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Nasopharyngeal Carcinoma In Situ
Two Cases of an Emerging Diagnostic Entity
Florence Cheung, M.B.B.S.1
Siu Wah Pang, M.B.B.S.1
Fei Hioe, M.B.B.S.1
Kin-Nam Cheung, M.Phil.1
Anne Lee, M.B.B.S.2
Tsz-kok Yau, M.B.B.S.2
Department of Clinical Pathology, Pamela Youde
Nethersole Eastern Hospital, Chai Wan, Hong
Kong, People’s Republic of China.
Department of Clinical Oncology, Pamela Youde
Nethersole Eastern Hospital, Chai Wan, Hong
Kong, People’s Republic of China.
BACKGROUND. The association of Epstein–Barr virus (EBV) with the oncogenesis of
nasopharyngeal carcinoma (NPC) is well established. Latent infection by EBV with
clonal proliferation has also been demonstrated in preinvasive lesions of NPC. In
situ hybridization for EBV-encoded RNA (ISH EBER) now serves as an ancillary test
in the definitive diagnosis of these lesions.
METHODS. Two cases of nasopharyngeal carcinoma in situ (NPCIS) are presented
in this study. Their biopsies were studied by ordinary light microscopy, the ISH
EBER technique, and immunostaining for bcl-2. Tissue samples from 100 high risk
subjects negative for NPC and NPCIS, who served as controls, were also studied
using the ISH EBER technique.
RESULTS. NPCIS was characterized by abnormal light microscopic appearance as
well as positive staining by the ISH EBER technique; these features were not
observed in samples from the 100 high risk subjects. Immunostaining for bcl-2
protein was positive but less specific. Postradiotherapy biopsies of the two patients
were negative for NPCIS.
CONCLUSIONS. With the help of the ISH EBER technique, the diagnosis of NPCIS
is now possible in routine surgical pathology. As this entity is rare, it is necessary
to have a high degree of suspicion when evaluating biopsies from high risk
individuals. Radiotherapy for patients with NPCIS is justified in view of the risk of
cancer progression and the possibility of a coexisting invasive carcinoma. Cancer
1998;83:1069 –73. © 1998 American Cancer Society.
KEYWORDS: nasopharyngeal carcinoma in situ, preinvasive lesions, in situ hybridization Epstein–Barr virus– encoded RNA.
The authors thank Ms. Teresa Chan for clerical
Address for reprints: Florence Cheung, M.B.B.S.,
Department of Clinical Pathology, Pamela Youde
Nethersole Eastern Hospital, 3 Lok Man Road, Chai
Wan, Hong Kong, People’s Republic of China.
Received December 15, 1997; revision received
March 11, 1998; accepted March 11, 1998.
© 1998 American Cancer Society
asopharyngeal carcinoma (NPC), an epithelial carcinoma that
arises from the nasopharyngeal mucosa, is one of the most common cancers in southern China and parts of Southeast Asia. In Hong
Kong, in the year 1992, it accounted for 6.3% of all newly diagnosed
cancers, ranking third among the most common cancers in males and
eighth among those of females.1 That Epstein–Barr virus (EBV) is
associated with the oncogenesis of NPC and lymphoepithelioma-like
carcinomas of other organs is well established.2,3 EBV DNA, RNA, and
proteins have been found in the tumor cells of almost all NPCs,
implying a latent infection.4,5 There is evidence that tumor cells are
clonal proliferations derived from a single EBV-infected cell.4 On the
other hand, latent infection by EBV does not occur in normal nasopharyngeal epithelial cells.6 NPC patients and healthy people who
later develop NPC have high levels of antibodies against EBV proteins.7,8 Immunoglobulin (Ig)A titer to EBV viral capsid antigen (VCA)
is now widely used as a screening test for high risk populations or
relatives of NPC patients.9 If this test is used in conjunction with
nasopharyngeal endoscopy and prompt biopsy, it can be anticipated
CANCER September 15, 1998 / Volume 83 / Number 6
Clinical Data on Two Patients with NPCIS
Case 1 F/45
Case 2 M/55
IgA EBV endoscopy
VCA titer findings
Blood-stained postnasal drip 1:40
Bulge at left roof
Bulge at right roof
NPCIS: nasopharyngeal carcinoma in situ; IgA: immunoglobulin A; EBV: Epstein–Barr virus; VCA: viral
capsid antigen.
that more and more early stage NPCs and preinvasive
lesions will be encountered in clinical practice. In the
past, diagnostic criteria for preinvasive lesions of NPC
based on morphology alone have been vague, subjective, or impracticable.10 –13 Now, with the help of more
specific and nonisotopic molecular techniques, these
lesions can be diagnosed with confidence.14,15 Precedented by the success of cervical carcinoma screening,
recognition of preinvasive NPC lesions holds the
promise of early treatment and hence better prognosis. However, purely preinvasive lesions have rarely
been documented, and it is possible that the time
interval for their progression to invasion is short.16
Previous reports of lesions classified as preinvasive
came from retrospective studies of archival material
only.14,15 It was at this state of the art that we encountered two cases of pure nasopharyngeal carcinoma in
situ (NPCIS) unassociated with an invasive component. Diagnosis was based on histologic features and
confirmed by nonisotopic in situ hybridization for
EBV-encoded RNA (ISH EBER). The patients received
radiotherapy, and postradiotherapy biopsy revealed
no lesions. These two cases are presented herein, with
data from control studies that were conducted on
nasopharyngeal tissue from 100 high risk patients who
presented to our hospital during the same period.
These 100 patients had raised IgA EBV VCA titers but
negative biopsies for invasive and in situ lesions histologically.
Patients and Specimens
Over a 2-year period (November 1995 to October
1997), a total of 1220 nasopharyngeal biopsies were
performed at our hospital. They were performed in
response to screening of asymptomatic individuals
with raised IgA EBV VCA titer, patients with signs and
symptoms suggestive of NPC, or NPC patients after
completion of radiotherapy. Of these, 94 new cases of
NPC and 2 cases of NPCIS were diagnosed. Clinical
data for the latter two patients are shown in Table 1.
All tissue samples were fixed in 10% buffered formalin
and embedded in paraffin, and sections were prepared for light microscopy. To confirm the neoplastic
nature of the 2 NPCIS cases, we performed ISH EBER
studies on them, as well as on negative biopsies from
100 high risk patients who served as controls. Their
IgA EBV VCA titers varied from 1:10 to 1:640 (according to an immunofluorescence assay). Immunohistochemical studies to detect bcl-2 protein were also performed on the two NPCIS cases to assess their degree
of staining.
Nonisotopic ISH EBER Technique
Tissue sections 4 mm thick were cut and mounted
onto slides coated with 3-aminopropyltriethoxysilane.
The sections were deparaffinized, rehydrated, and airdried. The dewaxed slides were digested with 2.5
mg/mL pepsin in 0.1N HCl for 30 minutes at 37oC.
After the digestion, sections were dehydrated in 95%
ethanol and air-dried. The hybridization was done by
incubating the sections with fluorescein-conjugated
DNA probe to EBV EBER mRNA (BioGenex, San
Ramon, CA) for 1 hour at 37oC. After incubation, the
sections were washed with 0.05 M Tris buffer saline
(pH 7.6), and fluorescein-conjugated probes were detected with alkaline phosphatase conjugated mouse
antifluorescein monoclonal antibody (Dako, Glostrup,
Denmark). The color reaction was performed with
nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate solution in the dark for 20 minutes.
Appropriate positive controls were included in each
assay. The sections were counterstained with 0.1%
methyl green.
Immunostaining for bcl-2 protein
Standard immunoperoxidase technique was performed on paraffin sections for the detection of bcl-2
oncoprotein by incubation with bcl-2 monoclonal antibody (Dako, Glostrup, Denmark) at a dilution of 1:30.
The sections were counterstained with hematoxylin.
Case 1 had 2 biopsies, and NPCIS was detected only in
the first. Case 2 had 4 biopsies within a period of 2
months, with near-stripping of the mucosal bulge during the fourth biopsy. NPCIS was present in all four
biopsies. There was no accompanying invasive component in any of the above specimens. Microscopically, NPCIS changes in these two patients were similar, with patchy involvement of both covering and
cryptal epithelium (Figs. 1 and 2). These areas were
characterized by epithelial cells with loss of polarity,
enlarged vesicular nuclei, and large eosinophilic nucleoli. The basement membrane was disrupted by infiltrates of lymphocytes and plasma cells dispersed
Nasopharyngeal Carcinoma In Situ/Cheung et al.
Case 1: (A) The nasopharyngeal mucosa shows changes characteristic of nasopharyngeal carcinoma in situ, involving the whole thickness of the
epithelium. (B) A high-power view shows carcinoma in situ cells with large, vesicular nuclei and prominent eosinophilic nucleoli (H & E).
Case 2: (A) Nasopharyngeal carcinoma in situ involves the mucosal crypt. (B) A high-power view highlights the loss of polarity and disruption of the
basement membrane by lymphocytic infiltrates (H & E).
among the epithelial cells. Junction with adjacent respiratory epithelium was either abrupt or indistinct.
ISH EBER technique demonstrated moderate nuclear staining of these carcinoma in situ (CIS) cells,
accentuating their intraepithelial distribution (Fig. 3).
Positive staining of the nuclei was concentrated along
the nuclear membranes. The nucleoli were not
stained, but instead highlighted by a ringlike outline.
The staining intensity was slightly less than that in
classical NPC cells used as controls. As expected, studies of the 100 negative biopsies that served as controls
showed absolutely no uptake by the nonneoplastic
Immunohistochemical staining for bcl-2 protein,
similar to a recent study,17 showed positive staining of
the basal cells in normal epithelium and mild-to-mod-
erate cytoplasmic staining of the CIS cells (Fig. 4).
Distinction of neoplastic from normal epithelial cells
was much inferior to that offered by ISH EBER technique.
Both patients were treated by radiotherapy to a
total dose of 66 gray (Gy) delivered in 33 daily fractions. Nasopharyngeal endoscopy was performed 8
weeks after completion of radiotherapy and revealed
no visible tumor. Biopsy at the site where a bulge was
previously present revealed no residual CIS changes in
either of the two patients, and the ISH EBER studies
were negative.
The demonstration of EBV DNA and RNA in preinvasive
lesions of the nasopharynx, and evidence confirming
CANCER September 15, 1998 / Volume 83 / Number 6
(A) An in situ hybridization Epstein–Barr virus– encoded RNA study of nasopharyngeal carcinoma in situ (NPCIS) from Case 2 shows ribbonlike staining
of an intraepithelial lesion. (B) A high-power view shows positively stained nuclei with ringlike nucleoli (methyl green counterstaining).
FIGURE 4. Immunohistochemistry for the detection of bcl-2 protein demonstrates positive staining of basal cells and, to a lesser degree, nasopharyngeal carcinoma in situ cells (hematoxylin counterstaining).
that they are premalignant clones of EBV-infected cells,
mark an important step in understanding NPC oncogenesis.15,18 Although they are not long-lasting, these lesions
do occupy a place in the multistep process that culminates in invasive cancer. With the provision of molecular
techniques, especially the ISH EBER technique, more
and more NPCIS lesions will be diagnosed in routine
surgical pathology, requiring appropriate patient management. However, it is necessary to have a high degree
of suspicion when looking for foci of abnormal epithelial
cells, especially in high risk subjects with raised IgA EBV
VCA, to detect these subtle lesions. In the two cases that
we present herein, NPCIS is characterized by epithelial
cells with loss of polarity, large vesicular nuclei, promi-
nent eosinophilic nucleoli, disrupted basement membrane, and lymphocytic infiltrates. The low-power configuration of these foci recaptures that of the normal
reticular epithelium overlying lymphoid follicles described by Ali in his study of nasopharyngeal mucosa.19
The high-power morphology of the NPCIS cells resembles that of undifferentiated classical NPC, except that
NPCIS has a lesser degree of nuclear pleomorphism.
Whether different histologic types of NPC are preceded
by different morphologic forms of NPCIS is still unknown. It is the recognition of these abnormal cells that
alerts the surgical pathologist to the need for ISH EBER
studies. The NPCIS cells show moderate nuclear staining
in a pattern that is slightly different from the strong,
uniform nuclear staining usually seen in NPC tumor
cells during ISH EBER. Whether this reflects a lesser
degree of EBER expression in preinvasive tumor cells
remains to be verified.
According to studies by Sam et al.,6 normal nasopharyngeal biopsies from 23 high risk subjects were
negative for latent markers of EBV. Within the context
of nasopharyngeal epithelium, latent infection has
only been demonstrated in neoplastic NPC cells and
preinvasive lesions. This was borne out by the negative results of our study of specimens from the 100
high risk control subjects. It is also our experience that
squamous metaplasia, with or without atypia, and postirradiation dysplasia that we occasionally encounter
are ISH EBER negative (data not shown). Hence, the
ISH EBER technique is highly specific in picking up
NPCIS lesions, with its all-or-none staining characteristic, as distinct from the immunostaining by bcl-2
Awareness of the existence of preinvasive NPC
Nasopharyngeal Carcinoma In Situ/Cheung et al.
lesions and the availability of confirmatory ancillary
studies will certainly increase the number of these
lesions that are diagnosed. This poses a management
dilemma: to treat or not to treat. There is currently
little clinical data to guide treatment decisions. We
recommend definitive treatment, the justification being that coexisting invasive NPC can never be completely ruled out, even with repeated biopsies. The risk
of progression to invasive NPC is too real to ignore.
According to the studies of Pathmanathan et al.,15 5 of
8 of their patients with preinvasive lesions developed
cancer within 12 months of biopsy. The nasopharynx
is not a site that is easily accessible to observation,
especially in view of the frequent submucosal infiltrations that occur in NPC. Besides the inherent risk of
delay in detecting invasive carcinoma, the psychologic
distress and anxiety caused is likely to be substantial if
an observation policy is adopted. Hence, we decided
to treat our patients to a total dose of 66 Gy in 33 daily
fractions. Histologic confirmation of complete remission was achieved in both, but longer follow-up is
required to assess the ultimate control rate. Although
highly responsive to radiotherapy, NPC is associated
with mortality and its treatment with unpleasant morbidity. Hopefully, endoscopic biopsy of high risk subjects coupled with molecular studies of suspicious
intraepithelial lesions will screen out the early lesions
and alleviate the suffering of some patients.
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