Immunohistochemical and Genetic Analysis of Non–
Small Cell and Small Cell Gallbladder Carcinoma and
Their Precursor Lesions
Anil V. Parwani, M.D., Ph.D., Joseph Geradts, M.D., Eric Caspers, M.D., G. Johan Offerhaus, M.D.,
Charles J. Yeo, M.D., John L. Cameron, M.D., David S. Klimstra, M.D., Anirban Maitra, M.D.,
Ralph H. Hruban, M.D., Pedram Argani, M.D.
Departments of Pathology (AVP, AM, RHH, PA), Surgery (CJY, JLC), and Oncology (CJY, RHH), The Johns
Hopkins Hospital, Baltimore, Maryland; Department of Pathology and Laboratory Medicine (JG), Roswell
Park Cancer Institute, Buffalo, New York; The Academic Medical Center (EC, GJO), Amsterdam, The
Netherlands; and Department of Pathology, Memorial Sloan-Kettering Cancer Center (DSK), New York
Gallbladder carcinomas can be highly lethal neo-
plasms. Relatively little is known about the genetic
abnormalities that underlie these tumors, particu-
larly with respect to their timing in neoplastic pro-
gression. The authors evaluated 5 noninvasive dys-
plasias and 33 invasive gallbladder carcinomas (6
small cell carcinomas, 27 non–small cell carcino-
mas, of which 16 were accompanied by an in situ
carcinoma component) for expression of the pro-
tein products of the p16, p53, Dpc4, and pRB tumor
suppressor genes by immunohistochemistry. Neo-
plasms were also evaluated for the presence of ac-
tivating K-ras oncogene mutations. Seventy-five
percent of non–small cell gallbladder carcinomas
demonstrated loss of p16 expression, whereas 63%
accumulated high levels of p53. Loss of Dpc4 and
pRB expression was less frequent, seen in 19% and
4% of the neoplasms, respectively. Thirty percent of
neoplasms harbored activating K-ras mutations. In
contrast, 100% of the small cell carcinomas of the
gallbladder demonstrated inactivation of the pRB/
p16 pathway; 67% showed loss of pRB expression,
and the other 33% lost p16 expression. Eighty-three
percent of small cell carcinomas accumulated high
levels of p53, whereas loss of Dpc4 expression and
activating K-ras mutations were not found. Among
15 evaluable in situ components, 13 harbored the
same alterations found in the invasive component.
Inactivation of p16 and p53 occur in the majority of
non–small cell gallbladder carcinomas. Dpc4 inac-
tivation and K-ras mutations occur in a significant
minority of cases. pRB loss is uncommon in non–
small cell gallbladder carcinoma, but virtually all
small cell carcinomas inactivate the p16/pRB path-
way, usually by retinoblastoma protein loss. It is
noteworthy that all of these alterations occur at the
level of carcinoma in situ.
KEY WORDS: Carcinoma, Gallbladder, Genetics,
Immunohistochemistry, Tumor-suppressor gene.
Mod Pathol 2003;16(4):299–308
Carcinoma of the gallbladder is a relatively uncom-
mon, poorly understood, but highly lethal malig-
nancy that tends to present at an advanced stage
(1–3). There are approximately 5000 newly diag-
nosed cases per year in the United States, with a
female predominance. The incidence of gallbladder
carcinoma demonstrates marked geographic varia-
tion; for example, it is the single largest cause of
cancer death for women in Chile but accounts for
?0.5% of cancers in women in the United States (1).
Much of the geographic variation correlates with
the tendency to form gallstones, a recognized risk
factor in gallbladder carcinogenesis. Other risk fac-
tors include an abnormal junction of the chole-
dochopancreatic ducts (AJCPD; in which the bile
duct and pancreatic duct unite well above the
sphincter of Oddi, promoting reflux of pancreatic
juice into the bile duct), familial adenomatous pol-
yposis, and ulcerative colitis (1, 4). Overall 5-year
survival is ?5% (1).
The molecular pathogenesis of gallbladder carci-
noma remains enigmatic. Loss of heterozygosity
(LOH; 5–8) and cytogenetic (9) studies of non–small
cell gallbladder carcinoma have identified several
Copyright © 2003 by The United States and Canadian Academy of
VOL. 16, NO. 4, P. 299, 2003 Printed in the U.S.A.
Date of acceptance: January 28, 2003.
Supported by the Margaret Lee Fund for Gallbladder and Bile Duct Cancer
research at The Johns Hopkins Hospital.
Address reprint requests to: Pedram Argani, M.D., The Johns Hopkins
Hospital, Surgical Pathology, Weinberg Building, Room 2242, 401 N.
loci of recurrent genetic loss. These loci likely har-
bor tumor suppressor genes that are inactivated
and include chromosome arms 17p, 5q, 9p, 13q,
and 18q. Alterations in several specific tumor sup-
pressor genes that map to these loci have been
identified (i.e., p53 at 17p13, p16 at 9p21, RB at 13q),
but the data remain cloudy to date as differing
studies have yielded differing results (5, 7, 8, 10–18).
Some of the variation in results of immunohisto-
chemical studies may be due to use of different
techniques and cutoff points for positivity in differ-
ent studies. The loci on 18q involved in gallbladder
carcinoma have not been identified, with the DCC
and DPC4 genes being prime candidates. Finally,
mutational activation of the K-ras gene has been
demonstrated in 5–59% of cases (7, 14–18), with
suggestions that this frequency may be increased in
Japanese patients, particularly those with AJCPD.
Small cell carcinomas of the gallbladder have been
studied even less frequently (19).
In the present study, we analyzed our large series
of resected non–small cell and small cell gallblad-
der carcinomas for Dpc4, pRB, p16, and p53 protein
expression and for K-ras gene mutation.
This study was approved by the Johns Hopkins
Institutional Review Board. The computerized files
of the Surgical Pathology Division of the Depart-
ment of Pathology of The Johns Hopkins Hospital
were searched over the years 1985–2000 for cases
coded as “gallbladder” and “carcinoma.” Cases
were selected for study on the basis of availability of
a paraffin-embedded, formalin-fixed tissue block.
An additional case (Case 31) was obtained from
Memorial Sloan-Kettering Cancer Center. For each
case, a representative formalin-fixed, paraffin-
embedded tissue block containing carcinoma and
normal tissue was chosen for labeling. For cases in
which an in situ carcinoma component was noted,
a block containing this component was specifically
chosen for study.
We identified five cases of gallbladder dysplasia
or carcinoma in situ unassociated with invasive
carcinoma (Table 1) and identified 33 cases of in-
vasive gallbladder carcinoma for which blocks were
available. Patient ages ranged from 37 to 86 years
(mean age ? 62.8 y; median ? 63.5 y), and the
male-female ratio was 13:25. Twenty-one of 34
evaluable gallbladders harbored gallstones.
Two of the pure dysplasia cases were low grade,
whereas three were high grade or carcinoma in situ.
Of the invasive carcinomas, 16 were associated with
an in situ component; of these, the in situ compo-
nent had a flat component in 10 and was purely
papillary in 6.
Twenty-seven of the invasive carcinomas of the
gallbladder were non–small cell carcinomas (Cases
1–27); these included 21 adenocarcinomas, 5 ade-
nosquamous carcinomas (defined as showing vari-
able mixture of malignant squamous and glandular
components; 1), and 1 purely squamous carci-
noma. Of these 27 neoplasms, the primary tumor
was evaluated in 24, and a metastasis was the only
tissue available in 3. Of these 24 primary tumors, 15
were moderately differentiated (defined as 40–94%
of the tumor forming glands; 1), and 9 were poorly
differentiated (defined as 5–39% of the tumor form-
ing glands; 1). Six other neoplasms contained a
small cell (high-grade neuroendocrine) carcinoma
component (Cases 28–33). Two of these six were
pure small cell carcinomas (Cases 29, 30), whereas
one was predominantly small cell carcinoma with
focal squamous differentiation (Case 31). One tu-
mor was associated with flat carcinoma in situ
(Case 28), whereas two other invasive carcinomas
had both small cell carcinoma and adenocarci-
noma components along with papillary adenocar-
cinoma in situ (Cases 32, 33).
Several patients’ tumors were associated with un-
usual clinical presentations or pathologic findings.
One patient’s gallbladder carcinoma arose within a
septate gallbladder (Case 8). Another patient (Case
20) had concurrent colorectal adenocarcinoma and
gallbladder carcinoma, whereas another patient’s
gallbladder carcinoma extended into the extrahe-
patic bile ducts, which harbored a carcinoid tumor
Patients tended to present at advanced stage.
Using AJCC criteria (20), 29 of 30 patients’ primary
tumors presented at Stage 3 or above. At least 23 of
30 primary tumor resections were associated with
TABLE 1. Clinical and Molecular Features of Five Patients with Pure Dysplasia or Carcinoma In Situ
Case NumberAgeSex Stonep53Dpc4p16pRBK-ras
MUT D (Val)
NL ? normal pattern (intact labeling for p16, Dpc4, and pRB, less than 30% expression of p53, wild type K-ras); OE ? overexpression; LOSS ? loss
of expression; LGD ? low-grade dysplasia; CIS ? carcinoma in situ; MUT ? K-ras mutation; Stone ? gallstone present in specimen.
positive surgical margins microscopically, indicat-
ing incomplete excision of the neoplasm.
Immunohistochemistry for p53 and Dpc4
Unstained 4-?m sections were cut from the se-
lected paraffin block and deparaffinized by routine
techniques. The slides were steamed for 20 minutes
in sodium citrate buffer (diluted to 1? from 10?
heat-induced epitope retrieval buffer; Ventana-Bio
Tek solutions, Tucson, AZ). After cooling for 5 min-
utes, the slides were labeled for 40 minutes at room
temperature with either a 1:100 dilution of a mono-
clonal antibody to Dpc4 (clone B8, Santa Cruz Bio-
technology, Santa Cruz, CA) or a 1:250 dilution of a
monoclonal antibody to p53 (clone DO-7, DAKO,
Carpinteria, CA) using the Bio Tek 1000 automated
stainer (Ventana). Labeling was detected by adding
biotinylated secondary antibodies, avidin-biotin
complex, and 3, 3'-diaminobenzidine. Sections
were then counterstained with hematoxylin. Dpc4
and p53 immunolabeling were evaluated jointly by
two authors (AVP, PA) using a multiobserver micro-
scope, with agreement on all cases. For p53 label-
ing, a percentage of positive nuclei was determined.
Carcinomas were divided into two groups: normal
(?30% nuclear labeling) and positive for high-level
accumulation of p53 protein (?30% nuclear label-
ing). The labeling cutoff was chosen based on pre-
vious studies that have demonstrated that this cut-
off point correlates best with the status of the p53
gene in colorectal carcinomas (21). For Dpc4 label-
ing, any area of uniform cytoplasmic labeling and
focal nuclear labeling was considered positive. In
statistical analysis, any carcinoma showing even
focal nuclear and cytoplasmic labeling was consid-
ered positive (expressor), whereas carcinomas
demonstrating no expression in a background of
intact expression by non-neoplastic cells (desmo-
plastic stroma, normal peribiliary glands, etc.,
which served as internal controls) were considered
negative (nonexpressors). The rationale for consid-
ering carcinomas that labeled only focally as posi-
tives is based on the study of Wilentz et al. (22),
which found that pancreatic tumors with this focal
staining pattern proved to have an intact DPC4
Immunohistochemistry for p16, pRB
Immunohistochemistry for p16 and pRB was per-
formed in the laboratory of one of the authors (JG)
and interpreted by three of the authors (AVP, PA,
and JG). Mouse monoclonal anti-RB antibody 3C8
was purchased from QED (San Diego, CA), and
mouse monoclonal anti-p16 antibody 16P07 was
obtained from LabVision/NeoMarkers (Fremont,
CA). Nonspecific mouse IgG was used as a negative
antibody control. Standard ABC peroxidase assays
were performed as described in detail elsewhere
(22–26). For detection of pRB, deparaffinized sec-
tions were incubated with anti-RB antibody 3C8 at
2 ?g/mL for 2 hours, after an antigen retrieval step
in 0.01 M citrate buffer (95–100° C). For detection of
p16, sections were incubated with the anti-p16
monoclonal antibody 16P07 at 1 ?g/mL at 4° C
overnight, after antigen retrieval in 0.1 M EDTA pH
8.0 (20 min at 95–100° C). The detection reactions
for both markers used the Vectastain Elite ABC kit
using conditions recommended by the manufac-
turer. Diaminobenzidine with hematoxylin coun-
terstain was used for color development. Negative
controls were labeled under identical conditions.
The following external positive controls were used:
normal colonic mucosa for pRB and p16 and a
p16-positive lung cancer xenograft for p16. In ad-
dition, non-neoplastic stromal cells served as inter-
nal positive controls for pRB and p16 in every tu-
Each case was scored for pRB and p16 reactivity
using previously published criteria (19, 23–26).
Briefly, sections were examined for evidence of nu-
clear labeling above any cytoplasmic background;
cytoplasmic labeling itself was disregarded. If there
was nuclear labeling in a diffuse or mosaic distri-
bution throughout the lesion, it was considered
“positive” (normal) for the respective protein. If all
of the neoplastic nuclei failed to label whereas ad-
mixed non-neoplastic cells reacted positively, the
lesion was scored as “negative” (abnormal). Cases
were scored initially by one author (JO), and scoring
was subsequently reviewed by two authors (AVP,
PA) using a multiobserver microscope with agree-
ment on all cases.
K-ras Gene Codon 12 Analysis
For the K-ras gene analysis, tumor was microdis-
sected from unstained paraffin-embedded section
slides to ensure ?50% neoplastic cells in the sam-
ple. K-ras codon 12 analysis was performed with
allele-specific oligonucleotide (ASO) hybridization
method, as described elsewhere (27). DNA was iso-
lated from microdissected tumor tissue by over-
night incubation at 56° C in proteinase K solution,
followed by inactivation of proteinase K at 95° C for
10 minutes. DNA was amplified by PCR using K-ras
codon 12 specific primers (27). PCR products were
then digested with MvaI, which only recognizes
wild-type K-ras codon 12. Subsequently, a second-
round PCR was performed on both the digested and
undigested first-round PCR products. Cell suspen-
sions with mutant–wild-type ratios of 1:100 and
1:1000 were used as positive controls in every PCR
procedure. The cell suspensions were made of the
human colon cancer cell line SW 480 with a ho-
Gallbladder Carcinoma Genetics (A. V. Parwani et al.)301
mozygous GGT to GTT mutation at codon 12 of
K-ras and the human colon cancer cell line HT 29
with wild type K-ras. Water was used as a negative
control, and placental DNA was used as a control
for nonspecific hybridization. After denaturation,
the undigested and digested (mutant-enriched)
PCR products were spotted onto a nylon membrane
and hybridized to each of the K-ras codon 12
stringency washes were carried out at 63° C, fol-
lowed by autoradiography. K-ras codon 12 muta-
tional analysis was performed twice, in indepen-
In all cases, K-ras codon 12 mutations identified
by ASO hybridization were confirmed by direct flu-
orescent sequencing using the dideoxy chain termi-
nation method (27). Mutant-enriched PCR prod-
ucts (second-round PCR products of ASO-test) were
purified using the Qiaquick PCR purification kit
(Qiagen Inc.). DNA was sequenced with the DNA
sequencing kit, BigDye-Terminator Cycle sequenc-
ing Ready Reaction (Applied Biosystems, War-
rington, United Kingdom). The reaction products
were analyzed on an ABI Prism 3100 Genetic Anal-
yser (Hitachi, Applied Biosystems). Selected wild-
type cases were also sequenced.
Pure Dysplasias (n ? 5)
Neither of the two low-grade dysplasias demon-
strated abnormal expression of p53, Dpc4, and p16
or pRB protein, and neither contained a K-ras gene
mutation. In contrast, all three flat carcinoma in
situ lesions demonstrated accumulation of p53 pro-
tein at high levels and demonstrated loss of p16
expression. pRB expression was intact and diffuse
in these three cases, and Dpc4 was detectable in all
three (Fig. 1). One of the three carcinoma in situ
lesions harbored a K-ras mutation, involving con-
version to valine (Table 1).
Invasive Non–Small Cell Carcinomas (n ? 27)
This group included 14 pure invasive non–small
cell carcinomas and 13 invasive non–small cell car-
cinomas associated with an in situ carcinoma com-
ponent. Loss of p16 expression and abnormal ac-
abnormalities noted in this group. Eighteen (75%)
of 24 evaluable tumors demonstrated loss of p16
expression. This included 10 of 12 carcinomas un-
associated with an in situ component and 8 of 12
the most common
FIGURE 1. Flat carcinoma in situ (A) demonstrating p53 overexpression (B); intact, diffuse pRB expression (C); and loss of p16 expression (D).
Note the intact patchy labeling of stromal and endothelial cells for p16.
302 Modern Pathology
carcinomas associated with an in situ component.
The six p16-positive tumors generally displayed a
weak to moderate nuclear immunoreactivity in
?50% of cells in a mosaic pattern. Of note, in the
case (Case 13) with concurrent gallbladder carci-
noma and bile duct carcinoid tumor, p16 expres-
sion was intact in the carcinoid tumor but was lost
in the poorly differentiated gallbladder carcinoma.
Seventeen of 27 carcinomas (63%) accumulated
p53 at high levels; this included 8 of 14 pure inva-
sive carcinomas and 9 of 13 invasive carcinomas
associated with an in situ component. Five of 27
invasive carcinomas (19%) demonstrated loss of
Dpc4 expression, including 3 of 14 pure invasive
carcinomas and 2 of 13 invasive carcinomas asso-
ciated with carcinoma in situ. Of note, Dpc4 expres-
sion was intact in the in situ and invasive carci-
noma of the gallbladder but absent in the invasive
colorectal carcinoma that were concurrently re-
sected (Case 20), supporting the clinical and mor-
phologic impression that these were independent
primary neoplasms. Only 1 of 27 non–small cell
carcinomas (4%) showed loss of pRB expression; in
this tumor the in situ component also demon-
strated loss of pRB expression. Most tumors with
intact pRB expression demonstrated extensive la-
beling for pRB, particularly those demonstrating
loss of p16 expression (Fig. 2).
K-ras gene mutations were identified in 8 of 27
invasive non–small cell carcinomas (30%). Of the
eight invasive adenocarcinomas harboring a K-ras
gene mutation, six were primary tumors and two
were metastases. Five mutations involved conver-
sion to aspartate, and three, to serine.
There were no significant differences in results
for any tested marker among cases classified as
moderately versus poorly differentiated.
In Situ Carcinoma Components Associated with
Invasive Non–Small Cell Carcinoma (n ? 13)
There was a remarkable concordance between
the immunohistochemical labeling of the in situ
and the invasive non–small cell carcinoma compo-
nents for p16, pRB, p53, and Dpc4 in the above
cases. We identified p16 loss in 7 of 11 (64%) evalu-
able in situ components; the invasive carcinomas
corresponding to all of these tumors had shown p16
loss. We identified accumulation of p53 at high
levels in 10 of 13 in situ lesions (77%) associated
with invasive carcinoma; this included all 9 cases in
which the invasive component accumulated p53 at
FIGURE 2. Infiltrating non–small cell carcinoma (A) demonstrating loss of p16 expression (B) and intact, diffuse pRB expression (C). Note the
intact labeling of endothelial cells in Figure 2B.
Gallbladder Carcinoma Genetics (A. V. Parwani et al.) 303
high levels. There was one case with discordant
findings: in this case, the papillary in situ carci-
noma component demonstrated high levels of p53,
whereas the invasive component showed no p53
expression. We identified loss of Dpc4 expression in
both of the in situ lesions associated with invasive
carcinomas that showed loss of Dpc4 expression (2
of 13 in situ lesions overall, 15%; Fig. 3), whereas we
found loss of pRB expression in the in situ compo-
nent of the one invasive carcinoma that showed
loss of pRB (1 of 13 cases overall, 8%).
Invasive Small Cell Carcinomas (n ? 6)
Accumulation of p53 at abnormally high levels
was the most common abnormality noted, identi-
fied in five of six invasive small cell carcinomas. All
six small cell carcinomas demonstrated evidence of
inactivation of the pRB/p16 pathway. In contrast to
non–small cell carcinomas, loss of pRB expression
more common in this tumor type, identified in four
of six cases (67%), whereas the other two small cell
carcinomas with intact pRB expression demon-
strated loss of p16 expression (Fig. 4). Dpc4 protein
expression was intact in all six tumors, whereas
K-ras gene mutations were not identified in any of
the three tumors evaluated.
Non–Small Cell Components Associated with
Small Cell Carcinomas (n ? 3)
In two of three cases, the results were concor-
dant. The flat carcinoma in situ associated with one
small cell carcinoma and the papillary in situ and
invasive adenocarcinoma associated with another
each demonstrated p53 accumulation at high levels
and p16 loss like their small cell components. In the
other case, only the small cell carcinoma compo-
nent demonstrated loss of p16 expression; the pap-
illary in situ carcinoma and invasive carcinoma
with which it was associated expressed p16.
The results of immunohistochemical assays are
summarized in Table 2.
We studied a large series of resected gallbladder
carcinomas for expression of the protein products
FIGURE 3. Papillary in situ and infiltrating carcinoma (A) demonstrating loss of Dpc4 expression in the in situ component (B) and in the invasive
component (C). Note the intact labeling of normal stroma and benign gallbladder epithelium (Fig. 3C).
304 Modern Pathology
of the p16, DPC4, RB, and p53 tumor suppressor
genes and for mutation of the K-ras oncogene.
Our immunohistochemical results for p53 gener-
ally parallel those previously reported in the litera-
ture. A number of prior studies have shown that
accumulation of p53 at high levels is frequent in
invasive non–small cell carcinomas of the gallblad-
der and occurs at the level of carcinoma in situ (7,
10, 11). Although most of these older studies have
used primary antibodies other than the D0–7 clone
(which is currently used by most laboratories), sev-
eral have shown good correlation of p53 expression
with p53 gene mutation (10) and LOH at 17p (7).
Our study is slightly different from those previously
published in that we show a slightly higher fre-
quency of high-level accumulation of p53 in in situ
carcinoma than in invasive carcinomas. In contrast,
most previous studies have shown progressive
overexpression of p53 as dysplastic lesions progress
to invasion (5, 7). Possible reasons for this differ-
ence could include variability in the antibody clone
or immunohistochemical technique used, as well as
criteria for distinction of low-grade dysplasia from
carcinoma in situ. For example, it is possible that
some of the lesions we designated low-grade dys-
plasia might be termed carcinoma in situ by others,
FIGURE 4. Small cell carcinoma (A) demonstrating loss of pRB expression (B) and intact, diffuse p16 expression (C). Note the intact labeling of
endothelial cells for pRB in Figure 4B.
TABLE 2. Immunolabeling and K-ras Mutation Analysis: Summary
Invasive Carcinoma Cases
Dysplasia Only Cases
In Situ Component of
Non-Small Cell Carcinomas
CIS Flat Papillary
ND ? not determined.
* The data are expressed as the number of abnormal results/number of normal and abnormal results. These data do not include the non-small cell
carcinoma components associated with the small cell carcinomas.
Gallbladder Carcinoma Genetics (A. V. Parwani et al.) 305
which would reduce the frequency of p53 alter-
ations somewhat. Nonetheless, because of the con-
cordance between in situ and invasive carcinoma,
our results suggest the possibility that gallbladder
carcinomas may arise via two pathways, with those
arising from flat carcinoma in situ overexpressing
p53 and with those that do not being less likely to
overexpress p53. The high frequency of p53 overex-
pression that we observed in small cell carcinomas
of the gallbladder (83%) is similar to that reported
To our knowledge, this is the first study to assess
Dpc4 protein expression in non–small cell gallblad-
der carcinomas. The 18% frequency of loss of pro-
tein expression that we observed in non–small cell
carcinomas is concordant with results from our
previous studies of biliary tract carcinomas. Previ-
ously, we showed that loss of Dpc4 expression is as
frequent in distal (intrapancreatic) bile duct carci-
nomas (55%) as it is in pancreatic adenocarcinoma.
In contrast, the frequency of Dpc4 loss was lower
(overall, 15%) in more proximal (perihilar and in-
trahepatic) bile duct carcinomas (28). Interestingly,
we observed loss of Dpc4 protein in a similar per-
centage of in situ gallbladder carcinomas associ-
ated with invasive carcinomas (2 of 13, or 15%),
suggesting that DPC4 inactivation occurs early in
these lesions. The 18% frequency of Dpc4 protein
loss in these tumors may account for a subset of the
approximately 30% frequency of LOH (5, 7) that has
been reported at chromosome 18q in prior allelo-
typing studies of gallbladder carcinomas.
The significant role of p16 alterations in the
emerged in the recent literature. Although studies
have shown a rate of mutation that ranges from
30.7 (13) to 80% (12), LOH at the p16 locus on
chromosome 9p has been reported to range be-
tween 0 and 68% (7, 8, 12). To our knowledge, only
two other studies have evaluated p16 protein im-
munolabeling in gallbladder carcinomas. Shi et al.
(29) have recently shown loss of p16 expression in
75.7% of gallbladder carcinomas, whereas Kim et
al. (13) showed loss of labeling in only 23%. Our
study is concordant with the results of Shi et al.
(29) in that we saw frequent loss of p16 protein
expression (69%). It is interesting that the two
adenocarcinomas associated with small cell car-
cinomas in this study maintained p16 expression,
suggesting that they are biologically different
from pure adenocarcinomas.
We found pRB loss to be infrequent in invasive
non–small cell gallbladder carcinomas; in fact,
most tumors expressed pRB diffusely. Diffuse pRB
expression in these tumors likely reflects the se-
quelae of p16 inactivation, because p16 negatively
regulates cellular levels of RB (30). In fact, inverse
relationship of p16 and pRB has been demonstrated
in several malignancies including bladder carcino-
mas, lung carcinomas, pancreatic carcinomas, and
gliomas (30–32), and, most recently, gallbladder
carcinomas (29). In our series, the inverse relation-
ship was also apparent; no invasive carcinoma
demonstrated loss of both p16 and pRB, whereas an
inverse relationship (loss of p16 or Rb, but not both)
was evident in 25 of 30 tumors. One would not
expect inactivation of both of these tumor suppres-
sor genes within the same tumor, because they
each function in the same growth-regulatory path-
way, and therefore there would be no selective
pressure to inactivate both.
In contrast, we found that pRB loss was common
(4 of 6 cases) in the small cell carcinomas available
for study. Although pRB expression has, to our
knowledge, never been evaluated in small cell car-
cinoma of the gallbladder before, pRB loss is com-
mon in pulmonary small cell carcinomas (33) and
has been reported in small cell carcinomas in ex-
trapulmonary sites (34, 35). Hence, gallbladder car-
cinomas appear to fit the paradigm that small cell
(high grade) neuroendocrine carcinomas of any site
are consistently characterized by loss of pRB ex-
pression. It is noteworthy that the two cases that
retained pRB showed loss of p16, indicating that all
small cell carcinomas of the gallbladder target the
The role of K-ras gene mutation in gallbladder
carcinomas is not clear from the literature. Several
groups have considered K-ras mutations to be “rare
and late events” (7) in gallbladder carcinomas.
Wistuba et al. (36) found K-ras gene mutations in
only 1 of 21 gallbladder carcinomas, and the muta-
tion was confined to the poorly differentiated area
of this tumor. This same group found a higher
frequency of K-ras gene mutation (25%) in gallblad-
der adenomas and used this finding to support the
idea that adenomas are not the precursor of most
invasive carcinomas (36). In contrast, studies from
Japan have shown a higher frequency of K-ras gene
mutation in gallbladder carcinomas and occur-
rence in precursor lesions. For example, Ajiki et al.
(15) found K-ras gene mutations in 73% of gallblad-
der dysplasias and 59% of invasive carcinomas. The
presence of K-ras gene mutations does correlate
with the anatomic finding of anomalous junction of
the choledochopancreatic ducts (AJCPD) outside of
the ampulla of Vater, which is more common in
Japan. Hanada et al. (10) showed that the rate of
K-ras mutation was higher in gallbladder carcino-
mas of patients with AJCPD than in gallbladder
carcinomas of patients without AJCPD (50% versus
6%, P ? .05). Our study shows an intermediate rate
of mutation in our cohort of 30%. Two of our three
metastatic carcinomas harbored K-ras gene muta-
tions; however, we demonstrated a mutation in one
patient with pure flat carcinoma in situ, indicating
that this genetic alteration may be an early event in
In summary, our results with p53, p16, and K-ras
are consistent with those of previous studies in the
literature that have shown a significant rate of al-
terations in these genes in gallbladder carcinoma.
Loss of p16 expression and accumulation of p53 at
high levels are the most common abnormalities
associated with these carcinomas. Our study is the
first to show Dpc4 protein loss in gallbladder car-
cinoma. We also show that loss of pRB expression is
rare in non–small cell gallbladder carcinoma but is
common in small cell carcinoma of the gallbladder.
Indeed, the p16/pRB pathway was inactivated in all
six small cell carcinomas studied. Perhaps most
important, using immunohistochemistry, we were
able to localize all of these alterations to the stage of
carcinoma in situ. Indeed, of 15 invasive carcino-
mas associated with an evaluable in situ compo-
nent, there was complete concordance between the
results in 13 cases. Along these lines, one case of
pure in situ carcinoma in our study contained three
distinct alterations: p53 accumulation, p16 loss,
and K-ras gene mutation.
Hence, carcinoma in situ of the gallbladder, al-
though clinically occult, is a genetically advanced
lesion. These results suggest that patients with pure
carcinoma in situ of the gallbladder should receive
careful clinical follow-up, particularly if the status
of the cystic duct margin is unclear. At the same
time, these results highlight our ignorance of mech-
anisms and markers of progression in this lethal
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Nuland SB: Lost in America: A Journey with
My Father, 212 pp, New York, Alfred A.
Knopf, 2003 ($24.00).
At the moment, Americans are intrigued with the
childhood struggles of other people, especially if
they are immigrants (or first generation prod-
ucts) from Ireland or holocaust-affected regions
of Europe. In spite of all the family deformations,
the narrators typically end up with successful
careers and fulfilled lives. And good writers from
the medical ranks enjoy a special regard within
this genre because their readers assume an
added and special dimension, that these authors
understand the chemistry and physics that make
people tick. Sherwin B. Nuland, M.D., an emeri-
tus clinical professor of surgery at Yale Univer-
sity, has already published several successful
socio-medical books, and his Lost in America is
bound to find a wide audience.
This short book can be pleasantly consumed
in a weekend. It is a completely factual account,
to the extent that there is no declaration other-
wise, even with regard to possible name changes
for peripheral characters such as Nuland’s girl-
friends. It reads like a novel in terms of gripping
opening, tension, and skillful timing in the re-
lease of information, all presented in a smooth
narrative style. Notwithstanding the title, the dis-
course is primarily autobiographical. His father,
Meyer Nudelman, was a Russian Jew who arrived
in 1907, on his own, and was never assimilated in
terms of American culture or even language. Sh-
erwin and his older brother Harvey officially
changed the spelling of their surname while the
author was in high school. After the premature
death of their mother, the boys were nurtured by
a maternal aunt, influenced by their maternal
grandmother, embarrassed and needled by their
father, all living in the same Bronx apartment.
The interactions are at times novel, pitiful, sol-
emn, wrenching, and even humorous, but never
boring. The climacteric of Meyer’s illness is ex-
Many of us would have been interested in
some family portraits, but there is no illustration
except for the unidentified, partial figures on the
dust-cover, presumably Sherwin and Meyer.
There are some troublesome paradoxes with re-
gard to medical education and practice. For in-
stance, Nuland dismisses the basic sciences (bio-
chemistry in particular) as being of minor
consequence to a medical education. He thus
joins the throng of naive freshman, but it does
not sit well with his more mature mantra about
being totally prepared to understand the patient,
at all levels.
Dr. Nuland tells us that he was driven to
write this book by an attempt to evaluate the
relationship with his departed father. He elects
not to analyze the overall thesis, ‘I will never
know the cost of being Meyer’s son.‘ We are left
to reach our own conclusion, and that is part of
the charm of his contribution.
Wilfred Niels Arnold
University of Kansas Medical Center
Kansas City, Kansas
308 Modern Pathology