- Email: [email protected]
An unusual bronchial carcinoid tumor: Light and electron microscopy
AN UNUSUAL BRONCHIAL CARCINOID TUMOR: LIGHT AND ELECTRON MICROSCOPY Elizabeth M. McDowell, B. Vet. Med., Ph.D.,* Se~gei P. Sorokin, M.D.,~ Richard F. Hoyt, Jr., Ph.D.,r and Benjamin F. TT~tmp, M.D.w Abstract An unusual bronchial carcinoid t u m o r was studied by light and electron microscopy. T h e t u m o r cells, which a p p e a r e d to be m o n o t o n o u s l y t m i t b r m in hematoxylin and eosin stained sections, were f o u n d to be morphologically h e t e r o g e n e o u s at the ultrastructural level with r e g a r d to the size, n u m b e r , and m o r p h o l o g y o f the e n d o c r i n e granules. Presumptive e h d o c r i n e granules were seen in all t u m o r cells, but some cells contained only small r o u n d granules (2000 ,~ largest diameter), o t h e r cells contained large r o u n d granules (some with as large a d i a m e t e r as 1.0/z), and sotne cells contained large p o l y m o r p h i c granules. Many o f the cells stained positively at the light microscopic level when selective stains for e n d o c r i n e cells were applied. All types o f grant.ties showed argyrophilia at the ttltrastructural level. N u m e r o u s clusters o f e n d o c r i n e cells were observed in the otherwise n o r m a l bronchial and bronchial glandular epithelium. T h e spectrunt o f granule morphologies, as seen in the t u m o r cells, was displayed in cells o f the intraepithelial clusters. Some nmcous cells and sparsely ciliated cells within these clusters contained argyrophilic granules. Multiple continuities existed hetween the epithelial e n d o c r i n e cell clusters and the u n d e r l y i n g t u m o r mass. T h e intraepithelial d u s t e r s r e p r e s e n t foci o f carcinoma in situ, the genesis o f which is discnssed.
It is generally believed that presumptively endocrine cells o f the bronchial and bronchiolar epitheliu m are the cells o f origin o f carcinoid a n d oat cell tumors o f the lung. Ha Peripheral p t t l m o n a r y t u m o r lets may also derive f r o m these cells? 's~8 E n d o c r i n e cells have been described in the epithelium o f n o r m a l bronchi, bronclfioles, and bronchial glands o f adult h u t n a n lung. 3' 9, ~9-.oGT h e cells are seen only rarely lying on the basal lamina. The)' usually occur singly, but occasionally g r o u p s o f cells are seenY ~ E n d o c r i n e cells are also present in res-
piratory epithelium o f h u m a n fetuses and neonates,.,_o, o.t, _oT-a~and at least t h r e e a p p a r e n t l y d i f f e r e n t types o f fetal e n d o c r i n e cells have been described on the basis o f morphological characteristics o f the dense core granulesY 2' _04.3~ However, in studies o f g r a t m l a r tnorphology, only one type o f cell has been described in adult h u m a n lungs. -~176 T h e granules are characterized by their u n i f o r m r o u n d shape, stnall size (approximately 1400 ,~, in diameter), and h o m o g e n e o u s dense matt'ices. T h e granules are s u r r o u n d e d by a single m e m -
Accepted for publication January 24, 1980. This work was supported ill part by contract NOI-CP-43237 awarded to Dr. Trum'p by the National Cancer Institute aud by research grant ItL19379 awarded to Dr. lloyt by the Iieart, Lung, and Blood Institute. This is contribution 819 from tile Cellular Pathobiology Laboratory, University of Mar):land School of Medicine. *Associate l'rofessor, Department of I'athology, University of Maryland School of Medicine, Bahimore, Maryland. ]'Associate Professor of Cell Biology, Depart,nent of Physiology, ttarvard University School of Public Health, Boston, Massachusetts. :[:Assistant l'rofessort Department of Anatomy, Boston Universit)"School of Medicine. Boston, Massachusetts. w
338
and Clmirman, Department of l'athology, University of .Maryland School of Medicine, Baltimore, Marylandl IIUMAN PATIIOLOGY--VOLUME 12, NUMBER 4 Ap,ql1981
AN UNUSUAL
brane, which is usually separated from the dense core by a narrow halo. This type of cell closely resembles one of the three types o f endocrine cells normally present in human fetal lungs. 2"-''2t' 3! Abnormally large granules (up to 2500 A in diameter) scattered among the smaller granules were described in a few cells in one human caseY t The ultrastructural characteristics o f pulmonary carcinoid tumors have been described in detail, va" 7, 9t4, a'_,-34 However, the most detailed reports with regard to the histogenesis of carcinoid tumors are those of Hage n and Capella et al. 14 Hage n described the ultrastructural morphology of six bronchial carcinoid tumors; the individual tumors were composed of one, two, or even three apparently different types of endocrine cells differing in the morphological and cytochemical characteristics of the granules. Two of the three types of cells found in the tumors were similar to endocrine cells normally found in the respiratory epithelium of human fetuses. In contrast, in the study o f Capella et al. 14 two distinct types of tumors were described. One type was composed entirely of small granule cells and the other, large granule cells; small and large granule cells were not observed in individual tumors. In the present report a carcinoid tumor is described, which in terms of the size and other morp'hological characteristics of the granules, was composed of at least three and possibly as many as six apparently different types o f endocrine cells. Abnormally large numbers of the same types of endocrine cells seen in the tumor were also present in the bronchial epithelium and in the epithelium of the neck of the bronchial glands, and multiple sites of continuity (invasion) were seen between foci of neoplastic endocrine cells in the respirator}, epithelium and the underlying carcinoid tumor. Case
Report
A 62 },ear old male was noted t o have a coin lesion in the left upper lobe when a routine chest x-ray view was taken. The history was unremarkable except for a peptic ulcer 10 years previo.usly and rheumatoid arthritis with shoulder and hip pain. T h e patient was in good condition with no weight loss or cnest pain, but hemoptysis was noted one week prior to admission to Union Memorial Hospital, Baltimore, in April 1977. Bronchial washings, sputum specimens, and a lung biopsy study were negau.'ve for malignant cells, but a second x-ray fihn revealed not only the coin lesion but also suspected metastases to lymph nodes. A left thoracotomy was performed and the upper left lobe was removed. In the region of the hilum of the lobe a firm pale yellow mass, 3.5 by 2.1 by 2.0 cm., was present, which partially occluded some branches of the bronchus. Multiple black firm circumscribed nodules resembling lyxnph nodes were found in the hilar region measuring up to 1.3 cm. in diameter. Some of these were directly adjacent to the
BRONCHIAL
C A R C I N O I D T U M O R - - M c D O w E L L ET AL.
tumor mass. One portion o f the lobe, adjacent to the hilum and extending to the periphery, was dark red and of a rubber}' consistency. Sections through this area revealed multiple firm calcified nodules measuring tip to 0.3 cm. in diameter. T h e remainder of the lobe had normal crepitance but on cut section revealed several small hard calcified nodules measuring up to 0.3 cm. in diameter. Healed granulomas were present in the lung parenchyma and associated lymph nodes. On the basis of routine light microscopy a diagnosis of bronchial carcinoid with metastases to adjacent lymph nodes was made. T h e patient was discharged on the eighteenth hospital day. MATERIALS
AND
METHODS
The tumor and associated bronchus were fixed in 4 per cent formaldehyde-1 per cent glutaraldehyde and stored in fixative at room temperature until required for study. 3~
L I G H T M I C R O S C O P Y AND L I G H T MICROSCOPIC HISTOCHEMISTRY
The tissue was embedded in paraffin and 8 /.L thick sections were stained with hematoxylin and eosin. T h e presence of mucosubstances was monitored using the alcian blue (pH 2.5)-PAS method. 36 PAS-lead hematoxylin and alcian blue after acid hydrolysis were used as selective stains for endocrine cellsY' 3s For the demonstration o f biogenic amines the diazoiaium method (using fast red salt B), Gibb's reaction and the afgentaffin silver reaction of Masson-Fontana were applied. ~9 A test for argyrophilia was performed by using the Grimelius procedure. 46 Schmorl's reaction was also used. 39
H I G H R E S O L U T I O N L I G H T AND T R A N S M I S S I O N E L E C T R O N MICROSCOPY
For high resolution light microscopy and for electron microscopy t h e tissues were cut into small pieces and washed overnight in 0.2 M sncrose cacodylate buffer (pH 7.2), postfixed with 1 per cent osmium tetroxide buffered with 0.1M s-collidine, and stained en bloc with uranyl acetate, at T h e tissues were dehydrated in a graded series of ethanols and embedded in Epon. Areas were chosen for thin sectioning by examining 1.0/* thick sections stained with toluidine blue. Thin sections were double stained on coated grids with uranyl magnesium acetate and lead citrate and were examined in a J E O L 100B electron microscope. For demonstration of argyrophilia at the ultrastructural level, very thin, aldehyde fixed sections of tumor and associated bronchus, cut by hand with a razor blade, were impregnated with silver nitrate and
339
HUMAN
PATHOLOGY--VOLUME
12, N U M B E R
4
April 1981
reimpregnated as directed for glutaraldehyde fixed tissues.*-' T h e initial silver impregnation was accomplished at 40 ~ C. in the clark for 120 hours. The sections were then fixed in osmium tetroxide, but staining with uranyl acetate en bloc was omitted. Some of the thin section's were not stained on grids with either uranyl or lead salts.
rated by thin bands of vasctflarized connective tissue (Fig. 1). In other areas the cell nests were large and the organoid pattern was less obvious. Nuclei were of a uniform size and nucleoli were not prominent. Mitoses and necrosis were not observed. Mucosubstances were not demonstrated in the tumor. Approximately one quarter of the cells were stained by alcian blue after acid hydrolysis. Stain deposits were coarsely granular in some cells, "but in others the stain was diffusely distributed throughout the cytoplasm. Some cells were deeply stained whereas others had intermediate or light staining. Similarly stained cells tended to be grouped together (Fig. 2). Very few scattered cells were stained by either Schmorl's reaction or the diazo method (Fig. 3). No cells stained with Gibb's or the Masson-Fontana procedure. Some cells (but many more than were Schmorl or diazo positive) stained with the Grimelius procedure; the argyrophilic cells tended to be clustered in groups. Argyrophilia was more widespread and intense when demonstrated by en bloc silver nitrate impregnation prior to embedding in glycol methacrylate. (For detailed de-
RESULTS L I G H T M I C R O S C O P Y AND L I G I I T MICROSCOPIC HISTOCtIEMISTRY
PAI~X.FFIN SECTIONS. Only tumor tissue was present in paraffin sections, and therefore the relationship of the tumor to the bronchial epitheliuxn could not be assessed using these preparations. In some areas the tumor had an organoid pattern typical of many carcinoid tumors. In sections stained with hematoxylin and eosin the cells appeared monotonously regular, grouped into compact cell nests sepaFigure "
~r ~ r
.
9
.
1 .
Figure
.
~ ~'
"
~ - ' :"
'
- ~ "~-"
. . . . ~ q. l~ l .~ ~ ~-" r " ' ".'.
"
~_'~\."-~'.
"
,
"
.
t
~
9
~
~
-"
~
9
-
~:
,~"
.,.'~
"'.,"
~
:~"
.-k-'-
"
1;:
a*m.~--~-
9-
2
.~7 5 "'~ .
7": .
,~
,
..
_
,.* ' .
--'~'-~ i
9
~
't~-"
Figure 3 Figure 4 Figure l. Carcinoid tumor. Compact nests of cells, which apl)ear monotonously regular, are separated by thin bands of vascularized connective tissue, presenting a typical organoid pattern. (Paralfin section stained with hematoxylin and eosin, l.ight micrograph. • Figure 2. Carcinoid tunlOl-. Stain deposits are coarsely granular in some cells whereas in others the stain is more diffuse. Stain intensity varies; note that similarly stained cells tend to be grouped together. Many cells are not stained. (Paralfiq section staiqed with alcian hlue after acid hydrolysis, l.ight micrograpli. • Figure 3. Carcinuid tumor. Two cell~ (left lower center) are diazopositive. T h e nuclei are counterstained with hemalum. (i'araffin section stained by diazo method, l.ight micrograph. • Figure epithelium. tumor and blue. I+ight
340
4. Abnormally llumerotts arid large clusters of cells are seen basalb," (arrows) in the otherwise normal ciliated bronchial T h e cells comp<+sing the dusters al+pear identical to those of tit,: tmt]erl)ing tumor (T). Note areas of continuity betweelt epithelial clusters in bronchus a,ld bronchial gland (arrowheads). BG, bronchial gland. (Epon section stained with toluidine micrograph. •
AN UNUSUAL BRONCIIIA1, CARCINOID TUMOR--McDOWELL EI" AL. scriptions of tiffs and l'AS-lead hematoxylin staining of the tumor, see the accoml)anying article on page 302.) E P O N S E C T I O N S S T A I N E D SVITII T O L U I D I N E
BLUE
A bronchus anti associated tumor were available for study. Abnormally numerous and large clusters of cells were seen basally situated in otherwise normal bronchial epithelium and at the base of the epithelium lining the opening to a bronchial gland. T h e cells composing the clusters appeared identical to cells composing the underlying tumor mass and in several places were seen to be continuous with the tumor (Fig. 4). ELECTRON MICROSCOPY TUMOR. The tumor was composed of round, oval, and stellate cells. The plasma membranes closely paralleled one another so that intercellular spaces were minimal. T h e cells were joined by small inconspicuous desmosomes. The cytoplasm was electron lucent, and many cells contained well developed stacks o f rough endoplasmic reticulum. T h e Golgi apparatus was inconspicuous. Large residual bodies containing lipofilscin were common and fat droplets wgre seen in sonte cells. Some tumor cells were binucleated. All the tumor cells contained characteristic granules, but the number and morphology o f the granules varied greatly from cell to ceil. The results are summarized in Table I. Many cells contained small round dense core granules with diameters up to 2000 A (small granule cells; Figs. 5, 14). The central homogeneous dense core was separated from the surrounding membrane
TABLE 1.
by a halo (Fig. 5, inset). Some cells contained many of these granules (Fig. 14), whereas others contairied relatively few (Fig. 5). Many other cells contained large round dense core granules, which sometimes crowded the cytoplasm (Fig. 5). Collectively these cells could be called large granule cells, but on the basis of granular morphology there appeared to be at least tour different types o f large granule cells. In one type the granular matrix was homogeneons and of intermediate density throughout the large granule population in a single cell (Fig. 6). The granules measured up to 5000 ,~ in diameter. In a second type of large granule cell, two varieties of large granules were distinguishable by the electron density of the granular nmtrices (Fig. 7). Granules with darkly stained cores measured up to 4000 ,~ in diameter and granules of intermediate density, up to 5000/~, in diameter (Fig. 7). In a third type of large granule cell, three varieties of large grannies were distinguished by granule matrices with electron lucent and low intermediate densities (Fig. 8). The electron lucent granules, which w e r e the largest of the three types of granules, measured up to 8000 ,~ in diamcter (Fig. 8). In a fourth type of cell as many as four varieties of large granules could be distinguished on the basis of matrix electron density (Fig. 5). T h e largest grannies had a low electron density and sometimes exceeded 1.0/x in diameter. Yet another cell type was identified on the basis of granular morphology. These cells were rare in the tumor and contained numerous large (up to 6000 A diameter) polymorphic (pleomorpbic) granules that were rotmd, ovoid, bean shaped, and cup shaped. Some variation in electron density was seen among the granules. Some of the grantfles contained a single electron lucent membrane bound vesicle, suggesting that they represented pockets o f cytoplasm, resulting from fortuitous cuts through cup shaped granules (see epithelial counterpart, Fig. 11 and inset).
U L T R A S T R U C T U R A L MORPHOLOGIES OF CELLS OF A B R O N C H I A L C A R C I N O I D T U M O R
Cell Type Small granule
Granular Morphology
Argyrophilia
Small round granules with homogeneous densely stained cores, up. to 20(1(I ~, (Fig. 5 and inset)
+
l.arge round gr:mnles~-:',cith homogencous cores of intermediate density, up to 5000 ~, (Fig. 6)
+
Type 2
Two varieties of large round granules; dark staining cores up to -t000 ,~, and cores of intermcdiate dcnsity, up to 5000 z~, (Fig. 7)
+
Type 3
l-hree varieties of large round granules; electron lucent cores up to 8000 t~, and smaller granules with cores of low and intermediate density (Fig. 8)
+
Type 4
Four varieties of large round granules; low density cores up to anct exceeding 1.0 xt ,find smaller gramdcs with lucent, intermediate, and densely stained cores (Fig. 5)
+
Large round, ovoid, bean shaped, or cup shaped granules; some variation in electron density but tending to be densely stained; (tp to 6(}01} t~, (see epithelia! cot, ntcrlmrt, Fig. I I)
+
l.arge granule Type I
Pol)morl~hic (pleonmrphic) granule
341
Figure 5
:,dllq~,
:-~-":~L.
....
",;
~ " ~ ' ~
9~
"
~
.0,._
: " ,- ' ' , ; , , '
," -~:.:..~-~';".~.
.........
:'~"
,-
~..
.,
....
.
:~;';;:~
~
, ~
".:--:
.....
.~I~.~:.~.~.., .~
~.
,.~
J
'
~:~-
Figure 8 Figure 9 Figure l0 Figure 5. Carcinoid tumor. Some cells contai*, onl) very few scattered small deuse core granules (arrows, and inset). Another cell contains numerous very large granules; on the basis of electron density of the gi'anular matrices.at least four varieties of large granules cau be distinguished, lnset: Enlargemeut of small dense core granules denoted b)" arrows. (Electron micrngraph. • Inset, • Figure 6. Carcinoid tumor. The matrix is relatively dense and homogenous throughout the large granules of this cell. (Electron micrograph. • 10,500.) Figure 7. Carcinoid tumor. The matrices of the large gra,mles in this cell are of dark and intermediate densities. (Electron micrograph. • 10,500.) Figure 8. Carciuoid tulnor. The matrices of the large.granules in this ceil demonstrate three degrees of electron densit)'. (Electron micrograph. • 11,000.) Figure 9. Carcinoid tumor. Silver grains (argyrophilia) are concentrated over large granules. Vet). few silver grains are present over cytoplasm or a mitochondrion "(lower left). Staining with lead and uran)l salts was omitted. (Electron micrograph, x27,000A Figure 10. Carcinoid tumor. Silver grains (arg)'rophilia) are concentrated over small granules. Silver grains are not deposited on the lipofi*scin grannie (lower center). Staining with uranyl salts was omitted. (Electron micrograph. •
AN UNUSUAL
BRONCHIAL
CARCINOID
TUMOR--McDoWELL
Ew AL.
Figure 11
Figure 12 Figure 11. Bronchial gland epitheliunl. Endocrine cell containing numerous large polymorphic grannies. Basal lamina is shown at base (left and center). Inset: An electron lucent vesicle lies within one pol)'morlfllic grannie (top right). This appearance resuhs from fortuitous cuts through cup shaped granules (top left). (Electron micrograPh, x 12,500. Inset, • 17,{)(10.)
Figure 12. Bronchial gland'el~ithelitnn. A cell within an epithelial cluster ofendocri,ae cells contains both mucous granules and dense core gram~les. Note aq aggregate"df tllicl~ filaments (Upl)er left) randomly dispersed in one cell containing small dense core granules. A cell containifig large granules is prc'scnt at bottom. Inset: Bronchial gland epitheliu,n. Silver grains (argyrnplfilia) are concentrated over a dense cdre granule (right), wbicl'~:lppcars to be within the same cell as a intlcous granule (left). (Electron micrograph, xg000. Inset, st,fined on grid with uran)l and lead saltg, x55,000.)
343
HUMAN
PATHOLOGY--VOLUME
12, N U M I ? , E R
4
April 1981
After impregnation with silver nitrate and exposure to a reducing agent, silver grains (argyroplfilia) were deposited on all granules of all morphological varieties; grains were not concentrated over nuclei, mitochondria, fat droplets, lipofilscin granules, or the cytoplasm (Figs. 9, 10). BRONCttlAL EPITtlELIUM. Clusters of endocrine cells, positively identified as such by light microscopy in glycol methacrylate sections, were composed of the same spectrum o f cell types as seen in the tumor. (see the accompanying article beginning on page 302). UItrastructnrally small granule cells, large granule cells containing granules as large as 1.0/z in diameter, and polymorphic granule cells were clearly identified in the bronchial epithelium (Figs. 11, 12, 14). An aggregate of thick filaments randomly dispersed and entrapping a few endocrine granules was seen in one epithelial small granule cell (Fig. 12). I,arge numbers of small round dense core granules, similar to the granules seen in endocrine cells in epitlzelium and in the tumor, were also observed in a few mucous cells that lay adjacent to and within the epithelial clusters of endocrine cells (Fig. 12). The long incubation in acidic medium at 40 ~ C. required to demonstrate argyrophilia at uhrastructural level tended to cause membrane disruption. Therefore, altl.mugh images were higldy suggestive of argyrophilic granules and mucous granules in the same cell, absolute proof is lacking (Fig. 12, inset). Profiles suggestive of endocrine granules were also seen rarely in ciliated ceils and in basally situated cells whose cytoplasm ms ~ ~
1 ;,.':.~:~.,",:.,. ,..., ....
:
e'_,..._-" : . . . .
.~
-
.,~.
""'~":"
- .". ",!,:'-
DISCUSSION
T h e majority of published reports describing the ultrastructural morphologies o f lung carcinoids, including peripheral carcinoids and puhnonary tumorlets, have shown the tumors to be composed exclusively of small granule cells resembling those normally seen in the adult respiratory epithelium (reviewed by Capella et al)~). Endocrine cells are sparsely distributed in the normal adult epitilelium, and cells with small round granules represent by far the majority of the endocrine cells present?" ,a. 1,a,.,t-'.,G Abnormally large granules up to 2500 ~, in diameter were descril)ed in a few endocrine cells of one human bronchus by McDowell et al. "-'~
",. "
-. "..~..-,,~ ' t i l e " .;_..~. .
':
contained well developed tonofilament bundles, i.e., basal cells. In the epithelial sample tlmt was incubated to demonstrate argyrophilic granules, an intraepithelial cyst fi)rmed by the apices o f the surrounding cells was found within a cluster o f endocrine cells. The apices of some of these cells were sparsely ciliated and a few cilia projected into the enclosed space. Some of these ciliated cells contained argyrophilic granules (Fig. 13 and inset). At focal points, nests of intraepithelial endocrine cells were continuous with the tumor mass through discontinuities in the basal lamina (Fig. 14 and inset).
.
'
- j ~ .
, , - ~
~
:.~-":
-
"
"
9
, :
" . f , 2, :~. 9 ~,,:~lr
.
,,,..: v~-.; - - , .
.
.
..-~
.
"..~,U,G",
: ',,
.
:.., .,..~
.
.,
~
,..:. ,. . .~, ,. : -,:' =,,
.
.
.
.
,_.,
9
F i g u r e 13. B r o n c h i a l gl:vld e p i t h e l i u m . Silver g r a i n s (argyrophilia) a r e c o n c e n t r a t e d o v e r large d e n s e cm'e g r a n u l e s (lower left), which a r c p r e s e n t in t h e ,tpcx o f a cell that b e a r s a few cilia. N o t e plCSCnCe o f t h r e e basal bodies at cell apex. Stained o n grid with ttr,myl a n d lead salts. Inset: E n l a r g e m e n t o f o n e o f t h e a r g y r o p h i l i c g r a n u l e s to s h o w silver g r a i n s m o r e cle:!rly. (Electron inicrogr:q)h. • 2.'~,()110. Inset. • 7,()(}U.)
344
AN. U N U S U A L B R O N C H I A L C A R C I N O I D TUMOR--McDOWELL ET AL.
Figure 14. Bronchial gland epitheliuna and underlying tunmr are c~mtinuotts through a breach (arrows) ill the basal lamina (BL). B, basal cells. T h e tumor is composed o f sfhall granule cells (SG) and large granule cells (I.G). Inset: l.ight micrograph of similar area. Note that the extension from au intraepithelial cluster of endocrine cells is continuous (arr~ws) with the tmdcrl)ing tumor mass. (Electron micrograph. • Inset, glycol methacr)late section stained with l'AS-lead hcmatoxylin.)
345
HUMAN PATIIOLOGY--VOLUME 12, NUMBER 4 April 1981 Very few puhnonary carcinoid tumors composed of mixed cell populations have ever bcen reported. The carcinoid tumor described in this report is sontewhat similar to tumors described by Hage H and Hosoda et al. aa in that it was composed of at least three apparently different types of endocrine cells as indicated b), the morphologies o f the granules. Hage ~ described cells containing slnall round granules, large round granules, and polymorphic (pleomorphic) granules in one carcinbid tumor of the six tuinors she studied. She drew attention to the fact that cells containing small round granules and large round granules are normally found in the bronchial epithelium of huntan fetuses, whereas cells containing polymorphic granules are not (Hage ~ a~). Capella et al. -04 did not find polymorphic granule cells in human fetal epithelium. Cells with polymorphic granules resenable enterochrontaffin cells of the gut 4a and are siinilar to cells composing ileal, appendiceal, and rectal carcinoids. 44 Polyntorphic granules morphologically similar to those in a pancreatic carcinoid were observed in cells of a puhnonary carcinoid b)' l'atchefsky et al..a4 In puhnonary carcinoid tumors these cells were said to be diazo positive and have been described as argentaffin positive or negative by different inx~estigators?l, 3.~.34 In tile tumor described here, as in the one described by Hosoda et al., aa polymnrphic grannie cells were rare. On the basis of granule size and geometry they probably correspond to the scattered population of coarsely granulated and solely PAS "positive cells illustrated in Fisure 6 on page 306. On the basis of the granule cytocnemistry, the occasional diazo positive cells in this case probably correspond to a different population o f rare anaine tluorescent-lead hematoxylin staining cells, illustrated in Figures 9 to 12 o f tile accompan)'ing article. Intmcytoplasmic inclusions consisting o f aggregates of thick microfilantents were recently described in cells o f a bronchial carcinoid and a duodeiml carcinoid.4r, 48 The chemical nature of these inclusions is presently unknown, but they appear to be identical to the aggregate of microfilaments noted in a single epithelial endocrine cell seen in this study (Fig. 12). The microfilaments appear similar to those described in whorl-like configurations in endocrine cells of the bronchial epithelium o f Syrian golden hantsters treated with. diethylnitrosamine, a compound that causes marked proliferation of these cells. 49 A few earlier studies have suggested an intraepithelial site of origin for presumptively endocrine puhnonary ttnnors. In studies of oat cell carcinomas and carcinoid tumors the superficial layer of coluntnar ciliated and mucous cells appeared normal and overlay focal areas of increased celhdarity within the basal layers of the epithelium conlposcd of clusters of cells (presumai)ly endocrine cells) that resembled the tumor?' ~o-~4In tile present case ntllnerous clusters of endocrine cells were fottnd basally, situated in bronchial epithelium that otherwise appeared normal. By
346
light microscopic histochemistry and electron microscopy, these intraepithelial endocrine cells were indistinguishable from those of tile underlying tumor. These dusters apparently represented focal areas of carcinoma in situ composed o f preneoplastic or neoplastic endocrine cells. Presumptive endocrine grannies were also demonstrated in a few epithelial cells .containing numerous large mncous granules, and argyrophilic granules were demonstrated in a few cells that bore sparse cilia. Occasional cilia have been reported projecting into enclosed spaces within bronchial carcinoid tumors. 14 The epithelial lining of the respiratory tract is of endodermal origin. Bronchial buds are originally lined by a simple epithelium that later differentiates into one that is pseudostratified. "~'~Basal cells, mucous cells, and ciliated cells are presumably derived from the simple epithelium. The origin o f endocrine cells in respiratory epithelium is less well established. One hypothesis was that endocrine cells were derived not from endoderm but from cells of the neural crest) 6 However, sufticient evidence now exists to suggest that endocrine cells o f the gastroenteropancreatic system do not arise from the neural crestY ''s A possible interpretation is that in some way, cells of cominon endodermal origin are programed in early fetal development to differentiate into basal, mncons, ciliated, or endocrine cells, perhaps inlluenced by nficroenvironmental factors. In normal gastrointestinal epithelium, transitional forms between argentaffin cells and ro.ucotls cells were described using light microscopic histochenfistry and electron microscopy, 59-c~Band presumptive endocrine grannles were observed at the uhrastructural level in mucous cells o f normal human bronchi. I"q Tateishi "~ fonnd that argyrophilic cells (presumptively endocrine ceils) in human bronchi and bronchioles were often found closely associated with areas of mucous goblet cell hyperplasia. These observations, inade on non-neoplastic epithelia together with the finding of ntucosubstances and presumptive endocrine granules in tumors of endodermal origin, provide collective evidence against a nenroectodermal origin of endocrine ceils in endodermally derived epithelia? 8 T h e presence of endocrine granules in mt, cous, ciliated, and even perhaps in basal cells, which were closely associated with t h e hyperplastic, ntetaplastic, or neoplastic intraepitheliai clusters of endocrine cells, in this case may have resulted from disturbances in the factors that norinally control cellular differentiation. These observations suggest that the large clnsters of abnormal intraepithelial endocrine cells may have derived fi'om scattered "indifferent ceils." Indifferent cells are tmdifferentiated cells that resemble fetal epithelial cells and that are distinct from basal and mucous cells. We believe that at least sonte of these indifferent ceils can arise in the adult bronchial epithelium fi'om division of the more differentiated cells that retain their capacity to divide, i.e.,
A N U N U S U A L B R O N C H I A L C A R C I N O I D TUMOR--McDoWELL ~T AL. blucous - e n d o c r i n e
Di Mucous
i
l ~ Basal
Ciliated
?
INDIFFER~Zf--~ / i | " .~
- m u c o u s ~"
~
~Ciliated
/
I
Endocrine
- endocrine
"
Ciliated
Figure 15. Possible inter-relationships between indifferent cells and other types ofcells in human bronchial epithelium. Indifferent cells appear to be undifferentiated cells, as indicated by their morphology. Whether subpopulations exist within the indifferent cell population, which are committed to become particular differentiated cell lines, is unknown. The question mark emphasizes that there is no experimental evidence to prove that endocrine cells dMde in the aduh respirator)" epithelium, ahhough images suggestive of mitoses have been seen in human fetal respirator)" epithelium.~ Mitoses are, of course, common in presumptive endocrine cells of oat cell ttimors. b a s a l a n d m u c o u s cells ( f o r d e t a i l e d d i s c u s s i o n s see M c D o w e l l et al. 64' 65), o r e v e n p e r h a p s e n d o c r i n e cells, a l t h o u g l ~ t h e r e is as y e t n o e x p e r i m e n t a l p r o o f t h a t e n d o c r i n e cells of, t h e n o r m a l a d u l t b r o n c h i a l e p i t h e l i u m c a n d M d e . T h i s s i t u a t i o n is n o t u n i q u e to t h e bronchial epitltelium. For example, undifferentiated cells a r i s e f r o m d i v i s i o n o f n o r m a l l y q u i e s c e n t d i f f e r e n t i a t e d e p i t h e l i a l cells in t h e p r o x i m a l t u b u l e s o f the kidney. Under appropriate stimuli the differentiate.d p r o x i m a l t u b u l a r cells d M d e a n d u n d i f f e r e n t i a t e d d a u g h t e r cells ( i n d i f f e r e n t cells) a r e p r o d u c e d . T h e s e cells s u b s e q u e n t l y d i f f e r e n t i a t e i n t o s p e c i a l i z e d p r o x i m a l t u b u l a r cells. W e s u g g e s t t h a t t h e s e i n d i f f e r e n t cells, i r r e s p e c tiv~ o f t h e p a r e n t cell(s), c a n d i f f e r e n t i a t e i n t o all t h e t y p e s o f cells t h a t n o r m a l l y c o n s t i t u t e t h e a d u l t b r o n ctfial e p i t h e l i u m , a p r o c e s s p o s s i b l y c o n t r o l l e d b y local m i c r o e n v i r o n m e n t a l i n f l n e n c e s . W e also s u g g e s t , o n t h e basis o f o u r p r e v i o n s a n d p r e s e n t o b s e r v a t i o n s , t h a t i n d i f f e r e n t cells, in a d d i t i o n to b e i n g a b l e to differentiate into basal, mucous, ciliated, and endoc r i n e cells, m a y d e v e l o p m i x e d p a t t e r n s o f d i f f e r e n t i a t i o n to p r o d u c e c i l i a t e d m u c o u s cells, 6a' 65 m u c o u s e n d o c r i n e cells, c i l i a t e d e n d o c r i n e cells, o r e v e n p e r h a p s c i l i a t e d m u c o u s - e n d o c r i n e cells ( a l t t m u g h w e d o n o t b e l i e v e t h a t we h a v e s e e n tiffs last t y p e o f cell yet). O u r h y p o t h e s i s is s u m m a r i z e d in F i g u r e 15, Perlmps the mechanism controlling cellular diff e r e n t i a t i o n was d i s t u r b e d in t h e p r e s e n t case, c a u s i n g a b n o r m a l l y l a r g e n u m b e r s o f i n d i f f e r e n t cells to d i f f e r e n t i a t e i n t o e n d o c r i n e cells, p r o d u c i n g a t first hyperplastic and metaplastic states that subsequently p r o g r e s s e d to n e o p l a s i a . S u c h c o n t r o l l i n g nir isms c o u l d i n c l u d e t h e i n i t i a t i n g c a r c i n o g e n ( s ) , w h i c h themselves might affect the phenotypic expression of t h e final t u m o r . I t is k n o w n , f o r e x a m p l e , t h a t u r a n i u m m i n e r s h a v e a n i n c r e a s e d i n c i d e n c e o f o a t cell c a r c i n o m a . 6c' ACKNO%VLEDGMENT T h e authors wish to thank Dr. Dezso K. Mercnyi, D e p a r t m e n t o f Pathology, Union Memorial Hospital, Baltimore, without whose cooperation this stud)' would not have been possible.
REFERENCES 1. Bensch, K. G., Gordon, G. B., and Miller, L. R.: Electron microscopic and biochemical studies on the bronchial carcinold tumor. Cancer, 18:592, 1965. 2. Toker, C.: Observations on the uhrastructure of a bronchial adenoma (carcinoid type). Cancer, 19:1943, 1966. 3. Gmelich, J. T., Bensch, K. G., and Liebow, A. A.: CelIs of Kuhclfitsky type in bronchioles and their relation to the origin of peripheral carcinoid tumor. Lab. Invest., 17:88, 1967. 4. Pariente, R., Even, P., and Brouet, G.: ~2tude uhrastructurale des carcinoides bronchiques. Presse Med., 75:183, 1967. 5. Hattori, S., Matsuda, M., Tateistfi, R., and Terazawa, T.: Electron microscopic studies oll human lung cancer cells. Gann, 58:283, 1967. 6. Hattori, S., Matsuda, M., Tateislfi, R., Tatsumi, N., and Terazawa, T.: Oat cell carcinoma of the hmg containing serotonin granules. Gann, 59:123, 1968. 7. Hattori, S., Matsuda, M., Tateishi, R., Nislfilmra, M., and Horai, T.: Oat cell carcinoma of the lung. Clinical and 9morphological studies in relation to its histogenesis. Cancer, 30:1014, 1972. 8. Hattori, S., Matsuda, M., Ikegami, H., Horai, T., and Takenaga, A.: Small cell carcinoma of the hmg: clinical and cytomorphological studies in relation to its response to chemotherapy. Carol, 68:321, 1977. 9. Bensch, K. G., Corrin, B., Pariente, R., and Spencer, H.: Oat cell carcinoma of the lung. Its origin and relationship to bronctfial carcinoid. Cancer, 22:1163, 1968. 10. Dube, V. E.: Peripheral bronchial carcinoid with a spindle-cell pattern. Arch. Path., 89:374, 1970. I 1. Hage, E.: Histochenfistry and fine structure of bronclfial carcinoid tumors. Virchows ArclL Abt..A Path. Anat., 361:121, 1973. 12. Bonikos, D. S., Bensch, K. G., and Jamplis, R. W.: Peripheral puhnouary carcinoid tumors. Cancer, 37:1977, 1976. 13. Churg, A.: Large spindle cell variant of peripheral bronclfial ~arcinoid tumor. Arch. Path. Lab. Med., 10l:216, 1977. 14. Capella, C., Gabrielli, M., Polak,J. M., Buffa, R., Solcia, E., and Bordi, C.: Uhrastructural and histological stud) of 11 bronchial carcinoids. Evidence for different types. Virchows Arch. Abt. A Path. Anat., 381:313, 1979. 15. Ranchod, M.: The h'istogenesis and devempment of puhnonary tumorlets. Cancer, 39:1135, 1977. 16. Churg, A., and Warnock, M. L.: Puhnonary tumorlet. A form of peripheral carcinoid. Cancer, 37:1469, 1976. 17. Torikata, C., Kawai, T., Yakmnaru, K., and Kageyama, K.: Histopathological studies on the tumorlet of the hmg with special reference to the cytogenesis of proliferating cells. Acta Path. Jap., 25:539, 1975. 18. Bonikos, D. S., Archibald, R., and Bensch, K. G.: On the origin of the so-called tumnrlets of the lung. Hmnan Path., 7:4ql, 1976.
347
IIUMAN PATIIOLOGY--VOLUME
12, N U M B E R 4
19. "l'erzakis, J. A., Sommers, S. C., and Andersson, B.: Neurosecrett,ry appearing cells of hunmn seglnental bronchi, l.ab. Invest., 26:127, 1972. 20. Tateishi, R.: Distribution of arg) rophil cells in adult bureau lungs. Arch. l'ath., 96:198, 1973. 21. McDowell, E. M., Barrett, L. A., and Trump, B. F.: Observations on small granule cells in the adult human bronchial epithelium and in carcinoid and oat cell tumors. Lab. Invest., 34:202, 1976. 22. llage, E.: Endocrine-like cells of the pulmonary epithelium. In Couplaild, R. E., and Fujita, T. (Editors): Chromaffin, Enterochromaffin and Related Cells. Amsterdam, Elsevier Scientific Publishing Co., 1976, pp. 317-332. 23. llage, E., Hage, J., and Jnel, G.: Endocrine-like cells of the pulmonary epithelium of the buman adult lung. Cell Tiss. Res., 178:39, 1977. 24. Capella, C., Hage, E., Solcia, E., and Uselllni, I,.: Uhrastructural similarity of endocrine-like cells of the In, man lung and some related cells of the gut. Cell Tiss. Res., 186:25, 1978. 25. Bassett, F., Poirier, j., l.cCrom, M., and Turiaf, J.: Etude uhrastructurale de l'6pithdlium bronchlolare humain. Z. 9 Zellforsch. Mikrosk. Anat., 116:425, 1971. 26. Bensch, K. G., Gordon, G. B., and Miller, L. R.: Studies on the bronchial counterpart of the Kuhchitsky (argentaffin) cell and innervation of bronchial glands. J. Uhrastruct. Res., 12:668, 1965.* 27. Campiche, M. A:, Gautier, A., 1lernandez, E. I., and Reymond, A.: An electron microscope stud)" of the fetal development of human lung. Pediatrics, 32:976, 1963. 28. Lauu'eryns, J. M., Pcuskens, J. C., and Cokelacrc, M.: Argyroplfil, fluorescent and granulated (peptide and amine producing?) AFG cells in human infant bronchial epithelium. Light and electron microscopic studies. Lil'e Sci., 9:1417, 1970. 29. Cutz, E., and Conch, P. E.: Endocrine-like cells in human fetal lungs: an electron microscopic study. Anat. Rec., 173:115, 1972. 30. Cutz, E., Chan, W., Wong, V., and Conen, P. E.: Ultrastructure and fluorescence histochemistry of endocrine (APUDtype) cells in tracheal mucosa of human and various animal species. Cell Tiss. Res., 158:425, 1975. 31. Hage, E.: Electron microscopic identification of several types of endocrine cells in the brouchial epithelium of human foetuses. Z. Zellforsch., 141:401, 1973. 32. Black, W. C.: Pulmonary oncocytoma. Cancer, 23:1347, 1969. 33. Hosoda, S., Nakamura, W., Suzuki, 1I., lloshino, M., and Karasawa, K.: A bronchial carcinoid having low serotonin concentration. Arch. Path., 90:320, 1970. 34. Patchefsky, A. S., Gordon, G., ttarrer, W. V., and Hoch, W. S.: Carcinoid tnmor of the pancreas. Ultrastructural observations of a lymph node metastasis anti comparison with bronchial carcinoid. Cani:er, 33:1349, 1974. 35. McDowell, E. hi., and Trump, B. F.: ilistologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med., 100:405, 1976. 36. Mowry, R. W., and Winkler, C. ti.: Coloration of acidic carbohydrates of bacteria and flmgi in tissue sections with special reference to capsules of Cryptococcns neoformans, pneumococci and staphylococci. Am. J. Pathol., 32:628, 1956. 37. Sorokin, S. P., and Hoyt, R. F.: l'AS-lead hematoxylin as a stain fi)r small-granule endocrine cell populations in the lungs. other pharyngeal Oerivat~ves and the gut. Anat. Rec., 192:2.t5, 1978. 38. Solcia, E., Vassallo, G., and Capella, C.: Selective staining of endocrine cells by basic dyes after acid hydrolysis, Stain Tech., 43:257, 1968. 39. Pearse, A. G. E.: Biogenic amines. In llistochemistD', Theoretical and Applied. Ed. 3. Baltimore, The Williams & Wilkins Co., 1972, Vol. 2, pp. 1103-1127. 9t0. Grilnelins, L.: A silver nitrate stain for cr2 cells in hunaan pancreatic islets. Acta Soc. Med. Upsalien, 73:243, 1968. 41. Farquhar, hi. G., and Pal'ade, G. E.: Celljt*nctlons in amphibian skin. J. Cell B!ol., 26:263. 1965. 9t2, Vassallo, G., Capella, C., and Solcia, E.: Grimelius silver stain
348
April 1981
43. 44.
45. 46. 47. 48. 't9. 50. 51. 52. 53. 54. 55. 56. 57.
58. 59. 60. 61. 62. 63.
64. 65.
66. 67.
for endocrine cell grannies as shown by electron microscopy. Stain Tech., 46:7, 1971. Pearse, A. G. E., Coulling, I., Weavers, B., and Friesen, S.: The endocrine polypeptide cells of the human stomach, duodenum and jejuuum. Gut, 11:649, 1970. Black, W. C., and Ilaffner, H. E.: Diffuse hyperplasia of gastric argTrophil cells and multiple carcinoid tutnors. A histochemical and ultrastrnctural stud)'. Cancer, 21:1080, 1968. Black, W. C.: Enterochromaffin cell types and corresponding carcinoid tumors, l~ab. Invest., 19:473, 1968. Williams, R. M.: A light and electron microscopic study of au ovarian and rectal carcinoid. Histopathology, 3:19, 1979. An, T.: Cytoplasmic inclusions in bronchial carcinoid. Human Path., 9:241, 1978. Carstens, P. II. B., and l~,roghamer, W. L.: Duodenal carcinoid with cytoplasmic whorls of microfilaments. J. Path., 124:235, 1978. Reznik-Schiiller, 1t.: Uhrastructural alterations of APUD cells during nitrosantine-induced lung carcinogenesis. J. Path., 121:79, 1977. Waher, J. B., and Pryce, D- M.: The histology of lung cancer. Thorax, 10:107, 1955. Watson, W. L., and Berg, J. w.: Oat cell lung cancer. Cancer, 15:759, 1962. Lisa, J. R., Trinidad, S., and Rosenblatt, M. B.: Site of origin, histogenesis and cytostructnre of bronchogenic carcinoma. Am. J. Clin. Path., 44:375, 1965. Kay, S.: Histologic and histogenetic observations on the peripheral adenoma of the lung. Arch. Path., 65:395, 1958. Salyer, D. C., Salyer, W. R., and Eggleston, J. C.: Bronchial carcinoid tumors. Cancer, 36:1522, 1975. Moore, K. I..: The Developing Human. Philadelphia, W. B. Saunders Company, 1973, p. 167. Pearse, A. G. E.: 5-lt)droxytryptophan uptake by dog thyroid "C"-cells and its possible significance in polypeptide hormone production. Nature (London), 211:598, 1966. l'earse, A. G. E.: The diffuse neuroendocrine system aud the APUD concept: related "eudocrine" peptides in brain, intestine, pituitary, placenta and anuran cutaneous glands. Med. Biol., 55:115, 1977. Sidhn, G. S.: The endodermzl origin of digestive and respiratdry tract APUD ceils, ttistopathologic evidence and a review of the literattlre. Am. j. Path., 96:5, 1979. Popoff, N. W.: Epithelial functional rejuvenation observed in the mucous cells of the gastro-intestinal tract of the parietal cells of the stomach. Arch. Path., 27:841, 1939. Schofield, G.: The argentaffin cells of the small intestine of guinea pig. Acta Anat. (Basel), 11:414, 1951. Schofield, G.: The argentaffin and mucous cells of the small and large intestines of the mouse. Acta Anat. (Basel), 16:1, 1952. Schofield, G.: The argentaffin and mucous cells of the human intestine. Acta Anat. (Basel), 18:256, 1953. Cheng, C., and Leblond, C. P.: Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat., 141:537, 1974. McDowell, E. M., Barrett, L. A., Glavin, F., llarris, C. C., and Trump, B. F.: The respiratory epithelium. 1. Iluman bronchus. J. Natl. Cancer Inst., 61:539, 1978. McDowell, E. M., Becci, P.J[, Schiirch, W., and Trump, B. F.: The respiratory epidaelium. Vll. Epiderlnoid metaplasia of hantster tracheal epithelium during regeneration following mechanical Mjury. J. Natl. Cancer Inst., 62:995, 1979. Saccomanno, G., Archer, V. E., Saunders, R. P., James, I.. A., anti Beckler, P. A.: l.ung cancer of ttranium miners on the Colorado Plateau. I leahh Physics, 10: I 195, 1964. Mcl)ougall, J.: Endocrine-like cells in the term iqal bronchioles and saccules of human fetal lung: an ultrastructural study. Thorax, 33:43, 1978. Department of Pathology University of hlaryland School of Medicine 10 South Pine Str6et Baltimore, Maryland 21201 (Dr. Trump)
It is generally believed that presumptively endocrine cells o f the bronchial and bronchiolar epitheliu m are the cells o f origin o f carcinoid a n d oat cell tumors o f the lung. Ha Peripheral p t t l m o n a r y t u m o r lets may also derive f r o m these cells? 's~8 E n d o c r i n e cells have been described in the epithelium o f n o r m a l bronchi, bronclfioles, and bronchial glands o f adult h u t n a n lung. 3' 9, ~9-.oGT h e cells are seen only rarely lying on the basal lamina. The)' usually occur singly, but occasionally g r o u p s o f cells are seenY ~ E n d o c r i n e cells are also present in res-
piratory epithelium o f h u m a n fetuses and neonates,.,_o, o.t, _oT-a~and at least t h r e e a p p a r e n t l y d i f f e r e n t types o f fetal e n d o c r i n e cells have been described on the basis o f morphological characteristics o f the dense core granulesY 2' _04.3~ However, in studies o f g r a t m l a r tnorphology, only one type o f cell has been described in adult h u m a n lungs. -~176 T h e granules are characterized by their u n i f o r m r o u n d shape, stnall size (approximately 1400 ,~, in diameter), and h o m o g e n e o u s dense matt'ices. T h e granules are s u r r o u n d e d by a single m e m -
Accepted for publication January 24, 1980. This work was supported ill part by contract NOI-CP-43237 awarded to Dr. Trum'p by the National Cancer Institute aud by research grant ItL19379 awarded to Dr. lloyt by the Iieart, Lung, and Blood Institute. This is contribution 819 from tile Cellular Pathobiology Laboratory, University of Mar):land School of Medicine. *Associate l'rofessor, Department of I'athology, University of Maryland School of Medicine, Bahimore, Maryland. ]'Associate Professor of Cell Biology, Depart,nent of Physiology, ttarvard University School of Public Health, Boston, Massachusetts. :[:Assistant l'rofessort Department of Anatomy, Boston Universit)"School of Medicine. Boston, Massachusetts. w
338
and Clmirman, Department of l'athology, University of .Maryland School of Medicine, Baltimore, Marylandl IIUMAN PATIIOLOGY--VOLUME 12, NUMBER 4 Ap,ql1981
AN UNUSUAL
brane, which is usually separated from the dense core by a narrow halo. This type of cell closely resembles one of the three types o f endocrine cells normally present in human fetal lungs. 2"-''2t' 3! Abnormally large granules (up to 2500 A in diameter) scattered among the smaller granules were described in a few cells in one human caseY t The ultrastructural characteristics o f pulmonary carcinoid tumors have been described in detail, va" 7, 9t4, a'_,-34 However, the most detailed reports with regard to the histogenesis of carcinoid tumors are those of Hage n and Capella et al. 14 Hage n described the ultrastructural morphology of six bronchial carcinoid tumors; the individual tumors were composed of one, two, or even three apparently different types of endocrine cells differing in the morphological and cytochemical characteristics of the granules. Two of the three types of cells found in the tumors were similar to endocrine cells normally found in the respiratory epithelium of human fetuses. In contrast, in the study o f Capella et al. 14 two distinct types of tumors were described. One type was composed entirely of small granule cells and the other, large granule cells; small and large granule cells were not observed in individual tumors. In the present report a carcinoid tumor is described, which in terms of the size and other morp'hological characteristics of the granules, was composed of at least three and possibly as many as six apparently different types o f endocrine cells. Abnormally large numbers of the same types of endocrine cells seen in the tumor were also present in the bronchial epithelium and in the epithelium of the neck of the bronchial glands, and multiple sites of continuity (invasion) were seen between foci of neoplastic endocrine cells in the respirator}, epithelium and the underlying carcinoid tumor. Case
Report
A 62 },ear old male was noted t o have a coin lesion in the left upper lobe when a routine chest x-ray view was taken. The history was unremarkable except for a peptic ulcer 10 years previo.usly and rheumatoid arthritis with shoulder and hip pain. T h e patient was in good condition with no weight loss or cnest pain, but hemoptysis was noted one week prior to admission to Union Memorial Hospital, Baltimore, in April 1977. Bronchial washings, sputum specimens, and a lung biopsy study were negau.'ve for malignant cells, but a second x-ray fihn revealed not only the coin lesion but also suspected metastases to lymph nodes. A left thoracotomy was performed and the upper left lobe was removed. In the region of the hilum of the lobe a firm pale yellow mass, 3.5 by 2.1 by 2.0 cm., was present, which partially occluded some branches of the bronchus. Multiple black firm circumscribed nodules resembling lyxnph nodes were found in the hilar region measuring up to 1.3 cm. in diameter. Some of these were directly adjacent to the
BRONCHIAL
C A R C I N O I D T U M O R - - M c D O w E L L ET AL.
tumor mass. One portion o f the lobe, adjacent to the hilum and extending to the periphery, was dark red and of a rubber}' consistency. Sections through this area revealed multiple firm calcified nodules measuring tip to 0.3 cm. in diameter. T h e remainder of the lobe had normal crepitance but on cut section revealed several small hard calcified nodules measuring up to 0.3 cm. in diameter. Healed granulomas were present in the lung parenchyma and associated lymph nodes. On the basis of routine light microscopy a diagnosis of bronchial carcinoid with metastases to adjacent lymph nodes was made. T h e patient was discharged on the eighteenth hospital day. MATERIALS
AND
METHODS
The tumor and associated bronchus were fixed in 4 per cent formaldehyde-1 per cent glutaraldehyde and stored in fixative at room temperature until required for study. 3~
L I G H T M I C R O S C O P Y AND L I G H T MICROSCOPIC HISTOCHEMISTRY
The tissue was embedded in paraffin and 8 /.L thick sections were stained with hematoxylin and eosin. T h e presence of mucosubstances was monitored using the alcian blue (pH 2.5)-PAS method. 36 PAS-lead hematoxylin and alcian blue after acid hydrolysis were used as selective stains for endocrine cellsY' 3s For the demonstration o f biogenic amines the diazoiaium method (using fast red salt B), Gibb's reaction and the afgentaffin silver reaction of Masson-Fontana were applied. ~9 A test for argyrophilia was performed by using the Grimelius procedure. 46 Schmorl's reaction was also used. 39
H I G H R E S O L U T I O N L I G H T AND T R A N S M I S S I O N E L E C T R O N MICROSCOPY
For high resolution light microscopy and for electron microscopy t h e tissues were cut into small pieces and washed overnight in 0.2 M sncrose cacodylate buffer (pH 7.2), postfixed with 1 per cent osmium tetroxide buffered with 0.1M s-collidine, and stained en bloc with uranyl acetate, at T h e tissues were dehydrated in a graded series of ethanols and embedded in Epon. Areas were chosen for thin sectioning by examining 1.0/* thick sections stained with toluidine blue. Thin sections were double stained on coated grids with uranyl magnesium acetate and lead citrate and were examined in a J E O L 100B electron microscope. For demonstration of argyrophilia at the ultrastructural level, very thin, aldehyde fixed sections of tumor and associated bronchus, cut by hand with a razor blade, were impregnated with silver nitrate and
339
HUMAN
PATHOLOGY--VOLUME
12, N U M B E R
4
April 1981
reimpregnated as directed for glutaraldehyde fixed tissues.*-' T h e initial silver impregnation was accomplished at 40 ~ C. in the clark for 120 hours. The sections were then fixed in osmium tetroxide, but staining with uranyl acetate en bloc was omitted. Some of the thin section's were not stained on grids with either uranyl or lead salts.
rated by thin bands of vasctflarized connective tissue (Fig. 1). In other areas the cell nests were large and the organoid pattern was less obvious. Nuclei were of a uniform size and nucleoli were not prominent. Mitoses and necrosis were not observed. Mucosubstances were not demonstrated in the tumor. Approximately one quarter of the cells were stained by alcian blue after acid hydrolysis. Stain deposits were coarsely granular in some cells, "but in others the stain was diffusely distributed throughout the cytoplasm. Some cells were deeply stained whereas others had intermediate or light staining. Similarly stained cells tended to be grouped together (Fig. 2). Very few scattered cells were stained by either Schmorl's reaction or the diazo method (Fig. 3). No cells stained with Gibb's or the Masson-Fontana procedure. Some cells (but many more than were Schmorl or diazo positive) stained with the Grimelius procedure; the argyrophilic cells tended to be clustered in groups. Argyrophilia was more widespread and intense when demonstrated by en bloc silver nitrate impregnation prior to embedding in glycol methacrylate. (For detailed de-
RESULTS L I G H T M I C R O S C O P Y AND L I G I I T MICROSCOPIC HISTOCtIEMISTRY
PAI~X.FFIN SECTIONS. Only tumor tissue was present in paraffin sections, and therefore the relationship of the tumor to the bronchial epitheliuxn could not be assessed using these preparations. In some areas the tumor had an organoid pattern typical of many carcinoid tumors. In sections stained with hematoxylin and eosin the cells appeared monotonously regular, grouped into compact cell nests sepaFigure "
~r ~ r
.
9
.
1 .
Figure
.
~ ~'
"
~ - ' :"
'
- ~ "~-"
. . . . ~ q. l~ l .~ ~ ~-" r " ' ".'.
"
~_'~\."-~'.
"
,
"
.
t
~
9
~
~
-"
~
9
-
~:
,~"
.,.'~
"'.,"
~
:~"
.-k-'-
"
1;:
a*m.~--~-
9-
2
.~7 5 "'~ .
7": .
,~
,
..
_
,.* ' .
--'~'-~ i
9
~
't~-"
Figure 3 Figure 4 Figure l. Carcinoid tumor. Compact nests of cells, which apl)ear monotonously regular, are separated by thin bands of vascularized connective tissue, presenting a typical organoid pattern. (Paralfin section stained with hematoxylin and eosin, l.ight micrograph. • Figure 2. Carcinoid tunlOl-. Stain deposits are coarsely granular in some cells whereas in others the stain is more diffuse. Stain intensity varies; note that similarly stained cells tend to be grouped together. Many cells are not stained. (Paralfiq section staiqed with alcian hlue after acid hydrolysis, l.ight micrograpli. • Figure 3. Carcinuid tumor. Two cell~ (left lower center) are diazopositive. T h e nuclei are counterstained with hemalum. (i'araffin section stained by diazo method, l.ight micrograph. • Figure epithelium. tumor and blue. I+ight
340
4. Abnormally llumerotts arid large clusters of cells are seen basalb," (arrows) in the otherwise normal ciliated bronchial T h e cells comp<+sing the dusters al+pear identical to those of tit,: tmt]erl)ing tumor (T). Note areas of continuity betweelt epithelial clusters in bronchus a,ld bronchial gland (arrowheads). BG, bronchial gland. (Epon section stained with toluidine micrograph. •
AN UNUSUAL BRONCIIIA1, CARCINOID TUMOR--McDOWELL EI" AL. scriptions of tiffs and l'AS-lead hematoxylin staining of the tumor, see the accoml)anying article on page 302.) E P O N S E C T I O N S S T A I N E D SVITII T O L U I D I N E
BLUE
A bronchus anti associated tumor were available for study. Abnormally numerous and large clusters of cells were seen basally situated in otherwise normal bronchial epithelium and at the base of the epithelium lining the opening to a bronchial gland. T h e cells composing the clusters appeared identical to cells composing the underlying tumor mass and in several places were seen to be continuous with the tumor (Fig. 4). ELECTRON MICROSCOPY TUMOR. The tumor was composed of round, oval, and stellate cells. The plasma membranes closely paralleled one another so that intercellular spaces were minimal. T h e cells were joined by small inconspicuous desmosomes. The cytoplasm was electron lucent, and many cells contained well developed stacks o f rough endoplasmic reticulum. T h e Golgi apparatus was inconspicuous. Large residual bodies containing lipofilscin were common and fat droplets wgre seen in sonte cells. Some tumor cells were binucleated. All the tumor cells contained characteristic granules, but the number and morphology o f the granules varied greatly from cell to ceil. The results are summarized in Table I. Many cells contained small round dense core granules with diameters up to 2000 A (small granule cells; Figs. 5, 14). The central homogeneous dense core was separated from the surrounding membrane
TABLE 1.
by a halo (Fig. 5, inset). Some cells contained many of these granules (Fig. 14), whereas others contairied relatively few (Fig. 5). Many other cells contained large round dense core granules, which sometimes crowded the cytoplasm (Fig. 5). Collectively these cells could be called large granule cells, but on the basis of granular morphology there appeared to be at least tour different types o f large granule cells. In one type the granular matrix was homogeneons and of intermediate density throughout the large granule population in a single cell (Fig. 6). The granules measured up to 5000 ,~ in diameter. In a second type of large granule cell, two varieties of large granules were distinguishable by the electron density of the granular nmtrices (Fig. 7). Granules with darkly stained cores measured up to 4000 ,~ in diameter and granules of intermediate density, up to 5000/~, in diameter (Fig. 7). In a third type of large granule cell, three varieties of large grannies were distinguished by granule matrices with electron lucent and low intermediate densities (Fig. 8). The electron lucent granules, which w e r e the largest of the three types of granules, measured up to 8000 ,~ in diamcter (Fig. 8). In a fourth type of cell as many as four varieties of large granules could be distinguished on the basis of matrix electron density (Fig. 5). T h e largest grannies had a low electron density and sometimes exceeded 1.0/x in diameter. Yet another cell type was identified on the basis of granular morphology. These cells were rare in the tumor and contained numerous large (up to 6000 A diameter) polymorphic (pleomorpbic) granules that were rotmd, ovoid, bean shaped, and cup shaped. Some variation in electron density was seen among the granules. Some of the grantfles contained a single electron lucent membrane bound vesicle, suggesting that they represented pockets o f cytoplasm, resulting from fortuitous cuts through cup shaped granules (see epithelial counterpart, Fig. 11 and inset).
U L T R A S T R U C T U R A L MORPHOLOGIES OF CELLS OF A B R O N C H I A L C A R C I N O I D T U M O R
Cell Type Small granule
Granular Morphology
Argyrophilia
Small round granules with homogeneous densely stained cores, up. to 20(1(I ~, (Fig. 5 and inset)
+
l.arge round gr:mnles~-:',cith homogencous cores of intermediate density, up to 5000 ~, (Fig. 6)
+
Type 2
Two varieties of large round granules; dark staining cores up to -t000 ,~, and cores of intermcdiate dcnsity, up to 5000 z~, (Fig. 7)
+
Type 3
l-hree varieties of large round granules; electron lucent cores up to 8000 t~, and smaller granules with cores of low and intermediate density (Fig. 8)
+
Type 4
Four varieties of large round granules; low density cores up to anct exceeding 1.0 xt ,find smaller gramdcs with lucent, intermediate, and densely stained cores (Fig. 5)
+
Large round, ovoid, bean shaped, or cup shaped granules; some variation in electron density but tending to be densely stained; (tp to 6(}01} t~, (see epithelia! cot, ntcrlmrt, Fig. I I)
+
l.arge granule Type I
Pol)morl~hic (pleonmrphic) granule
341
Figure 5
:,dllq~,
:-~-":~L.
....
",;
~ " ~ ' ~
9~
"
~
.0,._
: " ,- ' ' , ; , , '
," -~:.:..~-~';".~.
.........
:'~"
,-
~..
.,
....
.
:~;';;:~
~
, ~
".:--:
.....
.~I~.~:.~.~.., .~
~.
,.~
J
'
~:~-
Figure 8 Figure 9 Figure l0 Figure 5. Carcinoid tumor. Some cells contai*, onl) very few scattered small deuse core granules (arrows, and inset). Another cell contains numerous very large granules; on the basis of electron density of the gi'anular matrices.at least four varieties of large granules cau be distinguished, lnset: Enlargemeut of small dense core granules denoted b)" arrows. (Electron micrngraph. • Inset, • Figure 6. Carcinoid tumor. The matrix is relatively dense and homogenous throughout the large granules of this cell. (Electron micrograph. • 10,500.) Figure 7. Carcinoid tumor. The matrices of the large gra,mles in this cell are of dark and intermediate densities. (Electron micrograph. • 10,500.) Figure 8. Carciuoid tulnor. The matrices of the large.granules in this ceil demonstrate three degrees of electron densit)'. (Electron micrograph. • 11,000.) Figure 9. Carcinoid tumor. Silver grains (argyrophilia) are concentrated over large granules. Vet). few silver grains are present over cytoplasm or a mitochondrion "(lower left). Staining with lead and uran)l salts was omitted. (Electron micrograph, x27,000A Figure 10. Carcinoid tumor. Silver grains (arg)'rophilia) are concentrated over small granules. Silver grains are not deposited on the lipofi*scin grannie (lower center). Staining with uranyl salts was omitted. (Electron micrograph. •
AN UNUSUAL
BRONCHIAL
CARCINOID
TUMOR--McDoWELL
Ew AL.
Figure 11
Figure 12 Figure 11. Bronchial gland epitheliunl. Endocrine cell containing numerous large polymorphic grannies. Basal lamina is shown at base (left and center). Inset: An electron lucent vesicle lies within one pol)'morlfllic grannie (top right). This appearance resuhs from fortuitous cuts through cup shaped granules (top left). (Electron micrograPh, x 12,500. Inset, • 17,{)(10.)
Figure 12. Bronchial gland'el~ithelitnn. A cell within an epithelial cluster ofendocri,ae cells contains both mucous granules and dense core gram~les. Note aq aggregate"df tllicl~ filaments (Upl)er left) randomly dispersed in one cell containing small dense core granules. A cell containifig large granules is prc'scnt at bottom. Inset: Bronchial gland epitheliu,n. Silver grains (argyrnplfilia) are concentrated over a dense cdre granule (right), wbicl'~:lppcars to be within the same cell as a intlcous granule (left). (Electron micrograph, xg000. Inset, st,fined on grid with uran)l and lead saltg, x55,000.)
343
HUMAN
PATHOLOGY--VOLUME
12, N U M I ? , E R
4
April 1981
After impregnation with silver nitrate and exposure to a reducing agent, silver grains (argyroplfilia) were deposited on all granules of all morphological varieties; grains were not concentrated over nuclei, mitochondria, fat droplets, lipofilscin granules, or the cytoplasm (Figs. 9, 10). BRONCttlAL EPITtlELIUM. Clusters of endocrine cells, positively identified as such by light microscopy in glycol methacrylate sections, were composed of the same spectrum o f cell types as seen in the tumor. (see the accompanying article beginning on page 302). UItrastructnrally small granule cells, large granule cells containing granules as large as 1.0/z in diameter, and polymorphic granule cells were clearly identified in the bronchial epithelium (Figs. 11, 12, 14). An aggregate of thick filaments randomly dispersed and entrapping a few endocrine granules was seen in one epithelial small granule cell (Fig. 12). I,arge numbers of small round dense core granules, similar to the granules seen in endocrine cells in epitlzelium and in the tumor, were also observed in a few mucous cells that lay adjacent to and within the epithelial clusters of endocrine cells (Fig. 12). The long incubation in acidic medium at 40 ~ C. required to demonstrate argyrophilia at uhrastructural level tended to cause membrane disruption. Therefore, altl.mugh images were higldy suggestive of argyrophilic granules and mucous granules in the same cell, absolute proof is lacking (Fig. 12, inset). Profiles suggestive of endocrine granules were also seen rarely in ciliated ceils and in basally situated cells whose cytoplasm ms ~ ~
1 ;,.':.~:~.,",:.,. ,..., ....
:
e'_,..._-" : . . . .
.~
-
.,~.
""'~":"
- .". ",!,:'-
DISCUSSION
T h e majority of published reports describing the ultrastructural morphologies o f lung carcinoids, including peripheral carcinoids and puhnonary tumorlets, have shown the tumors to be composed exclusively of small granule cells resembling those normally seen in the adult respiratory epithelium (reviewed by Capella et al)~). Endocrine cells are sparsely distributed in the normal adult epitilelium, and cells with small round granules represent by far the majority of the endocrine cells present?" ,a. 1,a,.,t-'.,G Abnormally large granules up to 2500 ~, in diameter were descril)ed in a few endocrine cells of one human bronchus by McDowell et al. "-'~
",. "
-. "..~..-,,~ ' t i l e " .;_..~. .
':
contained well developed tonofilament bundles, i.e., basal cells. In the epithelial sample tlmt was incubated to demonstrate argyrophilic granules, an intraepithelial cyst fi)rmed by the apices o f the surrounding cells was found within a cluster o f endocrine cells. The apices of some of these cells were sparsely ciliated and a few cilia projected into the enclosed space. Some of these ciliated cells contained argyrophilic granules (Fig. 13 and inset). At focal points, nests of intraepithelial endocrine cells were continuous with the tumor mass through discontinuities in the basal lamina (Fig. 14 and inset).
.
'
- j ~ .
, , - ~
~
:.~-":
-
"
"
9
, :
" . f , 2, :~. 9 ~,,:~lr
.
,,,..: v~-.; - - , .
.
.
..-~
.
"..~,U,G",
: ',,
.
:.., .,..~
.
.,
~
,..:. ,. . .~, ,. : -,:' =,,
.
.
.
.
,_.,
9
F i g u r e 13. B r o n c h i a l gl:vld e p i t h e l i u m . Silver g r a i n s (argyrophilia) a r e c o n c e n t r a t e d o v e r large d e n s e cm'e g r a n u l e s (lower left), which a r c p r e s e n t in t h e ,tpcx o f a cell that b e a r s a few cilia. N o t e plCSCnCe o f t h r e e basal bodies at cell apex. Stained o n grid with ttr,myl a n d lead salts. Inset: E n l a r g e m e n t o f o n e o f t h e a r g y r o p h i l i c g r a n u l e s to s h o w silver g r a i n s m o r e cle:!rly. (Electron inicrogr:q)h. • 2.'~,()110. Inset. • 7,()(}U.)
344
AN. U N U S U A L B R O N C H I A L C A R C I N O I D TUMOR--McDOWELL ET AL.
Figure 14. Bronchial gland epitheliuna and underlying tunmr are c~mtinuotts through a breach (arrows) ill the basal lamina (BL). B, basal cells. T h e tumor is composed o f sfhall granule cells (SG) and large granule cells (I.G). Inset: l.ight micrograph of similar area. Note that the extension from au intraepithelial cluster of endocrine cells is continuous (arr~ws) with the tmdcrl)ing tumor mass. (Electron micrograph. • Inset, glycol methacr)late section stained with l'AS-lead hcmatoxylin.)
345
HUMAN PATIIOLOGY--VOLUME 12, NUMBER 4 April 1981 Very few puhnonary carcinoid tumors composed of mixed cell populations have ever bcen reported. The carcinoid tumor described in this report is sontewhat similar to tumors described by Hage H and Hosoda et al. aa in that it was composed of at least three apparently different types of endocrine cells as indicated b), the morphologies o f the granules. Hage ~ described cells containing slnall round granules, large round granules, and polymorphic (pleomorphic) granules in one carcinbid tumor of the six tuinors she studied. She drew attention to the fact that cells containing small round granules and large round granules are normally found in the bronchial epithelium of huntan fetuses, whereas cells containing polymorphic granules are not (Hage ~ a~). Capella et al. -04 did not find polymorphic granule cells in human fetal epithelium. Cells with polymorphic granules resenable enterochrontaffin cells of the gut 4a and are siinilar to cells composing ileal, appendiceal, and rectal carcinoids. 44 Polyntorphic granules morphologically similar to those in a pancreatic carcinoid were observed in cells of a puhnonary carcinoid b)' l'atchefsky et al..a4 In puhnonary carcinoid tumors these cells were said to be diazo positive and have been described as argentaffin positive or negative by different inx~estigators?l, 3.~.34 In tile tumor described here, as in the one described by Hosoda et al., aa polymnrphic grannie cells were rare. On the basis of granule size and geometry they probably correspond to the scattered population of coarsely granulated and solely PAS "positive cells illustrated in Fisure 6 on page 306. On the basis of the granule cytocnemistry, the occasional diazo positive cells in this case probably correspond to a different population o f rare anaine tluorescent-lead hematoxylin staining cells, illustrated in Figures 9 to 12 o f tile accompan)'ing article. Intmcytoplasmic inclusions consisting o f aggregates of thick microfilantents were recently described in cells o f a bronchial carcinoid and a duodeiml carcinoid.4r, 48 The chemical nature of these inclusions is presently unknown, but they appear to be identical to the aggregate of microfilaments noted in a single epithelial endocrine cell seen in this study (Fig. 12). The microfilaments appear similar to those described in whorl-like configurations in endocrine cells of the bronchial epithelium o f Syrian golden hantsters treated with. diethylnitrosamine, a compound that causes marked proliferation of these cells. 49 A few earlier studies have suggested an intraepithelial site of origin for presumptively endocrine puhnonary ttnnors. In studies of oat cell carcinomas and carcinoid tumors the superficial layer of coluntnar ciliated and mucous cells appeared normal and overlay focal areas of increased celhdarity within the basal layers of the epithelium conlposcd of clusters of cells (presumai)ly endocrine cells) that resembled the tumor?' ~o-~4In tile present case ntllnerous clusters of endocrine cells were fottnd basally, situated in bronchial epithelium that otherwise appeared normal. By
346
light microscopic histochemistry and electron microscopy, these intraepithelial endocrine cells were indistinguishable from those of tile underlying tumor. These dusters apparently represented focal areas of carcinoma in situ composed o f preneoplastic or neoplastic endocrine cells. Presumptive endocrine grannies were also demonstrated in a few epithelial cells .containing numerous large mncous granules, and argyrophilic granules were demonstrated in a few cells that bore sparse cilia. Occasional cilia have been reported projecting into enclosed spaces within bronchial carcinoid tumors. 14 The epithelial lining of the respiratory tract is of endodermal origin. Bronchial buds are originally lined by a simple epithelium that later differentiates into one that is pseudostratified. "~'~Basal cells, mucous cells, and ciliated cells are presumably derived from the simple epithelium. The origin o f endocrine cells in respiratory epithelium is less well established. One hypothesis was that endocrine cells were derived not from endoderm but from cells of the neural crest) 6 However, sufticient evidence now exists to suggest that endocrine cells o f the gastroenteropancreatic system do not arise from the neural crestY ''s A possible interpretation is that in some way, cells of cominon endodermal origin are programed in early fetal development to differentiate into basal, mncons, ciliated, or endocrine cells, perhaps inlluenced by nficroenvironmental factors. In normal gastrointestinal epithelium, transitional forms between argentaffin cells and ro.ucotls cells were described using light microscopic histochenfistry and electron microscopy, 59-c~Band presumptive endocrine grannles were observed at the uhrastructural level in mucous cells o f normal human bronchi. I"q Tateishi "~ fonnd that argyrophilic cells (presumptively endocrine ceils) in human bronchi and bronchioles were often found closely associated with areas of mucous goblet cell hyperplasia. These observations, inade on non-neoplastic epithelia together with the finding of ntucosubstances and presumptive endocrine granules in tumors of endodermal origin, provide collective evidence against a nenroectodermal origin of endocrine ceils in endodermally derived epithelia? 8 T h e presence of endocrine granules in mt, cous, ciliated, and even perhaps in basal cells, which were closely associated with t h e hyperplastic, ntetaplastic, or neoplastic intraepitheliai clusters of endocrine cells, in this case may have resulted from disturbances in the factors that norinally control cellular differentiation. These observations suggest that the large clnsters of abnormal intraepithelial endocrine cells may have derived fi'om scattered "indifferent ceils." Indifferent cells are tmdifferentiated cells that resemble fetal epithelial cells and that are distinct from basal and mucous cells. We believe that at least sonte of these indifferent ceils can arise in the adult bronchial epithelium fi'om division of the more differentiated cells that retain their capacity to divide, i.e.,
A N U N U S U A L B R O N C H I A L C A R C I N O I D TUMOR--McDoWELL ~T AL. blucous - e n d o c r i n e
Di Mucous
i
l ~ Basal
Ciliated
?
INDIFFER~Zf--~ / i | " .~
- m u c o u s ~"
~
~Ciliated
/
I
Endocrine
- endocrine
"
Ciliated
Figure 15. Possible inter-relationships between indifferent cells and other types ofcells in human bronchial epithelium. Indifferent cells appear to be undifferentiated cells, as indicated by their morphology. Whether subpopulations exist within the indifferent cell population, which are committed to become particular differentiated cell lines, is unknown. The question mark emphasizes that there is no experimental evidence to prove that endocrine cells dMde in the aduh respirator)" epithelium, ahhough images suggestive of mitoses have been seen in human fetal respirator)" epithelium.~ Mitoses are, of course, common in presumptive endocrine cells of oat cell ttimors. b a s a l a n d m u c o u s cells ( f o r d e t a i l e d d i s c u s s i o n s see M c D o w e l l et al. 64' 65), o r e v e n p e r h a p s e n d o c r i n e cells, a l t h o u g l ~ t h e r e is as y e t n o e x p e r i m e n t a l p r o o f t h a t e n d o c r i n e cells of, t h e n o r m a l a d u l t b r o n c h i a l e p i t h e l i u m c a n d M d e . T h i s s i t u a t i o n is n o t u n i q u e to t h e bronchial epitltelium. For example, undifferentiated cells a r i s e f r o m d i v i s i o n o f n o r m a l l y q u i e s c e n t d i f f e r e n t i a t e d e p i t h e l i a l cells in t h e p r o x i m a l t u b u l e s o f the kidney. Under appropriate stimuli the differentiate.d p r o x i m a l t u b u l a r cells d M d e a n d u n d i f f e r e n t i a t e d d a u g h t e r cells ( i n d i f f e r e n t cells) a r e p r o d u c e d . T h e s e cells s u b s e q u e n t l y d i f f e r e n t i a t e i n t o s p e c i a l i z e d p r o x i m a l t u b u l a r cells. W e s u g g e s t t h a t t h e s e i n d i f f e r e n t cells, i r r e s p e c tiv~ o f t h e p a r e n t cell(s), c a n d i f f e r e n t i a t e i n t o all t h e t y p e s o f cells t h a t n o r m a l l y c o n s t i t u t e t h e a d u l t b r o n ctfial e p i t h e l i u m , a p r o c e s s p o s s i b l y c o n t r o l l e d b y local m i c r o e n v i r o n m e n t a l i n f l n e n c e s . W e also s u g g e s t , o n t h e basis o f o u r p r e v i o n s a n d p r e s e n t o b s e r v a t i o n s , t h a t i n d i f f e r e n t cells, in a d d i t i o n to b e i n g a b l e to differentiate into basal, mucous, ciliated, and endoc r i n e cells, m a y d e v e l o p m i x e d p a t t e r n s o f d i f f e r e n t i a t i o n to p r o d u c e c i l i a t e d m u c o u s cells, 6a' 65 m u c o u s e n d o c r i n e cells, c i l i a t e d e n d o c r i n e cells, o r e v e n p e r h a p s c i l i a t e d m u c o u s - e n d o c r i n e cells ( a l t t m u g h w e d o n o t b e l i e v e t h a t we h a v e s e e n tiffs last t y p e o f cell yet). O u r h y p o t h e s i s is s u m m a r i z e d in F i g u r e 15, Perlmps the mechanism controlling cellular diff e r e n t i a t i o n was d i s t u r b e d in t h e p r e s e n t case, c a u s i n g a b n o r m a l l y l a r g e n u m b e r s o f i n d i f f e r e n t cells to d i f f e r e n t i a t e i n t o e n d o c r i n e cells, p r o d u c i n g a t first hyperplastic and metaplastic states that subsequently p r o g r e s s e d to n e o p l a s i a . S u c h c o n t r o l l i n g nir isms c o u l d i n c l u d e t h e i n i t i a t i n g c a r c i n o g e n ( s ) , w h i c h themselves might affect the phenotypic expression of t h e final t u m o r . I t is k n o w n , f o r e x a m p l e , t h a t u r a n i u m m i n e r s h a v e a n i n c r e a s e d i n c i d e n c e o f o a t cell c a r c i n o m a . 6c' ACKNO%VLEDGMENT T h e authors wish to thank Dr. Dezso K. Mercnyi, D e p a r t m e n t o f Pathology, Union Memorial Hospital, Baltimore, without whose cooperation this stud)' would not have been possible.
REFERENCES 1. Bensch, K. G., Gordon, G. B., and Miller, L. R.: Electron microscopic and biochemical studies on the bronchial carcinold tumor. Cancer, 18:592, 1965. 2. Toker, C.: Observations on the uhrastructure of a bronchial adenoma (carcinoid type). Cancer, 19:1943, 1966. 3. Gmelich, J. T., Bensch, K. G., and Liebow, A. A.: CelIs of Kuhclfitsky type in bronchioles and their relation to the origin of peripheral carcinoid tumor. Lab. Invest., 17:88, 1967. 4. Pariente, R., Even, P., and Brouet, G.: ~2tude uhrastructurale des carcinoides bronchiques. Presse Med., 75:183, 1967. 5. Hattori, S., Matsuda, M., Tateistfi, R., and Terazawa, T.: Electron microscopic studies oll human lung cancer cells. Gann, 58:283, 1967. 6. Hattori, S., Matsuda, M., Tateislfi, R., Tatsumi, N., and Terazawa, T.: Oat cell carcinoma of the hmg containing serotonin granules. Gann, 59:123, 1968. 7. Hattori, S., Matsuda, M., Tateishi, R., Nislfilmra, M., and Horai, T.: Oat cell carcinoma of the lung. Clinical and 9morphological studies in relation to its histogenesis. Cancer, 30:1014, 1972. 8. Hattori, S., Matsuda, M., Ikegami, H., Horai, T., and Takenaga, A.: Small cell carcinoma of the hmg: clinical and cytomorphological studies in relation to its response to chemotherapy. Carol, 68:321, 1977. 9. Bensch, K. G., Corrin, B., Pariente, R., and Spencer, H.: Oat cell carcinoma of the lung. Its origin and relationship to bronctfial carcinoid. Cancer, 22:1163, 1968. 10. Dube, V. E.: Peripheral bronchial carcinoid with a spindle-cell pattern. Arch. Path., 89:374, 1970. I 1. Hage, E.: Histochenfistry and fine structure of bronclfial carcinoid tumors. Virchows ArclL Abt..A Path. Anat., 361:121, 1973. 12. Bonikos, D. S., Bensch, K. G., and Jamplis, R. W.: Peripheral puhnouary carcinoid tumors. Cancer, 37:1977, 1976. 13. Churg, A.: Large spindle cell variant of peripheral bronclfial ~arcinoid tumor. Arch. Path. Lab. Med., 10l:216, 1977. 14. Capella, C., Gabrielli, M., Polak,J. M., Buffa, R., Solcia, E., and Bordi, C.: Uhrastructural and histological stud) of 11 bronchial carcinoids. Evidence for different types. Virchows Arch. Abt. A Path. Anat., 381:313, 1979. 15. Ranchod, M.: The h'istogenesis and devempment of puhnonary tumorlets. Cancer, 39:1135, 1977. 16. Churg, A., and Warnock, M. L.: Puhnonary tumorlet. A form of peripheral carcinoid. Cancer, 37:1469, 1976. 17. Torikata, C., Kawai, T., Yakmnaru, K., and Kageyama, K.: Histopathological studies on the tumorlet of the hmg with special reference to the cytogenesis of proliferating cells. Acta Path. Jap., 25:539, 1975. 18. Bonikos, D. S., Archibald, R., and Bensch, K. G.: On the origin of the so-called tumnrlets of the lung. Hmnan Path., 7:4ql, 1976.
347
IIUMAN PATIIOLOGY--VOLUME
12, N U M B E R 4
19. "l'erzakis, J. A., Sommers, S. C., and Andersson, B.: Neurosecrett,ry appearing cells of hunmn seglnental bronchi, l.ab. Invest., 26:127, 1972. 20. Tateishi, R.: Distribution of arg) rophil cells in adult bureau lungs. Arch. l'ath., 96:198, 1973. 21. McDowell, E. M., Barrett, L. A., and Trump, B. F.: Observations on small granule cells in the adult human bronchial epithelium and in carcinoid and oat cell tumors. Lab. Invest., 34:202, 1976. 22. llage, E.: Endocrine-like cells of the pulmonary epithelium. In Couplaild, R. E., and Fujita, T. (Editors): Chromaffin, Enterochromaffin and Related Cells. Amsterdam, Elsevier Scientific Publishing Co., 1976, pp. 317-332. 23. llage, E., Hage, J., and Jnel, G.: Endocrine-like cells of the pulmonary epithelium of the buman adult lung. Cell Tiss. Res., 178:39, 1977. 24. Capella, C., Hage, E., Solcia, E., and Uselllni, I,.: Uhrastructural similarity of endocrine-like cells of the In, man lung and some related cells of the gut. Cell Tiss. Res., 186:25, 1978. 25. Bassett, F., Poirier, j., l.cCrom, M., and Turiaf, J.: Etude uhrastructurale de l'6pithdlium bronchlolare humain. Z. 9 Zellforsch. Mikrosk. Anat., 116:425, 1971. 26. Bensch, K. G., Gordon, G. B., and Miller, L. R.: Studies on the bronchial counterpart of the Kuhchitsky (argentaffin) cell and innervation of bronchial glands. J. Uhrastruct. Res., 12:668, 1965.* 27. Campiche, M. A:, Gautier, A., 1lernandez, E. I., and Reymond, A.: An electron microscope stud)" of the fetal development of human lung. Pediatrics, 32:976, 1963. 28. Lauu'eryns, J. M., Pcuskens, J. C., and Cokelacrc, M.: Argyroplfil, fluorescent and granulated (peptide and amine producing?) AFG cells in human infant bronchial epithelium. Light and electron microscopic studies. Lil'e Sci., 9:1417, 1970. 29. Cutz, E., and Conch, P. E.: Endocrine-like cells in human fetal lungs: an electron microscopic study. Anat. Rec., 173:115, 1972. 30. Cutz, E., Chan, W., Wong, V., and Conen, P. E.: Ultrastructure and fluorescence histochemistry of endocrine (APUDtype) cells in tracheal mucosa of human and various animal species. Cell Tiss. Res., 158:425, 1975. 31. Hage, E.: Electron microscopic identification of several types of endocrine cells in the brouchial epithelium of human foetuses. Z. Zellforsch., 141:401, 1973. 32. Black, W. C.: Pulmonary oncocytoma. Cancer, 23:1347, 1969. 33. Hosoda, S., Nakamura, W., Suzuki, 1I., lloshino, M., and Karasawa, K.: A bronchial carcinoid having low serotonin concentration. Arch. Path., 90:320, 1970. 34. Patchefsky, A. S., Gordon, G., ttarrer, W. V., and Hoch, W. S.: Carcinoid tnmor of the pancreas. Ultrastructural observations of a lymph node metastasis anti comparison with bronchial carcinoid. Cani:er, 33:1349, 1974. 35. McDowell, E. hi., and Trump, B. F.: ilistologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med., 100:405, 1976. 36. Mowry, R. W., and Winkler, C. ti.: Coloration of acidic carbohydrates of bacteria and flmgi in tissue sections with special reference to capsules of Cryptococcns neoformans, pneumococci and staphylococci. Am. J. Pathol., 32:628, 1956. 37. Sorokin, S. P., and Hoyt, R. F.: l'AS-lead hematoxylin as a stain fi)r small-granule endocrine cell populations in the lungs. other pharyngeal Oerivat~ves and the gut. Anat. Rec., 192:2.t5, 1978. 38. Solcia, E., Vassallo, G., and Capella, C.: Selective staining of endocrine cells by basic dyes after acid hydrolysis, Stain Tech., 43:257, 1968. 39. Pearse, A. G. E.: Biogenic amines. In llistochemistD', Theoretical and Applied. Ed. 3. Baltimore, The Williams & Wilkins Co., 1972, Vol. 2, pp. 1103-1127. 9t0. Grilnelins, L.: A silver nitrate stain for cr2 cells in hunaan pancreatic islets. Acta Soc. Med. Upsalien, 73:243, 1968. 41. Farquhar, hi. G., and Pal'ade, G. E.: Celljt*nctlons in amphibian skin. J. Cell B!ol., 26:263. 1965. 9t2, Vassallo, G., Capella, C., and Solcia, E.: Grimelius silver stain
348
April 1981
43. 44.
45. 46. 47. 48. 't9. 50. 51. 52. 53. 54. 55. 56. 57.
58. 59. 60. 61. 62. 63.
64. 65.
66. 67.
for endocrine cell grannies as shown by electron microscopy. Stain Tech., 46:7, 1971. Pearse, A. G. E., Coulling, I., Weavers, B., and Friesen, S.: The endocrine polypeptide cells of the human stomach, duodenum and jejuuum. Gut, 11:649, 1970. Black, W. C., and Ilaffner, H. E.: Diffuse hyperplasia of gastric argTrophil cells and multiple carcinoid tutnors. A histochemical and ultrastrnctural stud)'. Cancer, 21:1080, 1968. Black, W. C.: Enterochromaffin cell types and corresponding carcinoid tumors, l~ab. Invest., 19:473, 1968. Williams, R. M.: A light and electron microscopic study of au ovarian and rectal carcinoid. Histopathology, 3:19, 1979. An, T.: Cytoplasmic inclusions in bronchial carcinoid. Human Path., 9:241, 1978. Carstens, P. II. B., and l~,roghamer, W. L.: Duodenal carcinoid with cytoplasmic whorls of microfilaments. J. Path., 124:235, 1978. Reznik-Schiiller, 1t.: Uhrastructural alterations of APUD cells during nitrosantine-induced lung carcinogenesis. J. Path., 121:79, 1977. Waher, J. B., and Pryce, D- M.: The histology of lung cancer. Thorax, 10:107, 1955. Watson, W. L., and Berg, J. w.: Oat cell lung cancer. Cancer, 15:759, 1962. Lisa, J. R., Trinidad, S., and Rosenblatt, M. B.: Site of origin, histogenesis and cytostructnre of bronchogenic carcinoma. Am. J. Clin. Path., 44:375, 1965. Kay, S.: Histologic and histogenetic observations on the peripheral adenoma of the lung. Arch. Path., 65:395, 1958. Salyer, D. C., Salyer, W. R., and Eggleston, J. C.: Bronchial carcinoid tumors. Cancer, 36:1522, 1975. Moore, K. I..: The Developing Human. Philadelphia, W. B. Saunders Company, 1973, p. 167. Pearse, A. G. E.: 5-lt)droxytryptophan uptake by dog thyroid "C"-cells and its possible significance in polypeptide hormone production. Nature (London), 211:598, 1966. l'earse, A. G. E.: The diffuse neuroendocrine system aud the APUD concept: related "eudocrine" peptides in brain, intestine, pituitary, placenta and anuran cutaneous glands. Med. Biol., 55:115, 1977. Sidhn, G. S.: The endodermzl origin of digestive and respiratdry tract APUD ceils, ttistopathologic evidence and a review of the literattlre. Am. j. Path., 96:5, 1979. Popoff, N. W.: Epithelial functional rejuvenation observed in the mucous cells of the gastro-intestinal tract of the parietal cells of the stomach. Arch. Path., 27:841, 1939. Schofield, G.: The argentaffin cells of the small intestine of guinea pig. Acta Anat. (Basel), 11:414, 1951. Schofield, G.: The argentaffin and mucous cells of the small and large intestines of the mouse. Acta Anat. (Basel), 16:1, 1952. Schofield, G.: The argentaffin and mucous cells of the human intestine. Acta Anat. (Basel), 18:256, 1953. Cheng, C., and Leblond, C. P.: Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat., 141:537, 1974. McDowell, E. M., Barrett, L. A., Glavin, F., llarris, C. C., and Trump, B. F.: The respiratory epithelium. 1. Iluman bronchus. J. Natl. Cancer Inst., 61:539, 1978. McDowell, E. M., Becci, P.J[, Schiirch, W., and Trump, B. F.: The respiratory epidaelium. Vll. Epiderlnoid metaplasia of hantster tracheal epithelium during regeneration following mechanical Mjury. J. Natl. Cancer Inst., 62:995, 1979. Saccomanno, G., Archer, V. E., Saunders, R. P., James, I.. A., anti Beckler, P. A.: l.ung cancer of ttranium miners on the Colorado Plateau. I leahh Physics, 10: I 195, 1964. Mcl)ougall, J.: Endocrine-like cells in the term iqal bronchioles and saccules of human fetal lung: an ultrastructural study. Thorax, 33:43, 1978. Department of Pathology University of hlaryland School of Medicine 10 South Pine Str6et Baltimore, Maryland 21201 (Dr. Trump)