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ANATOMIC PATHOLOGY
Original Article
In Situ Hybridization for Human
Papillomavirus DNA in Uterine
Adenosquamous Carcinoma with Glassy
Cell Features ("Glassy Cell Carcinoma")
MARY B. KENNY, M.B., ELIZABETH R. UNGER, M.D., PH.D.,
MARGIE L. CHENGGIS, M.T.(ASCP), AND MICHAEL J. COSTA, M.D.
Eighteen uterine adenosquamous carcinomas that showed focal
glassy cell features (33% to 85% of tumor histology) or predominant glassy cell features (> 85% of tumor histology) were studied
by in situ hybridization for human papillomavirus (HPV). Viral
DNA was present in neoplastic cells in five cases: type 18 in
four cases (two cervical adenosquamous carcinomas with predominant glassy cell features, two cervical adenosquamous carcinomas with focal glassy cell features) and type 16 in one case
(cervical adenosquamous carcinoma with predominant glassy cell
features). Positive intranuclear staining for HPV DNA was
present within areas of squamous and glandular differentiation
and within areas with glassy cell features. The mean age of
HPV(+) patients was less than HPV(-) patients (mean, 57
years, compared to 67 years). No significant association between
HPV status and prognosis or glassy cell features was detected.
Human papillomavirus types 16 and 18 are associated with adenosquamous carcinoma with predominant glassy cell features
or focal glassy cell features, "glassy cell carcinoma." Automated
colorimetric in situ hybridization is an effective method to detect
HPV DNA. (Key words: Human papillomavirus; Adenosquamous carcinoma; Glassy cell carcinoma; In situ hybridization)
Am J Clin Pathol 1992; 98:180-187
bridization (ISH) or Southern blot hybridization, varies
from 20% to 100%6 and seems to vary according to geographic location and histologic type.13"15 Different subtypes of HPV have been identified within the major
subtypes of cervical carcinoma as follows: type 16 predominantly, and types 18 and 31 occasionally, in squamous cell carcinoma 7 ' 9 "' 5 1 6 ; types 16 and 18 in
adenocarcinoma 7 ' 9 " 12 ' 516 ; types 16, 18, and 31 in adenosquamous carcinoma 9 ""' 215 ' 7 ; types 16and 18 in glassy
cell carcinoma. 912 ' 7 Human papillomavirus 18 in particular has been correlated with high-grade carcinomas,
higher incidence of nodal metastasis, younger age of patients, and more aggressive clinical behavior. 7 "' 7 1 8 Human papillomavirus 18 is also identified much less comFrom the Department of Pathology and Laboratory Medicine. Emory
monly in intraepithelial lesions (3%) than in invasive carUniversity, Atlanta, Georgia.
cinoma (22%), suggesting that a precursor intraepithelial
lesion may be short-lived and progress rapidly to an inReceived October 31, 1991; received revised manuscript and accepted
for publication February 24, 1992.
vasive carcinoma.17"19
Dr. Costa is affiliated with the Department of Pathology, University
Previous studies of adenosquamous carcinoma by hyof California. San Francisco, California.
Address reprint requests to Mary Kenny, Resident in Pathology: Debridization methods in the literature7"26 often have inpartment of Pathology and Laboratory Medicine. Room 783. Woodruff
cluded a few cases of glassy cell carcinoma, but did not
Memorial Building. Emory University School of Medicine, Atlanta,
mention the criteria used for the diagnosis of glassy cell
Georgia 30322.
Human papillomavirus has been associated with genital
neoplasia.1-6 In most instances, human papillomavirus
(HPV) types 6 and 11 are associated with low-grade cervical intraepithelial neoplasia and condyloma, and HPV
types 16, 18 and 31 are associated with high-grade intraepithelial neoplasia and invasive carcinoma. 5 ' 78 Human
papillomavirus DNA has been identified in all of the major
subtypes of invasive cervical carcinoma: squamous cell
carcinoma, adenocarcinoma, adenosquamous carcinoma
(ADSQ), adenoid cystic carcinoma, and neuroendocrine
carcinoma.6"13 The percentage of invasive carcinomas
containing HPV DNA, as demonstrated by in situ hy-
180
KENNY ET AL.
Glassy Cell Carcinoma
carcinoma. The aim of this study was to use ISH to probe
for HPV types 6, 11, 16, 18, 31, 33, and 35 in ADSQ of
the cervix with focal or predominant glassy cell features
(GCF), that is, "glassy cell carcinomas."
MATERIALS AND METHODS
Case Selection
All consecutive uterine adenocarcinomas and ADSQs
accessioned in the Surgical Pathology Department of
Grady Memorial Hospital (1982 to 1989) and all cervical
adenocarcinomas and ADSQs accessioned in the Surgical
Pathology Department of Emory University Hospital
(1985 to 1989) were retrieved from the computerized files.
The surgical pathology reports, all available hematoxylin
and eosin slides, representative periodic acid-Schiff, and
mucicarmine stains were reviewed by two of us (M.B.K.
and M.J.C.). Adenosquamous carcinoma was defined as
a malignant neoplasm showing both glandular and squamous differentiation (Figs. 1 and 2), or showing predominant GCFs (Fig. 3). Glassy cell features were defined according to the original criteria as proposed by Glucksmann
and associates27: (1) moderate amount of ground glass
cytoplasm that stained faintly blue with hematoxylin and
eosin, (2) fairly distinct periodic acid-Schiff-positive cell
membranes, and (3) large nuclei with prominent nucleoli.
Adenosquamous carcinomas with more than 85% of tumor histology showing GCFs were classified as adenosquamous carcinoma with predominant glassy cell features (PGCF); those ADSQs with 33% to 85% of tumor
histology showing GCFs were classified as ADSQ with
focal glassy cell features (FGCF). Slides also were reviewed
for the presence of intraepithelial neoplasia, koilocytosis,
and normal squamous epithelium.
Clinical information was obtained through the tumor
registry and by review of patient's computerized medical
records. The stage at presentation was determined using
both clinical and pathologic information and classified
according to FIGO. The data were analyzed using chisquare statistics.28 One representative block from each case
was selected for analysis by ISH. Biopsy material was used
whenever possible because of better and more even fixation.
In Situ
Hybridization
The colorimetric method of ISH, previously described
by Unger and colleagues,29 was used as adapted for automation, 30 using the Code-On Histomatic Slide Stainer
(Instrumentation Laboratories, Lexington, MA). Briefly,
5-/um-thick tissue sections were mounted on 3-aminopropyltriethoxysilane-coated (Sigma Chemical Co., St. Louis,
181
MO) slides, deparaffinized, dehydrated in absolute alcohol,
and digested with pepsin (Sigma Chemical Co.; 0.25 to 4
mg/0.1 N hydrochloride) for 20 minutes at 37 °C. After
digestion, the sections were washed with TRIS-saline-Brij
(0.1 mol/L TRIS hydrochloride, pH 7.5, 0.1 N sodium
chloride, 5 mmol/L magnesium chloride, and 2.5 mol/L
of 30% Brij 35) and dehydrated with 95% and absolute
alcohols. The dry tissues were covered with a hybridization
cocktail that contained 1.0 jttg/inL of a biotin-labeled DNA
probe. The cocktail has been described previously29 and
includes 36% deionized formamide, 5X standard saline
citrate (SSC) (5X SSC = 0.75 mmol/L sodium chloride
and 0.075 mmoL/L sodium citrate), 25 mmol/L sodium
phosphate, pH 6.5, IX Denhardt's solution (Denhardt's
solution = 0.02% each of povidone 40, ficoll 400, and
bovine serum albumin fraction V), 250 mg/L of sheared
denatured herring sperm DNA, and 10% weight/volume
dextran sulfate). The tissue and probe were simultaneously
denatured at 100 to 105 °C for 20 minutes and then cooled
to 37 °C for 2 hours of hybridization. The unhybridized
probe was removed by a series of graded salt washes: 2X
SSC/Brij/0.1% sodium dodecyl sulfate (SDS) and 0.2X
SSC/Brij/0.1% SDS at room temperature and 0.10X SSC/
Brij/0.1% SDS at 42 °C (IX SSC = 0.15 mmol/L of sodium chloride, 0.015 mmoL/L of sodium citrate; Brij
= 2.5 mol/L of 30% Brij 35). Slides were blocked with
3% bovine serum albumin in TRIS-saline-Brij and specifically hybridized biotin was detected with an avidinalkaline phosphatase conjugate (Dako Corp., Santa Barbara, CA). The conjugate was diluted 1:500 in 1% bovine
serum albumin TRIS-saline-Brij and incubated with the
sections for 20 minutes at 37 °C. Excess conjugate was
removed by washes in TRIS-saline-Brij, followed by color
development with McGadey reagent at 37 °C for 1 hour.
(McGadey reagent is comprised of 5-bromo-4-chloro-3
indoyl phosphate/nitroblue tetrazolium, as described
previously.29) Slides were counterstained with nuclear fast
red, covered with CrystalMount (Biomeda, Foster City,
CA), air dried, and mounted with Permount (Fisher Scientific, Fairlawn, NJ). These conditions resulted in moderate stringency, minimizing the extent of cross-hybridization and maintaining an adequate signal.
Clones for HPV types 6, 11, 16, 18, 31, 33, and 35
containing the complete viral genome in pBR322 or
pT713 were obtained as a gift from Bethesda Research
Laboratories, Gaithersburg, Maryland. The plasmid DNA
from each of these clones was isolated and purified using
standard techniques. Therefore, each HPV probe included
both vector and insert sequences. Unmodified plasmid
DNA (pBR322, Bethesda Research Laboratories) was used
as the negative control probe. Human placental DNA
(Sigma Chemical Co.) was used as the endogenous positive
control probe to control for optimization of digestion
Vol. 98 • No. 2
182
ANATOMIC PATHOLOGY
Original Article
FIG. 1 (i/p/w/<?//). Glandular
differentiation in adenosquamous carcinoma of cervix with focal glassy cell features (hematoxylin and eosin;
original
magnification,
X400).
FIG. 2 {upper right). Squamous differentiation with
several dyskeratotic cells in
adenosquamous carcinoma
with focal glassy cell features
(hematoxylin and eosin;
original
magnification,
X400).
FIG. 3 {lower). Glassy cell
area in adenosquamous carcinoma of cervix with predominant glassy cell features.
The tumor nuclei show
prominent nucleoli and fairly
distinct cell membranes (hematoxylin and eosin; original
magnification, X400).
?i_. G.f, , ^ y
conditions. All probes were labeled by nick translation
with Bio-11-dUTP (Bethesda Research Laboratories), as
previously described.30 Slides were evaluated by light microscopic examination. The hybridization was considered
satisfactory when (1) the endogenous positive control
probe resulted in a dark even signal over nearly every
nucleus, indicating adequate digestion and availability of
the target DNA, and (2) the negative control probe yielded
no signal.
A preliminary hybridization was performed with the
endogenous positive control probe to allow selection of
»_t.
the optimal conditions for digestion. Each case was subjected to "cocktail" probes of HPV 6/11, HPV 16/18,
and HPV 31/33/35. Any case that demonstrated signal
within the nucleus of neoplastic cells was considered positive, with confirmation of that positive signal and subtyping of the virus achieved by repeated hybridization
with separate probes for each viral type represented in the
screening cocktail, which gave preliminary positive results.
In this way, each positive result was confirmed by a second
hybridization. Sometimes equivocal results in the screening hybridization could not be confirmed in the subse-
A.J.C.P. • August l l »2
183
KENNY ET AL.
Glassy Cell Carcinoma
quent type-specific hybridizations. These cases were not
included as positive but could indicate the presence of
viral types not included in the assay.
RESULTS
Ninety cases were reviewed, and of these 27 were
ADSQ, 18 of which showed GCF. These 18 cases formed
the basis of this investigation. Of these 18 cases, 7 showed
PGCF (6 cervical, 1 endometrial) and 11 showed FGCF
(9 cervical, 2 endometrial). The age range of the patient
population was 26 to 87 years. Mode of presentation,
stage at presentation, and treatment are summarized in
Table 1.
Human papillomavirus was demonstrated in 5 of 18
carcinomas with GCF (28%). An additional case, an endometrial ADSQ with FGCF, was positive for HPV 33
in histologically benign endocervical glands, whereas the
carcinoma was negative (Table 1). Of the five carcinomas
positive for HPV in the neoplastic tissue, all were of cervical origin. Four cases were positive for HPV 18 (two
PGCF and two FGCF) and one was positive for HPV 16
(PGCF). There was no significant association between the
presence of HPV DNA and PGCF or FGCF. None of the
endometrial ADSQ with GCF were positive for HPV.
The intensity of the hybridization signal for HPV varied
among the cases but was low. Signal could be identified
as small nuclear dots in variable numbers of glandular
(Fig. 4), squamous (Fig. 5), or glassy cells (Fig. 6). The
low signal is attributable to the low copy number and can
be interpreted with confidence only when the negative
control hybridizations have no signal (Fig. 7). Glassy cells
positive for HPV were easily identified in the three positive
PGCF cases. Of 2 cases with FGCF, both showed positive
nuclei within areas of squamous and glandular differentiation. The loss of histologic detail resulting from the
hybridization procedure made areas of poorly differentiated squamous cell carcinoma difficult to distinguish
from glassy cell areas. This loss of morphology, combined
with the focal nature of GCF, made it impossible to determine definitively whether glassy cell areas in all cases
of ADSQ with FGCF were indeed positive for HPV. None
of the positive cases showed any evidence of koilocytotic
or dysplastic squamous epithelium. Normal squamous
epithelium present in three of six positive cases showed
no evidence of HPV infection.
There was a statistically significant difference between
the age of patients with HPV(+) and HPV(-) tumors (P
< 0.05). The mean age of HPV(+) cases was 57 years
(range, 26 to 73 years) versus HPV(-) cases, which was
67 years (range, 53 to 82 years).
Of the 5 HPV(+) cases, the stage at presentation was
as follows: 2 at Stage I, 2 at Stage III, and 1 at unknown
stage. Stage at presentation of 13 HPV(-) cases was as
follows: 1 at Stage I, 6 at Stage II, 1 at Stage III, 4 at Stage
IV, and 1 at unknown stage. Follow-up information was
available for 14 of 18 cases (78%) and follow-up time
ranged from 1 to 77 months (Table 1). The four patients
TABLE 1. IN SITU HYBRIDIZATION FOR HPV VIRAL DNA IN UTERINE ADENOSQUAMOUS
CARCINOMA SHOWING GLASSY CELL FEATURES
1 ° Site
HPV-Positive Cases
1.
C
2.
C
C
3.
4.
C
5.
C
6.
E
HPV- Negative Cases
7.
C
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
E
18.
E
c
c
c
c
c
c
c
c
c
GCF
HPV
Age
Pres
Stage
Rx
F/U
Status
P
P
P
F
F
F
18
18
16
18
18
33*
70
51
26
73
64
59
U
M
M&PAP
PAP
M
B
1
1
U
3
3
2
H
R
U
H&R
R
H&R
L
16
L
41
30
77
U
D-A
U
D-D
ND-A
ND-A
P
P
P
F
F
F
F
F
F
F
P
F
A#
A#
A
A
A
A
A
A
A
A
A
A#
68
62
73
62
53
82
73
81
68
71
61
55
M
PAP&B
PAP
PAP
B
B
PAP
PAP
B
M
B
M
2
4
U
1
2
2
2
3
4
4
2
4
R
R
U
R
R
U
R
R
N
N
H&R
H
28
2
L
17
34
L
16
21
1
3
60
2
ND-A
D-D
U
ND-A
ND-D
U
D-D
ND-A
D-D
D-D
ND-A
D-D
I c Site = primary site: C = cervix, E = endometrium". GCF = glassy cell features: P = predominant. F = focal; HPV = subtype of human papilloma virus detected: A = absence of HPV
DNA. * Viral DNA identified outside neoplastic tissue and # indicates equivocal staining that
may represent another untested HPV viral type: Prcs = presenting findings: M = gross lesion: B
= abnormal bleeding. PAP = abnormal Pap smear; Rx = therapy: H = hysterectomy; R
= radiotherapy; N = no therapy; U = unknown; F/U = follow-up time in months; L = lost to
follow-up: ND-A = alive without disease: ND-D = dead without disease: D-D = dead from
disease; D-A = alive with recurrent disease.
Vol. 9 8 - No. 2
ANATOMIC PATHOLOGY
184
Original Article
FIG. 4 (upper left). Intranuclear human papillomavirus
type 18 by in situ hybridization (single-headed arrow),
area of glandular differentiation in adenosquamous carcinoma of cervix with focal
glassy cell features. Doubleheaded arrows denote glandular lumina (nuclear fast red
counterstain; original magnification, X 1,000).
I
1/
FlG. 5 (upper right). Intranuclear human papillomavirus type 18 by in situ hybridization, area of squamous
differentiation in adenosquamous carcinoma of cervix with focal glassy cell features. The corresponding area
on adjacent hematoxylinand-eosin-stained sections
showed squamous differentiation (nuclear fast red
counterstain; original magnification, X 1,000).
<A$^
V \*
f*1fe%
j#
*
,?f-
FIG. 6 (lower left). Intranuclear human papillomavirus
type 18 by in situ hybridization, area of glassy cell features in adenosquamous carcinoma of the cervix with
predominant glassy cell features. The neoplastic cells
have prominent nucleoli
(nuclear fast read counterstain; original magnification,
X 1,000).
FIG. 7 (lower right). Negative
control probe, pBR, by in situ
hybridization, area of squamous differentiation in adenosquamous carcinoma of
the cervix with focal glassy
cell features (nuclear fast red
counterstain; original magnification, X 1,000).
A
LJ
•Ai
with HPV(-) tumors with Stage IV disease died of disease
within 1 to 3 months and their clinical course probably
reflects advanced stage of disease at diagnosis. Of the remaining HPV(—) cases, 5 were alive and disease free at
17, 21, 28, 60, and 77 months, respectively, 1 died of
disease at 16 months, and 1 died of other causes with no
evidence of disease at 34 months. Of the HPV(+) cases,
1 was alive and disease free at 30 months, 1 had recurrent
A.J.C.P. • August 1992
KENNY ET AL.
185
Glassy Cell Carcinoma
disease at 16 months (HPV 18(+) case) and 1 was dead
of disease at 41 months (HPV 18(+)). Human papillomavirus showed no significant association with clinical
outcome; however, our sample size was relatively small.
DISCUSSION
Glassy cell carcinoma, as originally reported by
Glucksmann and associates,27 was considered to be the
most undifferentiated form of mixed glandular and squamous carcinoma of the cervix and was associated with
poor prognosis and poor response to radiotherapy. Some
subsequent studies have suggested that "glassy cell carcinoma" is probably a part of the spectrum of histologic
differentiation of ADSQ, rather than a distinct subtype
of uterine carcinoma.31"45 Glassy cell carcinoma accounts
for 11 % of cervical adenocarcinomas/ADSQs,27'46 and
7.4% of endometrial ADSQs.44 If carcinomas with more
than 85% of tumor histology showing GCF are considered
to be glassy cell carcinomas, then 11% of cervical and 3%
of endometrial adenocarcinomas/ADSQs in our series
were glassy cell carcinomas, which is similar to other reports. 27 ' 4446
We elected to evaluate these cases for the presence of
HPV using an automated colorimetric method of ISH.
The advantages of ISH include the ability to detect small
numbers of positive cells and the ability to correlate a
positive signal with morphologic features and intracellular
distribution of the viral sequences. Routinely processed
formalin-fixed archival tissue can be studied,47 hybridization time is short (visual results can be obtained within 8
hours of cutting the sections), and biotinylated probes are
stable. The results of the colorimetric method are semiquantitative and permanent. Sensitivity is limited by the
variable preservation of nucleic acid in archival material
and by the requirement for morphologic preservation,
which may limit the availability of the target DNA to
participate in the hybridization reaction. This limit may
have resulted in an underestimation of the true incidence
of HPV DNA present in the tumor cells. However, this
method has been shown to detect easily 10 to 50 copies
of HPV per cell, and with careful optimization of the
digestion conditions, 1 or 2 copies of HPV/cell can be
detected.48 Furthermore, in a comparative study of this
method of ISH and the polymerase chain reaction for the
detection of HPV in penile lesions, the results of the two
assays were concordant in 20 of 27 cases (4 cases were
polymerase chain reaction positive/ISH negative; 3 cases
were polymerase chain reaction negative/ISH positive).49
Many authors have examined ADSQ and glassy cell
carcinomas by ISH for HPV. 8 -' 01316 - 202426 The percentage
of HPV(+) ADSQ ranges from 0 to 100%1324 and the
percentage of HPV(+) glassy cell carcinomas ranges from
0 to 25%. 913 Tase and colleagues9 identified 10 of 28 (36%)
ADSQs and 3 of 12 glassy cell carcinomas (25%) as HPV
16 or 18(+). King and others26 identified 9 of 23 (39%)
ADSQs and 1 of 6 (17%) glassy cell carcinomas as HPV
16 or 18(+). Our results using the automated colorimetric
method were similar. Overall, 5 of 18 cases of ADSQ (28%)
were HPV(+), all of cervical origin. Three of seven cases
of ADSQ with PGCF (43%) were positive for HPV (1
HPV 16, 2 HPV 18(+)). Two of 11 ADSQ with FGCF
(18%) were positive for HPV (all HPV 18). Our findings
correlate with those of others9,26 who used ISH with Tritium-labeled probes. Our series contained only three
ADSQ carcinomas of endometrial origin; however, none
were positive for HPV. The numbers are too small for
meaningful interpretation but suggest further investigation
is warranted to preclude the possibility that HPV is not
associated with endometrial ADSQ.
Previous studies of ADSQ and glassy cell carcinomas
using Southern blot hybridization found HPV in 0 to
100% of cases."' 21417 - 22 ' 23 The largest previous series of
ADSQ by Walker and co-workers,19 reported that 55% (6
of 11 cases) of ADSQ were HPV(+) (2 HPV 16, 3 HPV
18, 1 HPV 31). No specific mention of glassy cell carcinoma was made. The polymerase chain reaction method
also has been used to detect HPV DNA. With this sensitive
method, HPV DNA has been identified in 60% of normal
cervical epithelium, 100% of intraepithelial neoplasia, and
60% to 100% of invasive cervical carcinoma. 2550 " 52 In addition, multiple HPV types have been identified in individual cases. The exact significance of the high prevalence
of HPV DNA detected by this sensitive method is uncertain.
We found a statistically significant difference between
the mean age of HPV(+) and HPV(-) cases (P < 0.05).
In contrast, King and others,26 in a study of 85 cases of
cervical carcinoma, including 29 cases of ADSQ and glassy
cell carcinoma, found no statistical differences between
HPV 16(+), HPV18(+), and HPV (-) patients with respect to age, survival, recurrence, nodal metastasis, or tumor grade.
With reference to clinical outcome, we found no significant difference between HPV(+) and HPV(—) patients.
However, we were somewhat hampered by the fact that
two patients in each of the HPV(+) and HPV(-) groups
were lost to follow-up. In addition, 7 of our patients initially were found to have advanced disease (Stages III or
IV). Walker and co-workers,19 in a study of 100 invasive
cervical carcinomas, reported that patients with HPV
18(+) tumors had a worse outcome than those with HPV
16(+) or HPV(-) tumors, whereas Riou and colleagues25
noted in their study of 106 early-stage invasive cervical
carcinomas that lack of HPV was associated with poor
prognosis.
Vol. 98 • No. 2
186
ANATOMIC PATHOLOGY
Original Article
The mechanism by which HPV is associated with cervical carcinoma may involve integration of the HPV genome and binding of the E6-related protein product of
HPV 6, 11, 16, and 18 to the retinoblastoma suppressor
gene.53 The transcription product of this gene is inactive
in many malignant neoplasms, such as retinoblastoma or
osteogenic sarcoma. Furthermore, the E6 proteins of HPV
16 and 18, the subtypes we detected in our series, have
been shown to bind to the gene product of the tumor
suppressor gene p53.54 Confirmation of this possible
mechanism awaits demonstration of the importance of
modulation of suppressor protein products by HPV gene
products in cervical carcinoma.
The clinical and experimental data in the literature implicate HPV as the probable etiologic agent involved in
cervical carcinoma. However, the epidemiologic evidence
is still incomplete.55 We have demonstrated HPV in 28%
of the ADSQ studied by ISH (combined PGCF and
FGCF) and in 43% of the subgroup of ADSQ that showed
PGCF, confirming that HPV types 16 and 18 are associated with ADSQ with PGCF or FGCF "glassy cell carcinomas."
Acknowledgment. The authors thank Bonnie Helton for assistance in
preparing this manuscript.
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