LABORATORY
INVESTIGATION
T h e U n i t e d States 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 .
EMANUEL
Editor
RUBIN,
I V A N D A M J A N O V , Associate
Advisory
EMMANUEL
FÄRBER
Editor
Editors
JOHN L . FÄRBER
PETER A . WARD
Editorial
Board
GIUSEPPE
A.
ANDRES
ANTONIO
BARBARA
F.
ATKINSON
ROBERT
EARI.
BENDITT
L.
P.
MAXIMILIAN
CHARLES
C.
SEAN
B L JA
CAPEN
HAROLD
JAMES
BARRY
CAVALLO
G.
S.
DAVID
E.
CRAIGHEAD
RONALD
JOSEPH
A.
C. FAN TON E
SAMUEL
W.
GLORIA
R.
BURTON
PETER
FRENCH
GALLO
D.
J .
STANLEY
FRED
DELELLIS
GOLDBERG
L .
M.
RAM/I
JOHN
CEDRIC
S.
RAINE
KEITH
ABEL
K.
M.
REDDY
REID
A.
REIMER
L . ROBER TSON, J R .
DAVID
M.
DAN
GOLDEARB
ROBERT
ROBERTSON
I E G. SCARPELLI
MICHAEL
SCOPE
L .
SHELANSKI
E.
JOSEPH
GRISHAM
GARY
E . STRIKER
H.
CLIVE
R.
LEONARD
ROBERT
KENT
H E P I INS I A L L
JARETT
B.
j .
KIM
E.
VICTOR
ROBERT
GOULD
PIERCE
PURTILO
JANARDAN
LYNNE
MOSES
PHILLIPS
T .
GOLDBLATT
GORSTEIN
MCCLUSKEY
MOORE
TITO
COTRAN
MARTINEZ-HERNANDEZ
T .
SOLEZ
ROBERT
JENNINGS
JOHN
Q.
JOHNSON
BENJAMIN
NATHAN
KAUEMAN
EMIL
ROBERT
KISILEYSKY
GORDON
K.
KLINTWORTH
R.
MANJERI
F.
TAYLOR
L . T R E E S IA D
TROJANOWSKI
F.
A.
STEPHEN
DAVID
LAGLNOEE
RONALD
LANCE
A.
LIOTTA
JOHN
ALDEN
V.
LOUD
H .
TRUMP
UNANUE
S.
VENKA
1 ACHALAM
VOGEL
WEINSTEIN
YARDLEY
VOLUME 61, 1989
W I L L I A M S & W I L K I N S , Baltimore, M D 21202
Laboratory
Official Journal
Investigation
of
The United Slates and Canadian
Academy of Pathology,
(United States-Canadian
of the International
V o l u m e 61
Cover:
Division
Number 1
JULY
Inc.
Academy of
Pathology)
1989
B r i t t a n y - B e a g l e d o g w i t h h e r e d i t a r y c a n i n e spinal m u s c u l a r 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 c o u r t e s y o f M r . V i c t o r Raspa.
Editorial:
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
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 G e n e t i c S u s c e p t i b i l i t y t o A u t o i m m u n e Disease
Peter K.
Gregersen
Original Contributions:
P l a s m i n o g e n A c t i v a t o r I n h i b i t o r T y p e s 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 Trophoblasts
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
L o c a l i z a t i o n 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
L e u k o c y t e s i n M u r i n e Placenta. I m p l i c a t i o n s f o r M a t e r n a l - F e t a l I m m u nological Relationship
Raymond W. Red line a n d Christopher Y. Lit
Pneumopathies o f the Graft-Versus-Host Reaction: Alveolitis Associated w i t h an
Increased Level o f T u m o r Necrosis Factor m R N A and C h r o n i c I n t e r s t i tial Pneumonitis
Pierre F. Piguet, Georges E. Grau, Martine A. Collart, Pierre Vassalli, a n d
Yusuf
Kapanci
Role for T Lymphocytes in Silica-Induced Pulmonary I n f l a m m a t i o n
Andrea K.
Hubbard
Effect o f E n v i r o n m e n t a l P a r t i c u l a t e s o n C u l t u r e d H u m a n a n d B o v i n e E n d o 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 P a t h w a y
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 F a c t o r - 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
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 C a n i n e
Spinal Muscular A t r o p h y
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
Ultrastructural and Immunohistochemical Characterization of A u t o n o m i c Neurop a t h y i n G e n e t i c a l l y D i a b e t i c Chinese H a m s t e r s
Robert E. Schmidt, Donald
A. Plurad,
Santiago B. Plurad,
Barbara
E.
Cogsivell, Arthur R. Diani, a n d Kevin A. Roth
C-erbB-2
G e n e 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 o n H u m a n
Fetal E p i t h e l i a l Cells
Shigeo Mori, Tetsu Akiyama, Yukinori Yamada, Yasuyuki Morishita, Isamu Sugawara, Kumao Toyoshima, a n d Tadashi Yamamoto
N e w M o n o c l o n a l A n t i b o d y t h a t 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
Toshiyuki Maruyama,
Sumiko Tanaka, Beta Bozöky, Fumihiko Kobayashi,
and
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
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LABORATORY INVESTIGATION
Copyright © 1989 by T h e United States and Canadian Academy of Pathology, Inc.
Supernumerary Chromosome 1 in Interphase
Nuclei of Atypical Germ Cells in ParaffinEmbedded Human Seminiferous Tubules
HEINRICH WALT,
PATRICIA EMMERICH, THOMAS CREMER,
FRIDOLIN BANNWART
M A R I E - C L A U D E HOFMANN,
AND
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 s o - c a l l e d a t y p i c a l g e r m cells or cells of c a r c i n o m a in situ morphologically resemble neoplastic
cells i n s e m i n o m a . S i n c e seminomas show n u m e r i c a l a b e r r a t i o n s of chromosome 1 w e h a v e used a
D N A probe s p e c i f i c for chromosome region l q l 2 to determine w h e t h e r s u c h a b e r r a t i o n s c a n be
detected i n a t y p i c a l g e r m cell n u c l e i i n paraffin-embedded seminiferous tubules as w e l l . O n e - t h i r d
of i n t r a t u b u l a r n u c l e i , containing a t y p i c a l g e r m cells, consistently showed three h y b r i d i z a t i o n
s i g n a l s i n c o n t r a s t to t w o signals r e g u l a r l y observed i n n o r m a l intestine a n d i n spermatogonia. We
thus show that cytogenetic studies of p r e c a n c e r o u s cells c a n be performed directly on the tissue
w h e r e these c e l l s o r i g i n a t e .
A d d i t i o n a l k e y w o r d s : H u m a n p r e c a n c e r o u s testicular g e r m cells, In situ h y b r i d i z a t i o n , P a r a f f i n
sections.
E x p e r i m e n t a l data from a n i m a l tumors a n d from a n a l yses of h u m a n t e r a t o c a r c i n o m a - d e r i v e d cell lines suggest
that the c r u c i a l event i n the formation of testicular germ
cell tumors is preceeded by the activation of premeiotic
germ cells i n the seminiferous tubules or i n dysgeneic
gonads (8). S u c h a c t i v a t e d germ cells, representing stem
cells for a l l k i n d s of seminomatous a n d nonseminomatous tumors, are described as atypical germ cells ( A G C s ,
21) or as cells of c a r c i n o m a in situ or gonocytoma in situ,
respectively (27). S o m e authors have proposed that these
cells require a second h i t to t r a n s f o r m the in situ c a r c i noma into a n i n v a s i v e neoplasm (9). T h e potential of
A G C s to develop into manifestly malignant cells is high,
for nearly a l l patients w i t h s u c h diagnoses w i l l progress
into invasive c a n c e r i f orchiectomy is not performed (2,
26, 28). A G C s resemble cells of classical seminoma morphologically (25). F u r t h e r m o r e , similar to seminoma
A G C s produce h u m a n p l a c e n t a l - l i k e alkaline phosphatase (13, 15) a n d show hypotetraploid D N A contents
(19). Cytogenetic a n d D N A flow cytometric studies have
shown t h a t cells of s e m i n o m a a n d teratocarcinoma, respectively, have a n aneuploid number of chromosomes
(1, 10, 20, 22, 27). I n cytogenetic preparations of a
testicular biopsy from a p a t i e n t w i t h A G C s , nine mitotic
cells were found w i t h n u m e r i c a l a n d structural chromosome anomalies (17).
F o r better c h a r a c t e r i z a t i o n of early steps i n tumorigenesis it is essential to obtain more information about
specific chromosome aberrations present in A G C s . I n terphase cytogenetics (6, 14) provides a new tool to
investigate specific chromosome changes i n interphase
nuclei i n paraffin-embedded tissue sections (11) a n d
eliminates the need for mitotic cells that are indispensable for conventional cytogenetic analyses (7). B o t h
chromosomes 1 a n d 2 contain the gene loci for alkaline
phosphatase isozymes. Recently, we noted overexpression of these isozymes i n both seminiferous tubules w i t h
A G C s a n d seminoma cells (13). I f these particular chromosomes c a n be detected i n higher numbers i n such cells
t h a n i n normal diploid elements, one could expect e n hanced number of copies of the respective genes. O u r
previous investigations showed a n increased number of
chromosome 1 i n seminoma a n d nonseminomatous germ
cell tumors (10).
U s i n g in situ hybridization of a D N A probe specific
for chromosome region l q l 2 (5), we could detect numerical chromosomal aberrations i n interphase nuclei of
these precancerous cells i n paraffin sections from surgical specimens. F u r t h e r data suggest that this method
could be used to improve the yield of data from biopsies.
EXPERIMENTAL
DESIGN
B a s e d on our previous experiments w i t h xenotransplantable h u m a n testicular tumors a n d related cell lines
(11), we applied interphase cytogenetics to surgical specimens. T h i s was performed on 6-/xm serial sections w i t h
528
WALT
ET
AL.
LABORATORY
INVESTIGATION
phosphate-buffered 4% formaldehyde a n d embedded in
paraffin. Sections were hybridized w i t h a biotinylated
D N A probe for l q l 2 (5) that was visualized by a peroxidase reaction.
RESULTS
HISTOLOGY
AND DISCUSSION
OF SEMINIFEROUS
TUBULES
WITH
AGCs
Seminiferous tubules adjacent to a tumor showed v a r ious alterations. L u m i n a of most tubules, some of t h e m
being fibrotic, showed A G C s , Sertoli cells, a n d occasionally lymphocytic infiltrations. A G C s were clearly recognizable by their increased cell a n d nuclear size a n d a
particularly bright cytoplasm ( F i g . 1). M a n y spermatogonia in areas w i t h at least partial spermatogenesis were
found to be binucleated ( F i g . 2).
HYBRIDIZED PARAFFIN
SECTIONS
After hybridization treatment, sections were still well preserved. Occasionally, however, delicate elements, such as A G C s
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 r e s e n t e d 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 . 2. Binucleated spermatogonium (arrow) w i t h i n germinal epithelium of a patient w i t h a seminoma. T h i s cell borders tunica propria
(TP) and a Sertoli cell (S). Embedded i n Kulzer Technovit, hematoxy l i n 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 i n A and B present both two and three spots, indicating
chromosome 1. Stained w i t h Mayers hematoxylin. X 1 2 0 0 .
F I G . 1. Seminiferous tubule w i t h impaired spermatogenesis cont a i n i n g AGCs (arrow) w i t h i n a pathologically thickened tubular wall.
S, extratubular areas w i t h seminoma cells. Paraffin embedded, hemat o x y l i n and eosin. X252.
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 D N A 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
CHROMOSOME
1 I N H U M A N PRECANCEROUS TESTICULAR GERM
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 overexpression 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 A G C s 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
CELLS
529
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 nonseminoma (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.
METHODS
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
HYBRIDIZATION
SITU
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% glutaraldehyde in phosphate buffer and embedded in Kulzer Technovit 7100 (Kulzer, Wehrheim, F R G ) . Sections of 2 ßm were
stained with hemalum eosin and with the periodic acid-Schiff
reaction.
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 i n A {arrow), which is
magnified i n C. Note typical arrangement of chromosomes i n nuclei
indicated by small arrows. I n B, one chromosome was found i n a
particular nucleus, whereas i n C two spots were detected w i t h i n the
consecutive section of the same nucleus. Cell membranes are partially
damaged by the hybridization treatment. Stained w i t h Mayers hemat o x y l i n . A X390, B and C X890.
TABLE
Patient
LABELING PROCEDURE FOR D N A PROBE PUCI.77,
P R E T R E A T M E N T OF SECTIONS, HYBRIDIZATION, AND
PROBE DETECTION
Procedures of this part were identical to those described in
detail elsewhere (11). Probe p U C l . 7 7 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 l q l 2 (5). The probe was nick-translated with
b i o t i n - l l - d U T P as described (16).
Before in situ hybridization, a special pretreatment was
1. E V A L U A T I O N O F N U C L E A R
Tumor type
n
SIGNALS
Spots per nucleus
l
2
3
83
123
61
56
49
32
14
15
17
11
44
47
27
17
17
7
60
19
21
20
T o t a l of all nuclei
Percent
372
100
89
24
152
41
127
34
Nuclei i n n o r m a l tubules from
patient 2
Percent
196
121
75
100
62
38
1
2
3
4
5
Seminoma
Seminoma/teratocarcinoma
Teratocarcinoma
Teratocarcinoma
Teratocarcinoma
4
2
1
1
4
1
Nuclear signals w i t h i n seminiferous tubules were evaluated after in situ hybridization of paraffin sections w i t h probe p U C l . 7 7 (peroxidase
detection). Seminiferous tubules were adjacent to testicular germ cell tumors from five patients, n, Number of evaluated intratubular nuclei.
W A L T E T AL.
530
LABORATORY
INVESTIGATION
Date of acceptance: J u l y 19, 1989.
T h i s 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 t o : H e i n r i c h W a l t , Laboratory o f Reproduction and Development, Department of Gynecology and Obstetrics,
University Hospital, C H - 8 0 9 1 Zürich, Switzerland.
REFERENCES
F I G . 5. Segment of seminiferous tubule w i t h intact spermatogenesis. T h i s optical plane shows t w o nuclei o f spermatogonia w i t h t w o
spots near the tubular wall a n d 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 w i t h o u t
hybridization signal. Stained w i t h Mayer's hematoxylin. X790.
applied to tissue sections including five subsequent freezingthawing steps, overnight incubation at 65°C, removal of para f f i n (5 min xylene, 5 min 99% ethanol), 0.2 N H C l 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 D N A ,
and 2 Mg/ml labeled probe was applied to the specimen. A
coverglass was sealed, and probes and specimen were denaturated 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% f o r m a m i d e / l x S S C
for 15 min at room temperature and once in O . l x S S C for 30
min at 37°C. Hybridized probes were visualized by a peroxidase
reaction, as described previously (11). F o r counting spots (i.e.,
peroxidase reaction products at hybridized l q l 2 sites), identical
areas of three consecutive sections were photographed. Intratubular 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:
We
thank Jolanda
Brecher,
E g l i , a n d R i t a Moos for e x c e l l e n t t e c h n i c a l
Rita
assistance,
Prof. D . H a u r i a n d h i s t e a m f r o m t h e D e p a r t m e n t of
Urology for outstanding c o o p e r a t i o n , P r o f . J . C o s t a ( I n stitute of Pathology of t h e U n i v e r s i t y of L a u s a n n e ) for
c r i t i c a l c o m m e n t s on t h e m a n u s c r i p t .
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Zentralbl H a u t Geschlechtskrankheiten 154:861, 1988
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