoma; NOS, not otherwise specified; WHO, World Health Organization.
Since the mid-1960s, the secretion of gonadotropin by lung carcinomas has been r e p o r t e d anecdotally. 1~ Although all types of the lung carcinomas p r o d u c e d h u m a n chorionic gonadotropin (hCG), the large cell carcinoma (LCC), especially giant cell carcinoma (GC), was the most conspicuous in this regard. 7 In 1976, human chorionic gonadotropin (hCG) was localized in t u m o r tissue by immunofluorescence by McManus, Naughton, and Martinez-Hernandez. s Hattori et al 9 studied hCG in plasma and in t u m o r tissue in 35 cases of lung carcinomas and f o u n d hCG in plasma in 6% of patients and hCG in tissue extraction in 35% of patients. Wilson et al, 1° conversely, studied hCG-/3 by immunoperoxidase technique in paraffin sections of 61 lung carcinomas. They f o u n d positive reaction in 84% of lung carcinomas with all cell types except small cell carcinoma (SCC). This finding was disputed by Heyderman, Chapman, and Richardson, al who found hCG in only five of 40 lung carcinomas (12.5%). Although this controversy about hCG production in lung carcinomas was in progress, several case reports of primary choriocarcinoma of the lung appeared in the literature, a2-14The diagnosis was based on the existence o f bizarre tumor giant cells (syncytiotrophoblasts) and the positive reaction of the tumor cells for hCG by immunohistochemical technique. Given the prevalence of hCG production in lung carcinomas, especially GCs,
the distinction of this entity from the large cell lung carcinoma seems to be ambiguous. The question arises whether there exists a distinct class of lung tumor that has a unique combination of hCG production and bizarre giant cells. In this study, the authors tried to answer this question by examining various histological types of lung carcinomas with immunoperoxidase technique using antibodies against trophoblastic markers. H u m a n chorionic gonadotropin consists of two subunits, 0~ and /3. T h e ot-subunit is shared by other polypeptide h o r m o n e s and p r o d u c e d by various tumors. 15 The /3-subunit is unique to each h o r m o n e and differentiates the biological activities. Therefore, the expression of hCG-/3 by lung carcinomas is of interest. In addition to hCG-beta, the authors examined the expression of h u m a n placental lactogen (hPL) and pregnancy-specific/3Fglycoprotein (SP-1) to determine whether the expression of hCG-/3 is an isolated p h e n o m e n o n or whether the same t u m o r cells produce the spectrum of trophoblastic markers. All these markers are p r o d u c e d in benign and malignant syncytiotrophoblasts. The expression of these markers seems to show some differentiation in various trophoblastic tumors. 16'17 Therefore, it is interesting to see whether there exists a pattern of these markers in certain lung carcinomas.
MATERIALS AND METHODS From the Department of Pathology, Washington University School of Medicine, St, Louis, MO; and the Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center, and University of Kentucky College of Medicine, Lexington, KY. Accepted for publication April 19, 1995. Address correspondence and reprint requests to Kokichi Yoneda, MD, Pathology and Laboratory Medicine Service (113 CDD), Veterans Affairs Medical Center, 2250 Leestown Rd, Lexington, KY405111093. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2611-000855.00/0
Forty-four cases of lung carcinomas were selected from the surgical pathology file of the Veterans Affairs Medical Center (Lexington, KY). They consisted of seven biopsy specimens and 37 pulmonary resection (lobectomy or pneumonectomy) specimens. All the blocks were processed routinely for histological examination. The specimen was fixed in 10% buffered formalin for 2 hours to overnight. The specimens were processed routinely and embedded in paraffin. The biopsy specimens were three of squamous cell carcinoma in situ (CIS) of the bronchus and four SCCs. For each resection
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specimen, two to three blocks of tumor for each case were selected for study. Altogether 105 blocks were studied by immunoperoxidase staining. The authors followed the World Heahh Organization (WHO) classification of lung tumors TM in selection of the cases with the following exceptions: (1) squamous cell carcinoma (SQCC) was divided into two categories of well differentiated (SQCC WD) and poorly differentiated (SQCC PD) based on the presence of keratin by light microscopic examination of hematoxylin-eosin-stained sections. (2) Small cell carcinoma (SCC) was not divided into further categories. 19 (3) The authors did not select the cases from the category of solid adenocarcinoma with mucus formation because they wished to examine the difference between clearly distinct cell types. The classification of tumors selected was based on consensus. Three cases, on whose classification the authors did not agree, were not included in the current study. The following are the tumor types studied with respective number of cases in parenthesis: SQCC (n = 14) consisting of CIS (n = three), SQCC WD (n = five) and SQCC PD (n = six); adenocarcinoma (n = 10), consisting of bronchioloalveolar cell type (BAC, n = three), papillary type (PAP, n = four), and acinar type (AC, n = three); LCC (n = 16) consisting of LCC not otherwise specified (LCC NOS, n = five), clear cell type (LCC CLC, n = three), and giant cell carcinoma (LCC GC, n -- eight); and SGC (n = four). Polyclonal rabbit antibodies against hCG beta, hPL, and SP-1 were purchased from DAKO (Carpinteria, CA). Four serial 5-/~m tissue sections were cut, and the first sections were stained with hematoxylin-eosin. The other tissue sections were deparaffinized with xylene and then washed in ethanol. Enzymatic digestion to enhance antigen was not performed. After endogenous peroxidase was blocked by hydrogen peroxide, sections were incubated with normal rabbitblocking serum. Prediluted primary antibodies were applied to the sections for 30 minutes. The sections were washed and then incubated for 10 minutes in diluted biotinylated secondary antibody. After rinsing, sections were incubated with Vectastain Elite ABC reagent (Vector, Burlingame, CA). The color was developed using 3,3'-diaminobenzidine-hydrogen peroxide substrate. Placental tissue was used as controls. In addition, sections of the SQCC of the skin (n = six), esophagus (n = three), and larynx (n = six) were used as controls for SQCC WD. These controls were used to rule out cross-reactivity between antitrophoblastic glycoproteins and antikeratin. The scoring of grading sections was done independently by each author, and the scores were averaged. Hematoxylineosin-stained sections of all the blocks used for immunoperoxidase study were examined for tumor type and existence of giant cells. Giant cells were defined in this study as those cells with over three times in diameter of the average tumor cells. They are either mononucleated or multinucleated. Their presence was scored as follows: - , no giant cells; +, giant cells consist of up to 5% of tumor cell population; ++, giant cells consist of 5% to 25% of tumor cell population; + + + , giant cells constitute more than 25% of tumor cell population. The last category is one of the diagnostic criteria of giant cell carcinoma. The immunoperoxidase staining was similarly graded as follows: - , no staining; +, positive staining in up to 10% of cells; ++, positive staining in 11% to 25% of cells; + + + , positive staining in more than 25% of tumor cells.
RESULTS Results are summarized in Table 1. A m o n g the SCCs, CIS did n o t display any giant
cells. With immunostaining, one o f three specimens showed positive staining with anti-SP-1 in less than 10% of t u m o r cell population. No positive staining was observed with anti-hCG and anti-hPL. None of the five specimens of SQCC WD displayed giant cells. With immunostaining, two were negative with all three antibodies. Two showed positive staining with all three antibodies in less than 10% o f t u m o r cells. O n e showed positive staining with anti-hCG and anti-SP-1 in less than 10% o f t u m o r cells, but negative staining with anti-hPL. All the positive cells with immunostaining seemed to be keratinized on examination o f hematoxylin-eosinstained sections. To exclude the possibility of an artificial reaction o f keratin to these antibodies or crossreactivity of the antibodies with keratin, the authors p e r f o r m e d the immunostaining o f keratinizing squamous cell carcinomas of skin (n = 2), esophagus (n = 3), and larynx (n = 6). None of the control sections showed positive reaction with anyone of the three antibodies. All six cases of SQCC PD had giant tumor cells constituting up to 5% of minor cells. All six SQCC PD cases were negative with anti-hCG, hPL was positive in two cases in less than 10% of tumor cells. SP-1 was positive in 11% to 25% of tumor cells in four cases (Fig 1). For all categories of SQCC, hCG was positive in three of 14 cases (21%), hPL was positive in four of 14 cases (28%), and SP-1 was positive in nine o f 14 cases (64%). The positive reaction was not confined to giant cells. A m o n g the adenocarcinomas, giant cells were f o u n d only in papillary carcinomas (PAPs), but n o t in bronchioloalveolar cell (BAC) or acinar carcinomas (ACs). hCG was positive ill three o f three BACs, in three of four PAPs and n o n e of three ACs. O n e BAC and two PAPs showed positive reaction with anti-hCG in 11% to 25% of t u m o r cells (Fig 2). The rest of the cases with positive reaction showed staining in less than 10% o f t u m o r cells, hPL was positive only on one BAC in less than 10% of t u m o r cells. SP-1 was positive in two of three BACs, four o f four PAPs, and two of three ACs. O n e BAC and three PAPs showed positive reaction in 11% to 25% of the t u m o r cell population. A m o n g the LCCs, giant cells were f o u n d in LCC not otherwise specified (LCC NOS; giant cells less than 5% of t u m o r cell population) and in giant cell carcin o m a (LCC GC; giant cells greater than 50% o f t u m o r cell population). Clear cell carcinoma (LCC CLC) lacked any appreciable giant cell population, hCG was positive in five of five LCC NOSs, in three of three LCC CLCs, and seven of eight LCC GCs. The prevalence of reaction ranged from less than 10% o f t u m o r cell population in five o f five LCC NOSs (Fig 3) and four of eight LCC GCs (Fig 4) to greater than 25% of t u m o r cell population in two o f eight LCC GCs. All five cases of LCC CLC showed positive reaction in 11% to 25% of t u m o r cell population, hPL was positive in three of five NOSs, three o f three LCC CLCs and three o f eight LCC GCs. hPL was positive in 11% to 25% of t u m o r cell population in two of three LCC CLCs. SP-1 was positive in four of five LCC NOSs, three o f three LCC CLCs, and eight of eight LCC GCs. Two cases out of
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TABLE 1.
Immunoperoxidase Staining in Various Types of Lung Carcinomas hCG
SQCC (n -- 14) CIS (n = 3) WD (n = 5) PD (n = 6) A d e n o c a r c i n o m a (n = 10) BAC (n = 3) PAP (n = 4) AC (n = 3) LC (n = 16) LCNOS (n=5) L C C L C ( n = 3) LC GC (n = 8) SCC (n = 4) Percent positivity o f all t u m o r types
GC Score
-
+
3 2 6
+
hPL Positive (%)
++
-
+
++
+++
21 3
1 3
2 1
2 2
2 4 3
4
2 3 1
+
2 2 1
1 3 4
1 1
1 1 2
1
4
++
+++
1
2
5 4
0
3 1 3
80
93
2 5 0
54
31
Positive (%) 64
56
5 1 4
-
10
1 2 93
+ +++ -
Positive (%) 28
3 3 4 60
+/++ -
SP-1
4
1 1
2 2 0
72
NOTE. Scores: Giant cell: - , n o giant; +, giant cell consists o f u p to 5% of t u m o r cell population; + + , giant cell 5% to 5% of t u m o r cell population; + + , giant cell greater t h a n 25% o f t u m o r cell population. I m m u n o p e r o x i d a s e scores: - , n o staining; +, positive staining in u p to 10% of t u m o r cells; + + , positive staining in 11% to 25% of t u m o r cells; + + + , positive staining in greater t h a n 25% o f t u m o r cells. Abbreviations: HCG, h u m a n chorionic g o n a d o t r o p h i n beta; hPL, h u m a n placental lactogen; SP-1, p r e g n a n c y specific beta-l-glycoprotein; SQCC, s q u a m o u s cell carcinoma; CIS, c a r c i n o m a in situ; WD, well differentiated; PD, poorly differentiated; BAC, bronchioalveolar cell type; PAP, papillary type; AC, acinar type; LC, large cell carcinoma; NOS, n o t otherwise specified; CLC, clear cell type; GC, giant cell; SCC, small cell carcinoma.
eight LCC GCs showed positive reaction with anti-SP-1 in greater than 25% of t u m o r cell population. SP-1 was positive in 11% to 25% o f t u m o r cell population in two of three LCC CLCs and one of eight LCC GCs and was positive in less than 10% of tumor cell population in four of five LCC NOSs, one of three LCC CLCs and five of eight LCC GCs. Small cell carcinomas lacked t u m o r giant cells in the specimens examined in this study. Immunostaining with three antibodies was universally negative in SCCs. Encompassing all t u m o r types, a positive reaction with hCG was f o u n d in 24 of 44 cases (54%), a positive reaction with hPL in 14 of 44 cases (31%), and a positive
T h e aims of this study were twofold: (1) distribution of the trophoblastic cell marker expression in various cell types of lung carcinomas, and (2) examination of the relationship between the trophoblastic cell markers and t u m o r giants in lung carcinomas. Without resorting to statistical analysis, positive reaction with these antibodies in the carcinomas that lack giant cells
FIGURE 1. Poorly differentiated squamous cell carcinoma with SP-1 immunoperoxidase staining. A few cells show strong cytoplasmic reaction. (Immunoperoxidase stain; original magnification ×200.)
FIGURE 2. Papillary adenocarcinoma with hCG immunoperoxidase staining. Some giant cells show strong reaction in the peripheral cytoplasm. (Immunoperoxidase stain; original magnification x200.)
reaction with SP-1 in 32 of 44 cases (72%). All positive and negative controls stained appropriately.
DISCUSSION
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FIGURE3. Large cell carcinoma NOS, with hCG immunostaining. Scattered cells with strongly positive cytoplasmic reaction are noticed. (Immunoperoxidase stain; original magnification ×200.)
(SQCC WD, BAC, and LCC CLC) disproves the connection of tumor giant cells with trophoblastic cell markers. These data indicate clearly that all the categories of lung carcinomas except for SCCs express these trophoblastic markers at the tissue level, and these markers were positive in regular tumor cells. Small cell carcinomas in this study failed to show any positive reaction for these markers--similar to the results obtained by Wilson et al.10 Bondy,20 conversely, found hCG-/3 in serum of 19% of patients with SCC. Hattori et al9 did not find detectable hCG-/3 in the serum of their patients with SCC, but 21% of their SCC tissue samples showed a positive reaction to the hCG assay after extraction. Although Wilson et all0 do not specify the source of their samples, the discrepancy in hCG expression in SCC may be due to the sample size. The present study used small bronchial biopsies. Autopsy can provide larger specimens. However, the authors of this investigation chose not to study autopsy specimens, because the patients had received chemotherapeutic agents, and the effects that these agents might have on the cellular production of the hormones were unknown. In this study, hPL showed the lowest positivity and SP-1 the highest with the hCG in between. It is not clear whether this reflects genuine characteristics of lung carcinomas or the respective strength of the antibodies used. When tabulated as the positive expression of lung carcinoma categories, SQCC had the lowest positivity. The positive reaction is not concentrated in SQCC PD, which have more tumor giant cells. Positivity was found from CIS to SQCC PD. One point of interest was apparent positive reaction in keratinizing tumor cells. We used SQCCs from the other sites to exclude false-positive reaction by keratin. The control groups all showed negative reaction with the three antibodies, thus negating the possibility of false positivity because of keratin. Among the adenocarcinomas, only papillary carcinomas exhibited significant tumor giant cells. The positive reactivity, conversely, seems to be indistinguishable from that of the other adenocarcinomas, which lacked tumor
giant cells. As expected, the LCCs showed the highest and the most intense positive reactions, especially in GCCs. Some of the GCCs, however, lacked positive reaction altogether and showed only a patchy reaction in the tumor giant cells. All the clear cell carcinoma variants showed a moderate positive reaction with all three antibodies. As addressed by Wilson et al,10 this positive reaction raises the possibility of false positivity by intracellular glycogen. The authors of the present study have also tried immunostaining after diastase treatment of tissue sections, and the positive reaction was sustained, thus negating the false positivity by glycogen. Histological heterogeneity of lung carcinomas has been recognized for more than 50 years. 2L22This study tried to choose the cases with minimal histological variations. Small cell carcinoma and SQCC with CIS displayed uniform histological appearances of each cell type, partly because of the small size of the tissue samples of bronchial biopsy specimens. Squamous cell carcinomas usually exhibited a uniform histology with focal variation in differentiation. The distinction of the adenocarcinoma subtypes posed some problem. Most of the tumors showed small foci of other subtypes. The authors chose tissue blocks that showed rather uniform histology. This was also the case with the LCC NOS and the CLC LCC. Giant cell carcinoma presented a dilemma. There is no consensus regarding the diagnostic criteria for GC. The WHO classification states that "giant cell carcinoma is a large cell carcinoma containing a prominent component of highly pleomorphic multinucleated cells."~s Left undefined is " p r o m i n e n t " and "multinucleated" cells as criteria, which is not generally accepted. Varying percentages of a giant cell component have been proposed, ranging from 10% 23 tO 30% to 55%. 24 Further complicating the argument is the relationship of other histological types to GCC. WHO classification places GCC in the subtypes of LCC. Some found GCC more frequently in association with spindle cell carcinoma and adenocarcinoma. 23 Some authors suggest that tumors with components of LCC, adenocarcinoma, and SQCC should be excluded from
FIGURE 4. Giant cell carcinoma with hCG immunostaining. Some cells show strong reaction, whereas others do not. (Immunoperoxidase stain; original magnification ×200.)
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the category of GCC. z5 The investigators in this study chose to qualify the W H O classification of GCC as LCC with greater than 25% of t u m o r cell population consisting of giant cells. Chorionic gonadotropins are f o u n d in carcinomas of various sites beside trophoblastic tumors including urothelial carcinoma, 2~29 gastrointestinal adenocarcinomas, 3°''~1 and breast carcinomas. 8'32'3~ Although they are not as widely tested as hCG, h u m a n placental lactogen (hPL) and pregnancy-specific fll-glycoprotein (SPH e y1) are also f o u n d an various eplthehal tumo s. d e r m a n et a134f o u n d hPL in serum and tissue by immunoperoxidase in a few lung carcinomas. SP-1 was f o u n d in the serum of one of 32 patients with lung carcinoma by Grudzinska et al. 37 Tissue localization of SP-1 in lung carcinoma has not been d o c u m e n t e d to our knowledge. The prognostic significance of these markers in nongonadal tumors has been debated but remains unsettled, n o r does their presence correlate with the histological differentiation of the tumor• Interesting in this regard is the finding of hCG in noncancerous gastric mucosa. ~s Although the biological implication of this finding is not fully elucidated, some speculate that hCG expression may be linked to actively proliferating cells. ~s In this study, one of the three samples of SQCC CIS showed a positive reaction with SP-1. This may represent a similar reaction of bronchial mucosa to that o f gastric mucosa. Another possibility to be explored is that these placental glycoproteins may share some antigenicity with the other cellular glycoproteins, such as epidermal growth factor receptor, which is strongly expressed in lung carcinomas, especially in SQCC CIS. ~9 In conclusion, various histological types of lung carcinomas express placental glycoproteins by immunoperoxidase technique• T h e expression o f these markers is not limited to the t u m o r giant cells. The diagnosis of primary choriocarcinoma o f the lung, therefore, should be applied with caution• •
"
"
r
34-36
REFERENCES 1. Fusco FD, Rosen, SW: Gonadotropin-producing anaplastic large-cell carcinomas of the lung. N Engl J Med 275:50%515, 1966 2. Falman C, Colwell JA, Ryan RJ, et al: Gonadotropin secretion from a bronchogenic carcinoma: Demonstration by radioimmunoassay. N Engl J Med 277:1395-1399, 1967 3. Becket KL, CottrellJC, Moore CF, et al: Endocrine studies in a patient with a gonadotropin-secreting bronchogenic carcinoma. J Clin Endocrinol Metab 18:809-818, 1968 4. Rosen SW, Becket CE, Shlaff S, et al: Ectopic gonadotropin production before clinical recognition of bronchogenic carcinoma. N EnglJ Med 279:640-641, 1968 5. Dailey JE, Marcuse PM: Gonadotropin secreting giant cell carcinoma of the lung. Cancer 24:388-396, 1969 6. Weintraub BD, Rosen SW: Ectopic production of human chorionic somatomammotropin by nonotrophoblastic cancers. J Clin Endocrinol Metab 32:94-101, 1971 7. Rosen SW, Weintraub BD: Ectopic production of the isolated alpha subunit of the glycoproteins. N Engl J Med 290:1441-1447, 1974 8. McManus LM, Nanghton MA, Martinez-Hernandez A: Human chorionic gonadotropin in human neoplastic cells. Cancer Res 36:3476-3479, 1976 9. Hattori M, Fukase M, Yoshimi H, et al: Ectopic production of human chorionic gonadotropin in malignant tumors. Cancer 42:2328-2333, 1978 10. Wilson TS, McDowell EM, McIntire R, et al: Elaboration of
human chorionic gonadotropin by lung tumors: An immunocytochemical study. Arch Pathol Lab Med 105:169-173, 1981 11. Heyderman E, Chapman DV, Richardson TC: Biological markers in lung cancer: An immunocytochemical approach. Cancer Detect Prev 5:427-449, 1982 12. Pushchak MJ, Farhi DC: Primary choriocarcinoma of the lung. Arch Pathol Lab Med 111:477479, 1987 13. Sullivan LG: Primary choriocarcinoma of the lung in a man. Arch Pathol Lab Med 113:82-83, 1989 14. Sridhar KS, Saldana MJ, Thurer RJ, et al: Primary choriocarcinoma of the lung: A report of a case treated with multimodality therapy and review of the literature. J Surg Oncol 41:93-97, 1989 15. Heitz PV, yon Herbay G, Kloppel G, et al: The expression of subunits of human chorionic gonadotropin (hCG) by nontrophoblastic, nonendocrine, and endocrine tumors. Am J Clin Pathol 88:46%472, 1987 16. Kurman R, Young R, Norris H, et al: Immunocytochemical localization of placental lactogen and chorionic gonadotropin in the normal placenta and trophoblastic tumors, with emphasis on intermediate trophoblast and the placental site trophoblastic tumor. Int J Gynecol Pathol 3:101-121, 1984 17. Takayama M, Isaka K, Suzuki Y, et al: Comparative study of placental protein 19, human chorionic gonadotropin and pregnancyspecific beta-1 glycoprotein as immunohistochemical markers for extravillous trophoblast in pregnancy and trophoblastic disease. Histochemistry 93:16%173, 1989 18. Shimosato Y, Sobin LH, Spencer H, et al (editorial committee): The World Health Organization Histologic Typing of Lung Tumors (ed 2). A m J Clin Pathol 77:123-136, 1982 19. Yesner R: Classification of lung-cancer histology. N Engl J Med 312:652-653, 1985 20. Bondy PK: The pattern of ectopic hormone production in lung cancer. YaleJ Biol Med 54:181-185, 1981 21. Willis RA: Pathology ofTumours (ed 2). London, UK, Butterworth, 1953, p 357 22. Roggli VL, Vollmer RT, Greenberg SP, et al: Lung cancer heterogeneity: A blinded and randomized study of 100 consecutive cases. HUM PATHOL 16:569-579, 1985 23. Fishback NF, Travis WP, Moran CA, et al: Pleomorphic (spindle giant ceil) carcinoma of the lung: A clinico-pathologic correlation of 78 cases. Cancer 73:2936-2945, 1994 24. Mackay B, Lukeman J, Ordonez N: Tumors of the Lung. Philadelphia, PA, Saunders, 1991, pp 101-103 25. Ginsberg SS, Buzaid AC, Stern H, et al: Giant cell carcinoma of the lung. Cancer 70:608-610, 1992 26. KawamuraJ, Machida S, Yoshida O, et al: Bladder carcinoma associated with ectopic production of gonadotropin, Cancer 42:17732780, 1978 27. Yamase HT, Wurzel RS, Hieh PT, et al: Immunohistochemical demonstration of human chorionic gonadotropin in tumors of the urinary bladder. Ann Clin Lab Sci 15:414-417, 1985 28. ShahVM, Newman J, Crocker J, etal: Ectopic p-human chorionic gonadotropin production by bladder urothelial neoplasia. Arch Pathol Lab Med 110:107-111, 1986 29. Grammatico D, Grignon JD, Eberwein P, et al: Transitional cell carcinoma of the renal pelvis with choriocarcinomatous differentiation: Immunohistochemical and immunoelectron microscopic assessment of human chorionic gonadotropin production by transitional cell carcinoma of the urinary bladder. Cancer 71:1835-1841, 1993 30. Yakeishi Y, Mori M, Enjoji M: Distribution of t - h u m a n chorionic gonadotropin-positive cell sin noncancerous gastric mucosa and in malignant gastric tumors. Cancer 66:695-701, 1990 31. ConnellyJH,Johnston DA, BrunerJM: The prognostic value of human chorionic gonadotropin expression in cholorectal adenocarcinomas: An immunohistochemical study of 102 stage B2 and C2 nonmucinous adenocarcinomas. Arch Pathol Lab Med 117:824-826, 1993 32. Monteiro JCMP, Fergusson KM, McKinna JA, et al: Ectopic production of human chorionic gonadotropin like material by breast cancer. Cancer 53:957-962, 1984 33. Kuida CA, Braunstein GD, Shintaka P, et al: Human chorionic gonadotropin expression in lung, breast, and renal carcinomas. Arch Pathol Lab Med 112:282-285, 1988 34. Heyderman E, Chapman DV, Richardson TC, et al: Human
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chorionic gonadotropin and human placental lactogen in extragonadal tumors: An immunoperoxidase study often non-germ cell neoplasms. Cancer 56:2674-2682, 1985 35. Home CHW, Towler CM, Milne GD: Detection of pregnancy specific beta-l: Glycoprotein in formalin fixed tissues. J Clin Pathol 30:19-23, 1977 36. Kuhajda FP, Bohn H, Mendelsohn G: Pregnancy-specific beta-l-glycoprotein (SP-1) in breast carcinoma: Pathogenic and clinical considerations. Cancer 54:1392-1396, 1984
37. GmdzinskasJG, Coombes RC, RatcliffeJG, et al: Circulating levels of pregnancy specific beta: Glycoprotein in patients with testicular, bronchogenic and breast carcinomas. Cancer 45:102-103, 1980 38. Manabe T, Adachi M, Hirao ~ Human chorionic gonadotropin in normal, inflammatory, and carcinomatous gastric tissue. Gastroenterology 89:1319-1325, 1985 39. Yoneda K: Distribution of proliferating cell nuclear antigen and epidermal growth factor receptor in intraepithelial squamous cell lesions in human bronchus. Mod Pathol 7:480-486, 1994
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56%, and SP-1 in 93%. The positive reaction did not appear to be restricted to nor associated with the tumor giant cells. It was concluded that these trophoblastic cell markers are expressed in various types of lung carcinomas and that they are not associated with certain histological types or with tumor giant cells. HUM PATHOL 26:1201-1206. Copyright © 1995 by W.B. Saunders Company Key words: lung carcinomas, human chorionic gonadotropin, human placental lactogen, pregnancy-specific/~rglycoprotein. Abbreviations: hCG, human chorionic gonadotropin; hPL, human placental lactogen; SP-1, pregnancy-specific/$1-glycoprotein; CIS, carcinoma in situ; SCC, small cell carcinoma; SQCC, squamous cell carcinoma; WD, well differentiated; PD, poorly differentiated; BAC, bronchioloalveolar cell carcinoma; PAP, papillary carcinoma; AC, acinar carcinoma; LCC, large cell carcinoma; CLC, clear cell carcinoma; GC, giant cell carcinoma; NOS, not otherwise specified; WHO, World Health Organization.
Since the mid-1960s, the secretion of gonadotropin by lung carcinomas has been r e p o r t e d anecdotally. 1~ Although all types of the lung carcinomas p r o d u c e d h u m a n chorionic gonadotropin (hCG), the large cell carcinoma (LCC), especially giant cell carcinoma (GC), was the most conspicuous in this regard. 7 In 1976, human chorionic gonadotropin (hCG) was localized in t u m o r tissue by immunofluorescence by McManus, Naughton, and Martinez-Hernandez. s Hattori et al 9 studied hCG in plasma and in t u m o r tissue in 35 cases of lung carcinomas and f o u n d hCG in plasma in 6% of patients and hCG in tissue extraction in 35% of patients. Wilson et al, 1° conversely, studied hCG-/3 by immunoperoxidase technique in paraffin sections of 61 lung carcinomas. They f o u n d positive reaction in 84% of lung carcinomas with all cell types except small cell carcinoma (SCC). This finding was disputed by Heyderman, Chapman, and Richardson, al who found hCG in only five of 40 lung carcinomas (12.5%). Although this controversy about hCG production in lung carcinomas was in progress, several case reports of primary choriocarcinoma of the lung appeared in the literature, a2-14The diagnosis was based on the existence o f bizarre tumor giant cells (syncytiotrophoblasts) and the positive reaction of the tumor cells for hCG by immunohistochemical technique. Given the prevalence of hCG production in lung carcinomas, especially GCs,
the distinction of this entity from the large cell lung carcinoma seems to be ambiguous. The question arises whether there exists a distinct class of lung tumor that has a unique combination of hCG production and bizarre giant cells. In this study, the authors tried to answer this question by examining various histological types of lung carcinomas with immunoperoxidase technique using antibodies against trophoblastic markers. H u m a n chorionic gonadotropin consists of two subunits, 0~ and /3. T h e ot-subunit is shared by other polypeptide h o r m o n e s and p r o d u c e d by various tumors. 15 The /3-subunit is unique to each h o r m o n e and differentiates the biological activities. Therefore, the expression of hCG-/3 by lung carcinomas is of interest. In addition to hCG-beta, the authors examined the expression of h u m a n placental lactogen (hPL) and pregnancy-specific/3Fglycoprotein (SP-1) to determine whether the expression of hCG-/3 is an isolated p h e n o m e n o n or whether the same t u m o r cells produce the spectrum of trophoblastic markers. All these markers are p r o d u c e d in benign and malignant syncytiotrophoblasts. The expression of these markers seems to show some differentiation in various trophoblastic tumors. 16'17 Therefore, it is interesting to see whether there exists a pattern of these markers in certain lung carcinomas.
MATERIALS AND METHODS From the Department of Pathology, Washington University School of Medicine, St, Louis, MO; and the Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center, and University of Kentucky College of Medicine, Lexington, KY. Accepted for publication April 19, 1995. Address correspondence and reprint requests to Kokichi Yoneda, MD, Pathology and Laboratory Medicine Service (113 CDD), Veterans Affairs Medical Center, 2250 Leestown Rd, Lexington, KY405111093. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2611-000855.00/0
Forty-four cases of lung carcinomas were selected from the surgical pathology file of the Veterans Affairs Medical Center (Lexington, KY). They consisted of seven biopsy specimens and 37 pulmonary resection (lobectomy or pneumonectomy) specimens. All the blocks were processed routinely for histological examination. The specimen was fixed in 10% buffered formalin for 2 hours to overnight. The specimens were processed routinely and embedded in paraffin. The biopsy specimens were three of squamous cell carcinoma in situ (CIS) of the bronchus and four SCCs. For each resection
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specimen, two to three blocks of tumor for each case were selected for study. Altogether 105 blocks were studied by immunoperoxidase staining. The authors followed the World Heahh Organization (WHO) classification of lung tumors TM in selection of the cases with the following exceptions: (1) squamous cell carcinoma (SQCC) was divided into two categories of well differentiated (SQCC WD) and poorly differentiated (SQCC PD) based on the presence of keratin by light microscopic examination of hematoxylin-eosin-stained sections. (2) Small cell carcinoma (SCC) was not divided into further categories. 19 (3) The authors did not select the cases from the category of solid adenocarcinoma with mucus formation because they wished to examine the difference between clearly distinct cell types. The classification of tumors selected was based on consensus. Three cases, on whose classification the authors did not agree, were not included in the current study. The following are the tumor types studied with respective number of cases in parenthesis: SQCC (n = 14) consisting of CIS (n = three), SQCC WD (n = five) and SQCC PD (n = six); adenocarcinoma (n = 10), consisting of bronchioloalveolar cell type (BAC, n = three), papillary type (PAP, n = four), and acinar type (AC, n = three); LCC (n = 16) consisting of LCC not otherwise specified (LCC NOS, n = five), clear cell type (LCC CLC, n = three), and giant cell carcinoma (LCC GC, n -- eight); and SGC (n = four). Polyclonal rabbit antibodies against hCG beta, hPL, and SP-1 were purchased from DAKO (Carpinteria, CA). Four serial 5-/~m tissue sections were cut, and the first sections were stained with hematoxylin-eosin. The other tissue sections were deparaffinized with xylene and then washed in ethanol. Enzymatic digestion to enhance antigen was not performed. After endogenous peroxidase was blocked by hydrogen peroxide, sections were incubated with normal rabbitblocking serum. Prediluted primary antibodies were applied to the sections for 30 minutes. The sections were washed and then incubated for 10 minutes in diluted biotinylated secondary antibody. After rinsing, sections were incubated with Vectastain Elite ABC reagent (Vector, Burlingame, CA). The color was developed using 3,3'-diaminobenzidine-hydrogen peroxide substrate. Placental tissue was used as controls. In addition, sections of the SQCC of the skin (n = six), esophagus (n = three), and larynx (n = six) were used as controls for SQCC WD. These controls were used to rule out cross-reactivity between antitrophoblastic glycoproteins and antikeratin. The scoring of grading sections was done independently by each author, and the scores were averaged. Hematoxylineosin-stained sections of all the blocks used for immunoperoxidase study were examined for tumor type and existence of giant cells. Giant cells were defined in this study as those cells with over three times in diameter of the average tumor cells. They are either mononucleated or multinucleated. Their presence was scored as follows: - , no giant cells; +, giant cells consist of up to 5% of tumor cell population; ++, giant cells consist of 5% to 25% of tumor cell population; + + + , giant cells constitute more than 25% of tumor cell population. The last category is one of the diagnostic criteria of giant cell carcinoma. The immunoperoxidase staining was similarly graded as follows: - , no staining; +, positive staining in up to 10% of cells; ++, positive staining in 11% to 25% of cells; + + + , positive staining in more than 25% of tumor cells.
RESULTS Results are summarized in Table 1. A m o n g the SCCs, CIS did n o t display any giant
cells. With immunostaining, one o f three specimens showed positive staining with anti-SP-1 in less than 10% of t u m o r cell population. No positive staining was observed with anti-hCG and anti-hPL. None of the five specimens of SQCC WD displayed giant cells. With immunostaining, two were negative with all three antibodies. Two showed positive staining with all three antibodies in less than 10% o f t u m o r cells. O n e showed positive staining with anti-hCG and anti-SP-1 in less than 10% o f t u m o r cells, but negative staining with anti-hPL. All the positive cells with immunostaining seemed to be keratinized on examination o f hematoxylin-eosinstained sections. To exclude the possibility of an artificial reaction o f keratin to these antibodies or crossreactivity of the antibodies with keratin, the authors p e r f o r m e d the immunostaining o f keratinizing squamous cell carcinomas of skin (n = 2), esophagus (n = 3), and larynx (n = 6). None of the control sections showed positive reaction with anyone of the three antibodies. All six cases of SQCC PD had giant tumor cells constituting up to 5% of minor cells. All six SQCC PD cases were negative with anti-hCG, hPL was positive in two cases in less than 10% of tumor cells. SP-1 was positive in 11% to 25% of tumor cells in four cases (Fig 1). For all categories of SQCC, hCG was positive in three of 14 cases (21%), hPL was positive in four of 14 cases (28%), and SP-1 was positive in nine o f 14 cases (64%). The positive reaction was not confined to giant cells. A m o n g the adenocarcinomas, giant cells were f o u n d only in papillary carcinomas (PAPs), but n o t in bronchioloalveolar cell (BAC) or acinar carcinomas (ACs). hCG was positive ill three o f three BACs, in three of four PAPs and n o n e of three ACs. O n e BAC and two PAPs showed positive reaction with anti-hCG in 11% to 25% of t u m o r cells (Fig 2). The rest of the cases with positive reaction showed staining in less than 10% o f t u m o r cells, hPL was positive only on one BAC in less than 10% of t u m o r cells. SP-1 was positive in two of three BACs, four o f four PAPs, and two of three ACs. O n e BAC and three PAPs showed positive reaction in 11% to 25% of the t u m o r cell population. A m o n g the LCCs, giant cells were f o u n d in LCC not otherwise specified (LCC NOS; giant cells less than 5% of t u m o r cell population) and in giant cell carcin o m a (LCC GC; giant cells greater than 50% o f t u m o r cell population). Clear cell carcinoma (LCC CLC) lacked any appreciable giant cell population, hCG was positive in five of five LCC NOSs, in three of three LCC CLCs, and seven of eight LCC GCs. The prevalence of reaction ranged from less than 10% o f t u m o r cell population in five o f five LCC NOSs (Fig 3) and four of eight LCC GCs (Fig 4) to greater than 25% of t u m o r cell population in two o f eight LCC GCs. All five cases of LCC CLC showed positive reaction in 11% to 25% of t u m o r cell population, hPL was positive in three of five NOSs, three o f three LCC CLCs and three o f eight LCC GCs. hPL was positive in 11% to 25% of t u m o r cell population in two of three LCC CLCs. SP-1 was positive in four of five LCC NOSs, three o f three LCC CLCs, and eight of eight LCC GCs. Two cases out of
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TABLE 1.
Immunoperoxidase Staining in Various Types of Lung Carcinomas hCG
SQCC (n -- 14) CIS (n = 3) WD (n = 5) PD (n = 6) A d e n o c a r c i n o m a (n = 10) BAC (n = 3) PAP (n = 4) AC (n = 3) LC (n = 16) LCNOS (n=5) L C C L C ( n = 3) LC GC (n = 8) SCC (n = 4) Percent positivity o f all t u m o r types
GC Score
-
+
3 2 6
+
hPL Positive (%)
++
-
+
++
+++
21 3
1 3
2 1
2 2
2 4 3
4
2 3 1
+
2 2 1
1 3 4
1 1
1 1 2
1
4
++
+++
1
2
5 4
0
3 1 3
80
93
2 5 0
54
31
Positive (%) 64
56
5 1 4
-
10
1 2 93
+ +++ -
Positive (%) 28
3 3 4 60
+/++ -
SP-1
4
1 1
2 2 0
72
NOTE. Scores: Giant cell: - , n o giant; +, giant cell consists o f u p to 5% of t u m o r cell population; + + , giant cell 5% to 5% of t u m o r cell population; + + , giant cell greater t h a n 25% o f t u m o r cell population. I m m u n o p e r o x i d a s e scores: - , n o staining; +, positive staining in u p to 10% of t u m o r cells; + + , positive staining in 11% to 25% of t u m o r cells; + + + , positive staining in greater t h a n 25% o f t u m o r cells. Abbreviations: HCG, h u m a n chorionic g o n a d o t r o p h i n beta; hPL, h u m a n placental lactogen; SP-1, p r e g n a n c y specific beta-l-glycoprotein; SQCC, s q u a m o u s cell carcinoma; CIS, c a r c i n o m a in situ; WD, well differentiated; PD, poorly differentiated; BAC, bronchioalveolar cell type; PAP, papillary type; AC, acinar type; LC, large cell carcinoma; NOS, n o t otherwise specified; CLC, clear cell type; GC, giant cell; SCC, small cell carcinoma.
eight LCC GCs showed positive reaction with anti-SP-1 in greater than 25% of t u m o r cell population. SP-1 was positive in 11% to 25% o f t u m o r cell population in two of three LCC CLCs and one of eight LCC GCs and was positive in less than 10% of tumor cell population in four of five LCC NOSs, one of three LCC CLCs and five of eight LCC GCs. Small cell carcinomas lacked t u m o r giant cells in the specimens examined in this study. Immunostaining with three antibodies was universally negative in SCCs. Encompassing all t u m o r types, a positive reaction with hCG was f o u n d in 24 of 44 cases (54%), a positive reaction with hPL in 14 of 44 cases (31%), and a positive
T h e aims of this study were twofold: (1) distribution of the trophoblastic cell marker expression in various cell types of lung carcinomas, and (2) examination of the relationship between the trophoblastic cell markers and t u m o r giants in lung carcinomas. Without resorting to statistical analysis, positive reaction with these antibodies in the carcinomas that lack giant cells
FIGURE 1. Poorly differentiated squamous cell carcinoma with SP-1 immunoperoxidase staining. A few cells show strong cytoplasmic reaction. (Immunoperoxidase stain; original magnification ×200.)
FIGURE 2. Papillary adenocarcinoma with hCG immunoperoxidase staining. Some giant cells show strong reaction in the peripheral cytoplasm. (Immunoperoxidase stain; original magnification x200.)
reaction with SP-1 in 32 of 44 cases (72%). All positive and negative controls stained appropriately.
DISCUSSION
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FIGURE3. Large cell carcinoma NOS, with hCG immunostaining. Scattered cells with strongly positive cytoplasmic reaction are noticed. (Immunoperoxidase stain; original magnification ×200.)
(SQCC WD, BAC, and LCC CLC) disproves the connection of tumor giant cells with trophoblastic cell markers. These data indicate clearly that all the categories of lung carcinomas except for SCCs express these trophoblastic markers at the tissue level, and these markers were positive in regular tumor cells. Small cell carcinomas in this study failed to show any positive reaction for these markers--similar to the results obtained by Wilson et al.10 Bondy,20 conversely, found hCG-/3 in serum of 19% of patients with SCC. Hattori et al9 did not find detectable hCG-/3 in the serum of their patients with SCC, but 21% of their SCC tissue samples showed a positive reaction to the hCG assay after extraction. Although Wilson et all0 do not specify the source of their samples, the discrepancy in hCG expression in SCC may be due to the sample size. The present study used small bronchial biopsies. Autopsy can provide larger specimens. However, the authors of this investigation chose not to study autopsy specimens, because the patients had received chemotherapeutic agents, and the effects that these agents might have on the cellular production of the hormones were unknown. In this study, hPL showed the lowest positivity and SP-1 the highest with the hCG in between. It is not clear whether this reflects genuine characteristics of lung carcinomas or the respective strength of the antibodies used. When tabulated as the positive expression of lung carcinoma categories, SQCC had the lowest positivity. The positive reaction is not concentrated in SQCC PD, which have more tumor giant cells. Positivity was found from CIS to SQCC PD. One point of interest was apparent positive reaction in keratinizing tumor cells. We used SQCCs from the other sites to exclude false-positive reaction by keratin. The control groups all showed negative reaction with the three antibodies, thus negating the possibility of false positivity because of keratin. Among the adenocarcinomas, only papillary carcinomas exhibited significant tumor giant cells. The positive reactivity, conversely, seems to be indistinguishable from that of the other adenocarcinomas, which lacked tumor
giant cells. As expected, the LCCs showed the highest and the most intense positive reactions, especially in GCCs. Some of the GCCs, however, lacked positive reaction altogether and showed only a patchy reaction in the tumor giant cells. All the clear cell carcinoma variants showed a moderate positive reaction with all three antibodies. As addressed by Wilson et al,10 this positive reaction raises the possibility of false positivity by intracellular glycogen. The authors of the present study have also tried immunostaining after diastase treatment of tissue sections, and the positive reaction was sustained, thus negating the false positivity by glycogen. Histological heterogeneity of lung carcinomas has been recognized for more than 50 years. 2L22This study tried to choose the cases with minimal histological variations. Small cell carcinoma and SQCC with CIS displayed uniform histological appearances of each cell type, partly because of the small size of the tissue samples of bronchial biopsy specimens. Squamous cell carcinomas usually exhibited a uniform histology with focal variation in differentiation. The distinction of the adenocarcinoma subtypes posed some problem. Most of the tumors showed small foci of other subtypes. The authors chose tissue blocks that showed rather uniform histology. This was also the case with the LCC NOS and the CLC LCC. Giant cell carcinoma presented a dilemma. There is no consensus regarding the diagnostic criteria for GC. The WHO classification states that "giant cell carcinoma is a large cell carcinoma containing a prominent component of highly pleomorphic multinucleated cells."~s Left undefined is " p r o m i n e n t " and "multinucleated" cells as criteria, which is not generally accepted. Varying percentages of a giant cell component have been proposed, ranging from 10% 23 tO 30% to 55%. 24 Further complicating the argument is the relationship of other histological types to GCC. WHO classification places GCC in the subtypes of LCC. Some found GCC more frequently in association with spindle cell carcinoma and adenocarcinoma. 23 Some authors suggest that tumors with components of LCC, adenocarcinoma, and SQCC should be excluded from
FIGURE 4. Giant cell carcinoma with hCG immunostaining. Some cells show strong reaction, whereas others do not. (Immunoperoxidase stain; original magnification ×200.)
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TROPHOBLASTIC MARKERS IN LUNG CARCINOMA (Boucher & Yoneda)
the category of GCC. z5 The investigators in this study chose to qualify the W H O classification of GCC as LCC with greater than 25% of t u m o r cell population consisting of giant cells. Chorionic gonadotropins are f o u n d in carcinomas of various sites beside trophoblastic tumors including urothelial carcinoma, 2~29 gastrointestinal adenocarcinomas, 3°''~1 and breast carcinomas. 8'32'3~ Although they are not as widely tested as hCG, h u m a n placental lactogen (hPL) and pregnancy-specific fll-glycoprotein (SPH e y1) are also f o u n d an various eplthehal tumo s. d e r m a n et a134f o u n d hPL in serum and tissue by immunoperoxidase in a few lung carcinomas. SP-1 was f o u n d in the serum of one of 32 patients with lung carcinoma by Grudzinska et al. 37 Tissue localization of SP-1 in lung carcinoma has not been d o c u m e n t e d to our knowledge. The prognostic significance of these markers in nongonadal tumors has been debated but remains unsettled, n o r does their presence correlate with the histological differentiation of the tumor• Interesting in this regard is the finding of hCG in noncancerous gastric mucosa. ~s Although the biological implication of this finding is not fully elucidated, some speculate that hCG expression may be linked to actively proliferating cells. ~s In this study, one of the three samples of SQCC CIS showed a positive reaction with SP-1. This may represent a similar reaction of bronchial mucosa to that o f gastric mucosa. Another possibility to be explored is that these placental glycoproteins may share some antigenicity with the other cellular glycoproteins, such as epidermal growth factor receptor, which is strongly expressed in lung carcinomas, especially in SQCC CIS. ~9 In conclusion, various histological types of lung carcinomas express placental glycoproteins by immunoperoxidase technique• T h e expression o f these markers is not limited to the t u m o r giant cells. The diagnosis of primary choriocarcinoma o f the lung, therefore, should be applied with caution• •
"
"
r
34-36
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chorionic gonadotropin and human placental lactogen in extragonadal tumors: An immunoperoxidase study often non-germ cell neoplasms. Cancer 56:2674-2682, 1985 35. Home CHW, Towler CM, Milne GD: Detection of pregnancy specific beta-l: Glycoprotein in formalin fixed tissues. J Clin Pathol 30:19-23, 1977 36. Kuhajda FP, Bohn H, Mendelsohn G: Pregnancy-specific beta-l-glycoprotein (SP-1) in breast carcinoma: Pathogenic and clinical considerations. Cancer 54:1392-1396, 1984
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