L A B O R A T O R Y I N V E S T I G A T I O N

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J O H N H . Y A R D L E Y

VOLUME 61, 1989

W I L L I A M S & W I L K I N S , B a l t i m o r e , M D 2 1 2 0 2

Laboratory Investigation Official Journal of The United Slates and Canadian Academy of Pathology, Inc. (United States-Canadian Division of the International Academy of Pathology)

V o l u m e 61 N u m b e r 1 J U L Y 1989

Cover: Br i t tany -Beag le d o g w i t h h e r e d i t a r y canine spinal muscular a t r o p h y (see C o r k et al., p p . 6 9 - 7 6 ) . P h o t o g r a p h courtesy o f M r . V i c t o r Raspa.

E d i t o r i a l : F u n c t i o n a l a n d M o r p h o l o g i c Expressions o f T r o p h o b l a s t 1

I-Tien Yeh a n d Robert J . Kurman Biology of Disease :

H L A Class I I P o l y m o r p h i s m : I m p l i c a t i o n s f o r Genetic Suscept ibi l i ty t o A u t o i m ­m u n e Disease 5 Peter K. Gregersen

O r i g i n a l Contr ibut ions : Plasminogen A c t i v a t o r I n h i b i t o r Types 1 a n d 2 i n H u m a n T r o p h o b l a s t s : P A I - 1 Is

an I m m u n o c y t o c h e m i c a l M a r k e r o f I n v a d i n g T r o p h o b l a s t s 20 Ronald F. Feinberg, Lee-Chuan Kao, Julia E. Haimowitz, John T. Qiieenan, J r . , Tze-Chein Wun, Jerome F. Strauss III, a n d Hcnvey J . Kliman

Loca l i za t i on o f Fetal M a j o r H i s t o c o m p a t i b i l i t y C o m p l e x A n t i g e n s a n d M a t e r n a l Leukocytes i n M u r i n e Placenta. Impl i ca t i ons f o r M a t e r n a l - F e t a l I m m u ­nolog ica l Re lat ionship 27 Raymond W. Red line a n d Christopher Y. Lit

Pneumopathies o f the Gra f t -Versus -Host React ion : A lveo l i t i s Associated w i t h an Increased Leve l o f T u m o r Necrosis Factor m R N A a n d C h r o n i c I n t e r s t i ­t i a l P n e u m o n i t i s 37 Pierre F. Piguet, Georges E. Grau, Martine A. Collart, Pierre Vassalli, a n d Yusuf Kapanci

Role f o r T L y m p h o c y t e s in S i l i ca - Induced P u l m o n a r y I n f l a m m a t i o n 46 Andrea K. Hubbard

Effect o f E n v i r o n m e n t a l Particulates on C u l t u r e d H u m a n and Bov ine Endo ­t h e l i u m : C e l l u l a r I n j u r y V i a an O x i d a n t - d e p e n d e n t Pathway 53 Joe G. N. Garcia, Ronald F. Dodson, a n d Karleen S. Callahan

C y t o t o x i c i t y o f T u m o r Necrosis Factor -a f o r H u m a n U m b i l i c a l V e i n E n d o t h e l i a l Cells 62 Lucia Schuger, James Varani, Rory M. Marks, Steven L . Kunkel, Kent J.John­son, a n d Peter A. Ward

Changes i n N e u r o n a l Size a n d N e u r o t r a n s m i t t e r M a r k e r i n H e r e d i t a r y Canine Spinal M u s c u l a r A t r o p h y 69 Linda C. Cork, Richard J . Altschuler, Paul J . Bruha, Jeannette M. Morris, Donald G. Lloyd, Harry L . Loats, John W. Griffin, a n d Donald L . Price

U l t r a s t r u c t u r a l a n d I m m u n o h i s t o c h e m i c a l Charac ter i za t i on o f A u t o n o m i c N e u r o ­pathy i n Genet ica l ly Diabet ic Chinese Hamsters 77 Robert E. Schmidt, Donald A. Plurad, Santiago B. Plurad, Barbara E. Cogsivell, Arthur R. Diani, a n d Kevin A. Roth

C-erbB-2 Gene P r o d u c t , a M e m b r a n e P r o t e i n C o m m o n l y Expressed on H u m a n Fetal E p i t h e l i a l Cells 93 Shigeo Mori, Tetsu Akiyama, Yukinori Yamada, Yasuyuki Morishita, Isamu Sug-awara, Kumao Toyoshima, and Tadashi Yamamoto

New M o n o c l o n a l A n t i b o d y that Specifically Recognizes M u r i n e I n t e r d i g i t a t i n g a n d L a n g e r h a n s Cells 98 Toshiyuki Maruyama, Sumiko Tanaka, Beta Bozöky, Fumihiko Kobayashi, a n d Hirotugu Uda

L a b o r a t o r y I n v e s t i g a t i o n ( I S S N 0 0 2 3 - 6 8 3 7 ) is t h e o f f i c i a l j o u r n a l o f T h e U n i t e d S t a t e s a n d C a n a d i a n A c a d e m y o f P a t h o l o g y , I n c . , a n d is p u b l i s h e d m o n t h l y b y

W i l l i a m s & W i l k i n s , 4 2 8 E . P r e s t o n S t . , B a l t i m o r e , M D 2 1 2 0 2 . A n n u a l d u e s i n c l u d e S 3 0 f o r j o u r n a l s u b s c r i p t i o n . S e c o n d c l a s s p o s t a g e p a i d at B a l t i m o r e , M D a n d

at a d d i t i o n a l m a i l i n g o f f i c e s . P O S T M A S T E R , s e n d a d d r e s s c h a n g e s ( f o r m 3 5 7 9 ) to L A B O R A T O R Y I N V E S T I G A T I O N . 4 2 8 E . P r e s t o n S t r e e t . B a l t i m o r e , M D

2 1 2 0 2 . S u b s c r i p t i o n r a t e s S 9 0 ( S I 2 0 f o r e i g n . S I 6 6 J a p a n ) ; i n s t i t u t i o n s S I 5 5 ( S i 8 5 f o r e i g n , S 2 3 1 J a p a n ) ; i n - t r a i n i n g S 6 5 ( S 9 5 f o r e i g n , S 1 4 1 J a p a n ) ; s i n g l e c o p y

S I 3 ( S 1 6 f o r e i g n ) . J a p a n e s e r a t e s i n c l u d e a i r f r e i g h t . ( P r i c e s s u b j e c t t o c h a n g e . ) C o p y r i g h t © 1 9 8 9 b y T h e U n i t e d S t a t e s a n d C a n a d i a n A c a d e m y o f P a t h o l o g y . I n c .

0 0 2 3 - 6 8 3 7 / 8 9 / 6 1 0 5 - 0 5 2 7 $ 0 2 . 0 0 / 0 L A B O R A T O R Y I N V E S T I G A T I O N Copyright © 1989 by The United States and Canadian Academy of Pathology, Inc.

Vol. 61, No. 5, p. 527 , 1989 Printed in U.S.A.

Supernumerary Chromosome 1 in Interphase Nuclei of Atypical Germ Cells in Paraffin-Embedded Human Seminiferous Tubules

H E I N R I C H W A L T , P A T R I C I A E M M E R I C H , T H O M A S C R E M E R , M A R I E - C L A U D E H O F M A N N , AND F R I D O L I N B A N N W A R T

Institute of Pathology, University of Zürich, University Hospital, CH-8091 Zürich and Institute of Pathology, Stadtspital, Triemli, CH-8063 Zürich, Switzerland and Institute of Human Genetics

and Anthropology, University of Heidelberg, D-6900 Heidelberg, Federal Republic of Germany

T h e so-cal led a t y p i c a l germ cells or cells of carcinoma in situ morphologically resemble neoplastic cells i n seminoma. S i n c e seminomas show numerical aberrations of chromosome 1 we have used a D N A probe specif ic for chromosome region l q l 2 to determine whether such aberrations can be detected i n a t y p i c a l germ cell nuclei i n paraffin-embedded seminiferous tubules as w e l l . One- third of intratubular nuc le i , containing atypical germ cells , consistently showed three hybridization signals i n contrast to two signals regularly observed in normal intestine and i n spermatogonia. We thus show that cytogenetic studies of precancerous cells can be performed directly on the tissue w h e r e these cel ls originate .

Addit ional k e y w o r d s : H u m a n precancerous testicular germ cells, In situ hybridization, P a r a f f i n sections.

specific chromosome aberrations present in A G C s . I n ­terphase cytogenetics (6, 14) provides a new tool to investigate specific chromosome changes in interphase nuclei in paraffin-embedded tissue sections (11) and eliminates the need for mitotic cells that are indispen­sable for conventional cytogenetic analyses (7). Both chromosomes 1 and 2 contain the gene loci for alkaline phosphatase isozymes. Recently, we noted overexpres-sion of these isozymes in both seminiferous tubules with A G C s and seminoma cells (13). I f these particular chro­mosomes can be detected in higher numbers in such cells than in normal diploid elements, one could expect en­hanced number of copies of the respective genes. Our previous investigations showed an increased number of chromosome 1 in seminoma and nonseminomatous germ cell tumors (10).

Us ing in situ hybridization of a D N A probe specific for chromosome region l q l 2 (5), we could detect numer­ical chromosomal aberrations in interphase nuclei of these precancerous cells in paraffin sections from sur­gical specimens. Further data suggest that this method could be used to improve the yield of data from biopsies.

E X P E R I M E N T A L D E S I G N Based on our previous experiments with xenotrans-

plantable human testicular tumors and related cell lines (11), we applied interphase cytogenetics to surgical spec­imens. T h i s was performed on 6-/xm serial sections with

Exper imenta l data from animal tumors and from anal ­yses of human teratocarcinoma-derived cell lines suggest that the crucial event in the formation of testicular germ cell tumors is preceeded by the activation of premeiotic germ cells i n the seminiferous tubules or in dysgeneic gonads (8). S u c h activated germ cells, representing stem cells for al l kinds of seminomatous and nonseminoma­tous tumors, are described as atypical germ cells ( A G C s , 21) or as cells of carcinoma in situ or gonocytoma in situ, respectively (27). Some authors have proposed that these cells require a second hit to transform the in situ carci ­noma into a n invasive neoplasm (9). T h e potential of A G C s to develop into manifestly malignant cells is high, for nearly al l patients w i t h such diagnoses wil l progress into invasive cancer i f orchiectomy is not performed (2, 26, 28). A G C s resemble cells of classical seminoma mor­phologically (25). Furthermore , similar to seminoma A G C s produce h u m a n placental - l ike alkaline phospha­tase (13, 15) a n d show hypotetraploid D N A contents (19). Cytogenetic and D N A flow cytometric studies have shown that cells of seminoma and teratocarcinoma, re­spectively, have a n aneuploid number of chromosomes (1, 10, 20, 22, 27). I n cytogenetic preparations of a testicular biopsy from a patient with A G C s , nine mitotic cells were found with numerical and structural chromo­some anomalies (17).

For better characterization of early steps i n tumori-genesis it is essential to obtain more information about

528 W A L T ET AL. L A B O R A T O R Y I N V E S T I G A T I O N

phosphate-buffered 4% formaldehyde and embedded in paraffin. Sections were hybridized with a biotinylated D N A probe for l q l 2 (5) that was visualized by a peroxi­dase reaction.

R E S U L T S A N D D I S C U S S I O N

H I S T O L O G Y O F S E M I N I F E R O U S T U B U L E S W I T H A G C s

Seminiferous tubules adjacent to a tumor showed var­ious alterations. L u m i n a of most tubules, some of them being fibrotic, showed A G C s , Sertoli cells, and occasion­ally lymphocytic infiltrations. A G C s were clearly recog­nizable by their increased cell and nuclear size and a particularly bright cytoplasm (Fig. 1). M a n y spermato­gonia in areas with at least partial spermatogenesis were found to be binucleated (Fig. 2).

H Y B R I D I Z E D P A R A F F I N S E C T I O N S

After hybridization treatment, sections were still well pre­served. Occasionally, however, delicate elements, such as AGCs or cells of seminoma, displayed disruptures of the cell mem­brane and/or the cytoplasm. Small intestine mucosa served as normal control and displayed predominantly two spots per cell nucleus (11). The number of chromosome lql2-targets was aberrant in neoplastic interphase nuclei in sections from both seminoma and teratocarcinoma (Fig. 3). In consecutive sections through seminiferous tubules, interphase nuclei with one, two, three, and four hybridization spots were clearly recognizable (Fig. 4). In 34% of 372 intratubular nuclei analyzed, three spots were found. In 41% two spots and in 24% one spot were noted in nuclei. Four nuclei presented four signals each. In contrast, nuclei with three signals could not be detected in tubuli with normal seminiferous epithelium from patient 2; 121 out of 196 nuclei (62%) were counted with two spots, and 75 (38%) showed only one signal (Table 1). An area with normal spermatogenesis is p resented in Figure 5. S p e r m a t o g o n i a showed two spots per nucleus, whereas spermatids only presented one signal. Nuclei from spermatids, being similarly opaque to sperm heads, did

F I G . 1 . Seminiferous tubule w i t h impaired spermatogenesis con­taining AGCs (arrow) w i t h i n a pathologically thickened tubular wall . S, extratubular areas w i t h seminoma cells. Paraff in embedded, hema­toxyl in and eosin. X252.

F I G . 2. Binucleated spermatogonium (arrow) w i t h i n germinal epi­thel ium of a patient w i t h a seminoma. This cell borders tunica propria (TP) and a Sertoli cell (S). Embedded in Kulzer Technovit, hematox­y l in and eosin, Nomarski-contrast. X 1 1 0 0 .

F I G . 3. Area of seminoma (A) adjacent to seminiferous tubules w i t h AGCs. B, differentiated part (cartilage) of teratocarcinoma of the same patient. Nuclei in A and B present both two and three spots, indicating chromosome 1. Stained w i t h Mayers hematoxylin. X1200 .

not yield a detectable hybridization signal, probably indicating insufficient probe penetration in these cells.

Numerical aberrations, i.e., gains or losses of whole chro­mosomes often together with polyploidization events, are the most consistent and probably earliest gross chromosomal ab­normalities of malignant human gliomas (3). Similar to malig­nant gliomas, germ cell tumors show higher than diploid chro­mosome numbers, indicating the importance of abnormal chro­mosome segregation events in these tumors as well. Similarly to the data presented by Oosterhuis (22) using DNA flow cytometry our earlier chromosomal analysis suggests that the higher metastatic potential of nonseminomatous tumors is correlated with a range of 55 and 65 chromosomes, whereas

V o l . 6 1 , No. 5, 1989 C H R O M O S O M E 1 I N H U M A N PRECANCEROUS T E S T I C U L A R G E R M C E L L S 529

seminomas have generally higher numbers of chromosomes (70-80) (1, 10, 29).

Boveri (4) was the first to predict that segregational errors during mitosis might contribute to tumor formation from single somatic cells. Recent investigations have extended Boveri's early concept to the molecular level and indicated that the loss of certain chromosomes or chromosome segments may unmask recessive mutations at specific loci (12). Conversely, the gain of specific chromosome segments may contribute to the over-expression of dominantly acting tumor-related genes located within these segments, including oncogenes. Similarly, as dem­onstrated by isoelectric focusing, we found overexpression of the four isozymes of the alkaline phosphatase in germinal epithelium with AGCs and in seminoma, compared with normal tissues and those of nonseminomatous germ cell tumors (13). The genes for these isozymes are located on chromosomes 1

F I G . 4. Seminiferous tubule w i t h AGCs. Nuclei present one, two, three, and four spots, localizing hybridized chromosomes 1. B is a consecutive section through the same area in A {arrow), which is magnified i n C. Note typical arrangement of chromosomes in nuclei indicated by small arrows. I n B, one chromosome was found i n a particular nucleus, whereas in C two spots were detected w i t h i n the consecutive section of the same nucleus. Cell membranes are partial ly damaged by the hybridization treatment. Stained w i t h Mayers hema­toxyl in . A X390, B and C X890.

and 2. Whether overexpression of alkaline phosphatase iso­zymes is directly related to the number of one or both of these chromosomes is not yet clear.

If one speculates that seminoma can progress into nonsem­inomatous tumors, as suggested by Monaghan and Raghavan (18, 23) and very recently by Oosterhuis (22), this transition should be accompanied by a loss of part of the hypertriploid genome. Considering the high chromosome numbers typical of seminoma and the lower numbers generally found in nonsem-inoma (1, 10, 29) one might expect further activation through loss of chromosomes during progression to highly metastatic forms. In addition to numerical aberrations structural chro­mosome aberrations are likely to play an important but even less understood role in these types of tumors.

M E T H O D S

T I S S U E P R E P A R A T I O N F O R M O R P H O L O G Y A N D IN SITU H Y B R I D I Z A T I O N

Seminiferous epithelium from five patients with a testicular tumor (one seminoma, one teratocarcinoma with prominent portions of seminoma, and three teratocarcinoma without parts of seminoma) and normal intestinal tissue from ariother patient were fixed conventionally with 4% formaldehyde in phosphate buffer for 5 hours. The latter tissue served as a diploid somatic tissue control. For structural controls, 3-/um sections were produced and others of 6 ßm were prepared for in situ hybrid­ization by attaching them onto microscope slides pretreated by aminoalkylsilane (24). To allow histological analyses of entire nucleic areas, series of two or three consecutive sections per optical slide were prepared, and identical areas were photo­graphed. In addition, for optimal structural analysis tissues were fixed with a mixture of 2% formaldehyde and 0.2% glu-taraldehyde in phosphate buffer and embedded in Kulzer Tech-novit 7100 (Kulzer, Wehrheim, F R G ) . Sections of 2 ßm were stained with hemalum eosin and with the periodic acid-Schiff reaction.

L A B E L I N G P R O C E D U R E F O R D N A P R O B E P U C I . 7 7 , P R E T R E A T M E N T O F S E C T I O N S , H Y B R I D I Z A T I O N , A N D P R O B E D E T E C T I O N

Procedures of this part were identical to those described in detail elsewhere (11). Probe pUCl.77 was a generous gift from Howard Cooke. It was derived from human satellite D N A fraction I I I and represents a tandemly organized repeated se­quence from lql2 (5). The probe was nick-translated with biot in- l l -dUTP as described (16).

Before in situ hybridization, a special pretreatment was

T A B L E 1. E V A L U A T I O N OF N U C L E A R S I G N A L S

Spots per nucleus Patient Tumor type n

l 2 3 4

1 Seminoma 83 32 44 7

2 Seminoma/teratocarcinoma 123 14 47 60 2

3 Teratocarcinoma 61 15 27 19

4 Teratocarcinoma 56 17 17 21 1

5 Teratocarcinoma 49 11 17 20 1

Total of all nuclei 372 89 152 127 4

Percent 100 24 41 34 1

Nuclei i n normal tubules from 196 121 75

patient 2 Percent 100 62 38

Nuclear signals w i t h i n seminiferous tubules were evaluated after in situ hybridization of paraffin sections w i t h probe pUCl .77 (peroxidase detection). Seminiferous tubules were adjacent to testicular germ cell tumors from five patients, n, Number of evaluated intratubular nuclei.

530 W A L T E T AL. L A B O R A T O R Y I N V E S T I G A T I O N

F I G . 5. Segment of seminiferous tubule w i t h intact spermatogene­sis. This optical plane shows two nuclei of spermatogonia w i t h two spots near the tubular wall and only one signal i n more prominent nuclei of spermatocytes {arrowhead) or i n smaller ones of spermatids (above). A spermhead is recognizable {small arrowhead) but without hybridization signal. Stained w i t h Mayer's hematoxylin. X790.

applied to tissue sections including five subsequent freezing-thawing steps, overnight incubation at 65°C, removal of par­a f f i n (5 min xylene, 5 min 99% ethanol), 0.2 N HCl for 20 min, and proteinase K digestion (50 pg/m\ phosphate buffered sa­line) at 37°C for 10 min. After the pretreatment sections were dehydrated and finally air dried.

The hybridization mixture containing 60% formamide, 2 x S S C , 5% dextrane sulfate, 100 vg/m\ herring sperm DNA, and 2 Mg/ml labeled probe was applied to the specimen. A coverglass was sealed, and probes and specimen were denatur-ated simultaneously at 76°C for 10 min in a moist chamber. Hybridization was performed overnight at 40°C. Unbound probe was removed by washing twice in 50% formamide/lxSSC for 15 min at room temperature and once in O.lxSSC for 30 min at 37°C. Hybridized probes were visualized by a peroxidase reaction, as described previously (11). For counting spots (i.e., peroxidase reaction products at hybridized l q l 2 sites), identical areas of three consecutive sections were photographed. Intra-tubular nuclei of two to ten seminiferous tubules of each patient were evaluated by their hybridization signals and compared with tubules presenting normal spermatogenesis (Table 1). Cells of seminoma and differentiated parts of teratocarcinoma as well as of normal human small intestine were treated iden­tically.

Acknowledgments: W e thank J o l a n d a Brecher, R i t a E g l i , and R i t a Moos for excellent technical assistance, Prof. D . H a u r i and his team from the Department of Urology for outstanding cooperation, Prof. J . Costa ( I n ­stitute of Pathology of the Univers i ty of Lausanne) for critical comments on the manuscript .

Date of acceptance: July 19, 1989. This work was supported by the Krebsliga des Kantons Zürich, the

Roche Research Foundation, Basel, and the Bundesministerium für Forschung und Technologie, Federal Republic of Germany.

Address reprint requests to: Heinrich Wal t , Laboratory of Repro­duction and Development, Department of Gynecology and Obstetrics, University Hospital , CH-8091 Zürich, Switzerland.

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