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Annals of Oncology 10 Suppl. 4: S300-S304, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Review.
Biliopancreatic malignancy: Future prospects for progress
Jenny J.L. Ho & Young S. Kim
Gastrointestinal Research Laboratory (151 M2), Veterans Medical Center and Dept. of Medicine, University of California, San Francisco, California,
U.S.A.
Summary
Several key areas are targeted by novel therapies. Growth factors
and their receptors are overexpressed in a high percentage of
pancreatic tumors. These factors are critical to tumor cell growth
and development. They also promote tumor growth by stimulating
angiogenesis. Mutations in other molecules that regulate cell
growth, such as the ras protein and the tumor suppressor p53,
contribute to a state of continously stimulated cell proliferation.
Other types of molecules such as mucins are also altered or
overexpressed in tumors. Mucins are immunosuppressive and are
important in tumor cell metastasis.
A number of promising new therapeutic strategies are now being
tested. Ribozymes or antisense nucleic acids can prevent synthesis
Introduction
The pancreas, liver, gallbladder and bile ducts share a common
embryonic origin. These organs also resemble each other in another
respect. Patients with cancers of these organs have extremely poor
5-year survival rates. At this time the best hope for patient survival
is still early diagnosis followed by surgical intervention.
Conventional chemotherapy and radiation have had very little
impact on mortality rates over the past decade, especially in those
patients with late stage disease. Recent advances in the following
four fronts could have significant impact on future mortality rates:
1. Identification of risk factors
2. Early diagnosis
3. Creation of additional candidates for curative resection
4. Prevention, identification and treatment of metastases
Identification of risk factors
Identification of risk factors serves two purposes. The first
purpose is to allow us to design public education programs
that will focus on prevention. The second purpose is to
pinpoint the population that would most benefit from
periodic testing. Because the incidence of pancreatic cancer
is so low large scale screenings are not practical with
currently available tests. A screening of 10,162 persons over
40 years of age conducted in Japan found only four cases
(0.04%) of the disease [1]. The identification of the risk
factors and genetic alterations that are associated with
biliopancreatic cancers, and especially those factors that act
during the early stages of tumorigenesis, will assist in
identifying those who are at greatest risk.
Although a family history of pancreatic adenocarcinoma is
not common in patients with pancreatic cancer they are
statistically more likely than controls to have a close relative
with pancreatic cancer. 5-10% of patients have a first-degree
relative with the disease [2]. This suggests that in familial
of growth factor receptors or ras protein. Monoclonal antibodies
block interaction between receptor and its ligand. Newly developed
drugs prevent tyrosine phosphorylation of growth factor receptors
or famesylation of ras protein. Gene therapy is another approach
that is under investigation. Transduction or transfection of genes for
wild-type tumor suppressors could correct defects in growth
regulation. Vaccines developed against tumor antigens provide
hope for the control of not only the primary tumor, but also of the
metastatic lesions as well.
Key words: gene therapy, immunotherapy, risk factors, signal
transduction
pancreatic cancer the predisposition for the disease is
inherited through the germline. A detailed examination of the
genetic history of family members may provide information
on the process of tumorigenesis. Although the gene(s)
associated with familial pancreatic cancer is(are) not yet
known there is information about the genes that are probably
not involved. The incidence of germline mutations in DPC4
[2] or K-ras [3] in patients with a family history of cancer is
not significantly different from that of patients with no
family history. Expression of p21WAF-l and HER-2/neu in
these patients was also not significantly different from those
with no family history [3]. However, there was a
significantly lower incidence of p53 expression [3]. It is
known that certain other inherited disorders predispose
individuals to pancreatic cancer. One of these is hereditary
pancreatitis [4]. Other conditions are reviewed by Lumadue,
etal [4].
The rate of pancreatic cancer increases with age. About
80% of the cases occur between ages 60 and 80 [5].
Occurrence in individuals less than 40 yrs of age is rare.
This has socioeconomic implications in countries with aging
populations. As the proportion of the population over 60 yrs
of age grows we may expect to encounter an increase in the
incidence of pancreatic cancer. In the United States the
incidence is higher in African-Americans than in whites.
There is a higher incidence in men than in women, in both
the African-American and white populations. However,
there has been an increase in the incidence and mortality rate
in American women since 1974 [5].
Certain of the risk factors associated with lifestyle are
amenable to change. Cigarette smoking is one of the best
substantiated behavioral risk factors associated with
pancreatic cancer [5]. A recent report done on Swedish
women is of interest [6]. It showed an apparent inverse
relationship between age at first birth and the risk of
301
pancreatic cancer. However, the authors noted that young
age at first birth in Sweden may be associated with increased
frequency of smoking. The increase in incidence of
pancreatic cancer in American women that was noted above
may be related to a general increase in the rate of smoking in
American women. There is a positive association of
pancreatic cancer with consumption of salted food [7] and of
red meat [5]. Association with either alcohol or coffee
consumption is somewhat ambiguous [5, 8]. On the other
hand, consumption of whole grain/high fiber diets [9] and of
fruits and vegetables [5,10] are associated with reduced risk.
Recently, there has been considerable interest in the
protective components of green tea [11]. Public
announcements about the connection between lung cancer
and smoking have resulted in a drop in the number of
Americans smokers. Reinforcing the public's awareness of
the importance of diet and smoking in the etiology of many
types of diseases is a cost-effective means of prevention.
Early diagnosis
Because of the non-specific and ambiguous nature of the
symptoms associated with pancreatic and biliary tract
cancers the patient usually does not come into contact with
the physician until the disease is well advanced. Serum based
immunoassays of mucins (such as CA19-9 and SPan-1) and
other tumor antigens showed initial promise for early
diagnosis but have now been demonstrated to lack sensitivity
and specificity [12,13]. They retain usefulness in follow-up
and assessing prognosis. The lack of specificity of many of
the present tests results, in part, from the presence of the
antigens in normal pancreas. For example, the CA19-9 and
SPan-1 tests identify carbohydrate structures on the MUC1
type of mucin [14], a type of mucin that is prevalent in
normal pancreas. Recently, we have shown that another type
of mucin, MUC3, is present in very low levels in normal
pancreas [15]. Its expression is greatly increased in
pancreatic tumors. Part of the MUC3 cDNA sequence was
determined with anti-sera directed against deglycosylated
pancreatic cancer mucins [16]. The preferential expression
of MUC3 in tumors shows promise for a more specific
diagnostic test.
Currently there is a large body of literature on the
identification of K-ras and p53 gene mutations in body
fluids. Up to 95% of exocrine pancreatic tumors contain
mutations of the K-ras gene at codon 12 [ 17]. These types of
mutations occur early in pancreatic carcinogenesis in an
animal model [18]. Mutations are also present in 75% of
small human pancreatic tumors (1.2-3 cm; [19]) and 75% of
intraductal lesions, thought to be precursors of adenocarcinomas [20]. K-ras mutations are also detectable in bile
of 79% of patients with biliary duct cancer but not in
patients with gallbladder cancer or benign biliary diseases
[21]. Of those patients who had no apparent malignant cells
in their bile upon cytological examination 71% had K-ras
mutations of DNA in their bile as detected by mutant-allelespecific-amplification [21]. Detection of mutations in K-ras
in DNA found in peripheral blood [22] and stool [23] may
be the basis of simple non-invasive screening tests.
However, caution needs to be taken in the interpretation of
a positive test, because of the presence of K-ras mutations in
benign pancreatic conditions [24].
Creation of additional candidates for curative resection
Curative resection of small localized tumors is still the best
hope for survival. Recently, the operative mortality rate has
decreased significantly [25]. However, most patients are not
good candidates for resection at presentation. Some of these
individuals have large invasive tumors. Also surgery does
not deal with distant or undetected metastases and residual
tumor cells that could lead to relapse. Conventional
neoadjuvant treatments have not significantly altered the
proportion of patients that will ultimately be able to undergo
curative resection. It may be that novel therapeutic
approaches will succeed in converting additional patients to
resectability. Application of some of the more recent
advances in gene therapy, chemotherapy based on chemicals
that specifically target signal transduction pathways and
immunotherapy may provide new hope in improving
mortality rates.
What are some of the key sites that are being targeted with
these new therapeutic modalities? The importance of
vascularization to the growth of pancreatic (reviewed by
Pluda and Parkinson, [26]) and other types of tumors has
gained considerable public attention recently. Angiogenesis
is also important to tumor cell dissemination [27]. In
addition, several steps in the regulation of tumor cell
function have now been identified as being aberrant in
pancreatic tumor cells. In particular, these include the
overexpression • of the epidermal growth factor (EGF)
receptor family [28] including erbB-2/neu [29]. As noted
above mutations in condon 12 of K-ras occur frequently in
pancreatic tumors. Mutations in K-ras often lead to the
constant activation of the ras protein. Mutations in the tumor
suppressor p53, an important factor in cell cycle control, also
occur frequently in pancreatic tumors [30]. Mucins are also
an important therapeutic target. They inhibit the cells of the
immune system and participate in the process of metastasis
(reviewed by Ho and Kim, [31]. Their expression on the
surface of tumor cells makes them ideal antigens to be
targeted by monoclonal antibodies and vaccines.
Results from studies, many of which have been performed
on animal models, suggest that inhibition of tumorassociated angiogenesis will result in tumor regression.
Vascular endothelial growth factor (VEGF) is one of the
major angiogenic inducers [32]. One strategy to negate the
influence of VEGF has been to transfect anti-sense VEGF
cDNA. Melanoma cells transfected with the anti-sense
cDNA formed small tumors with little vascularization and
extensive necrosis in nude/SCID mice [33]. Moreover, antisense transfected cells formed few lung colonies. On the
other hand, melanoma cells transfected with sense VEGF
formed well vascularized tumors and numerous tumor
colonies in the lung. Other forms of gene therapy that target
angiogenesis are reviewed by Kong and Crystal [34].
Another means of inactivating VEGF is through the use of
a neutralizing antibody. Such an antibody inhibited both
primary and metastatic tumor growth of fibrosarcoma cells
in a nude mouse system [35]. Endostatin and angiostatin are
two antiangiogenic peptides that are effective in controlling
tumor growth. Endostatin is a 20 kilodalton portion of theCterminus of collagen XVIII. E. coli derived endostatin
caused primary tumors to regress to dormant microscopic
lesions [36]. Angiostatin is an internal fragment of
302
plasminogen. Recombinant angiostatin suppressed growth of
primary and metastatic Lewis lung carcinoma tumors in an
animal system [37].
Several approaches have been used against the EGF family
of receptors. Transfection of a ribozyme to specifically
cleave receptor mRNA [38] reduced the level of the
receptor. The receptor tyrosine kinase can be inactivated by
specific inhibitors [39]. Application of anti-growth factor
receptor monoclonal antibodies has been reviewed by Fan
[40]. Recently, the antibody, Herceptin [41], which is
directed against ErbB-2/neu (HER-2), has recently been
approved by the FDA for the treatment of advanced breast
cancer. An antibody against the EGF receptor induced
antibody-dependent cellular cytotoxicity (ADCC) in
pancreatic cancer patients [42]. Cells expressing a particular
receptor can also be destroyed by targeting that receptor with
a recombinant form of the ligand that had been modified by
inclusion of a protein toxin [43]. Inhibition of the activity of
the EGF receptor tyrosine kinase not only slows growth of
colonic tumor cells but it can also induce apoptosis of the
cells [44]. Neutralizing antibodies that block interaction
between EGF or erbB-2/neu ligand and their receptors also
down-regulated expression of VEGF in human epidermoid
carcinoma cells [45]. Treatment with a monoclonal antibody
can also improve the effectiveness of conventional
chemotherapy. For example, treatment with Herceptin
enhanced the anti-tumor activity of paclitaxel and
doxorubicin in human breast cancer xenografts [41].
It is attractive to theorize that the replacement of a defective
tumor suppressor genes or the blocking of a constantly active
gene product will be sufficient to reverse the malignant
characteristics of a tumor cell, if not kill it. However, it must
be remembered that cells that can be identified as being
malignant already have a number of genetic mutations [46].
For example, in one study all cases of pancreatic cancers that
contained p53 mutations also had codon 12 K-ras gene
mutations [30]. On the other hand, gene replacement may
also have a bystander effects. Replacement with wild-type
p53 by transduction with a viral vector into colonic cancer
cells decreased expression of RNA for VEGF [47]. The
reduction in VEGF expression was dose dependent. Several
approaches have been tested to alter the effects of K-ras and
p53 mutations: transfection of anti-sense K-ras gene
fragment into pancreatic cancer cells [48]; administration of
a viral vector bearing the wild-type p53 gene into the
peritoneum of nude mice to inhibit growth of primary
pancreatic xenograft tumors and to reduce peritoneal tumor
deposits [49]; and transduction of pancreatic cells with the
p21WAFl gene, the product of which is a downstream
effector of p53 function [50]. Famesylation of ras is required
for its activity. Inhibitors specific for farnesyltransferase are
another means of preventing ras activity [51]. Pancreatic
cancer patients have been vaccinated with mutant ras
peptides [52]. Vaccinated patients exhibited a transient Tcell response. p53 is also a target for vaccine development
[53].
Immunotherapy has always had appeal because immune
recognition is highly specific. Antibodies and immune cells
will target only those cells presenting a particular antigen,
which ideally are only the tumor cells. Immune responses are
effective against the metastases (known and unknow) as well
as against the primary tumor. There has been a recent study
using I31I labeled monoclonal antibodies for the treatment of
patients with non-resectable liver metastases of colon cancer
cells [54]. Two main obstacles to the effectiveness of
antibodies in the past have been the delivery of a sufficient
effective dose and, paradoxically, the extreme specificity of
the antibodies. In the first case, delivery of a large number
of macromolecules such as antibodies to tumor cells is
limited by barriers imposed by the blood supply, vessel wall,
and interstitium [55]. A growing understanding of these
barriers will help in the design of new delivery methods. A
recent detailed analysis of the killing effectiveness of
immunoconjugates to toxins showed that as few as 1000
molecules of immunotoxin per cell were necessary to kill
tumor cells [56]. Thus immunotoxins can be effective even
at relatively low doses.
Monoclonal antibodies can also be too specific. Because of
tumor cell heterogeneity not all cells in a tumor will express
the targeted antigen. One means of circumventing this
problem is with the use of immunoconjugates to high energy
radioiostopes such as I3II [54,57]. These radioisotopes will
affect neighboring cells as well as the targeted cells.
Antibody-directed enzyme prodrug therapy (ADEPT), where
a prodrug is administered systemically to be activated by
enzymes that have been pre-targeted by antibodies [58],
results in a local production of active drug. As
concentrations build in the interstitium the drug will also
affect neighboring cells not targeted by antibody/enzyme.
We are in the process of determining if increasing the level
of a targeted antigen (Nd2 mucin-associated antigen) and the
number of cells expressing that antigen [59] will enhance
antibody binding and uptake. Up-regulation of EGF receptor
enhanced antibody dependent cell mediated cytotoxicity
(ADCC) in one recent clinical study of pancreatic cancer
patients [42].
There is considerable interest in involving the patient's own
immune system in the process of tumor control. On the one
level there are bispecific antibodies that react with both a
tumor antigen and an antigen present on cells of the patient's
immune system. One study examined bispecific antibodies
that target MUC1 mucin and a surface antigen of killer cells
[60]. These antibodies effectively mediated interaction
between bile duct carcinoma cells and lymphokine activated
killer cells. Another means of targeting lymphokine activated
killer cells to bile duct cells was to chemically conjugate
staphylococcal enterotoxin A to a MUC1 specific antibody
[61]. It has also been observed that human/murine chimeras
of monoclonal antibodies directed against mucins are much
more effective as mediators of ADCC than the native murine
antibody [62, 63].
Another means of involving the patient's immune system is
by vaccines. Vaccine targets include mutated oncogenes [52,
53] and mucins. Mucin carbohydrates [64], protein [65] and
DNA [66] have been used. Recently, the dendritic cell was
demonstrated to be a highly effective antigen-presenting cell
(reviewed by Pardoll, [67]). In addition, they also express
high levels of molecules that activate T-cells. Dendritic cells
pulsed in vitro with a variety of antigens have proved
effective in producing specific anti-tumor effects in vivo,
including the inhibition or regression of metastatic tumors
[68,69]. Use of whole tumor cells or lysates, as opposed to
specific tumor antigens, circumvents the problem of tumor
cell heterogeneity. Tumor cells secreting cytokines such as
303
granulocyte-macrophage colony stimulating factor (GMCSF) are very effective in inducing an immune response
(reviewed by Pardoll, [67]). One of the actions of GM-CSF
is to promote dendritic cell differentiation at the site of
vaccination. Recently, a number of pancreatic cancer cells
lines have been developed that stably express GM-CSF [70].
Because these types of cells can be expanded indefinitely
they will be useful as a continuing source of target antigens
for vaccine development.
Identification, prevention, and treatment of metastases
As more becomes known about what distinguishes a
pancreatic metastasis new diagnostics tests can be designed.
One study reports that the immunophenotype of metastases
of epithelial malignancies closely resembles that of the
primary tumor [71]. In an animal model differences were
rare in lymph node and liver metastases of pancreatic tumor
cells, although they occurred more frequently in peritoneal
metastases [72]. Detection of pancreatic metastases in
regional lymph nodes by polymerase chain reaction that
targets K-ras mutations in codon 12 has been reported [73].
Detection of breast cancer micrometastases in axillary lymph
nodes by reverse transcriptase-polymerase chain reaction
targeted MUC1 mucin mRNA [74]. As discussed above
MUC1 is also an applicable antigen for the pancreas and bile
duct. Because other types of mucins such as MUC3 are
expressed atypically in primary pancreatic tumors [15] they
may also be appropriate target antigens to identify pancreatic
metastases.
In breast cancer the number of microvessels is an
independent predictor of metastatic disease [27].
Angiogenesis forms highly permeable new vessels that can
be easily penetrated by tumor cells. Novel anti-angiogenic
treatment discussed in an earlier section may also be
effective in reducing the likelihood of metastases and also
their growth if established. Hepatic metastases is a common
cause of treatment failure after curative resection in
pancreatic cancer. One major challenge is to prevent their
establishment. Carbohydrate structures of mucins on the
surface of tumor cells act as ligands in the metastasis of
pancreatic tumor cells (reviewed in ref. [31]). The principal
ligand is the sialylated Lewis' structure. Antibodies that
recognize this epitope, such as 19-9 and Span-1 [14], may be
useful in reducing liver metatases. Such an antibody has
been shown to be effective in reducing the development of
liver metastases of pancreatic tumor cells in an animal model
[75]. Regional delivery of cytotoxic drugs is another strategy
to prevent establishment and growth in new sites such as the
liver and peritoneal cavity. In one clinical study 5fluorouracil was continuously infused through the hepatic
artery and portal vein for 28-35 days [76]. This treatment
greatly improved 3-year patient survival and reduced the rate
of hepatic metastases. In an animal study a retroviral p53
vector was injected into the peritoneum of nude mice. This
resulted in a significant inhibition of peritoneal tumor
deposits of Bxpc3 pancreatic cancer cells [49]. In another
example of regional gene therapy a thymidine kinase
retroviral vector was injected into the peritoneum of nude
mice with established peritoneal metastases of human
pancreatic cancer cells [77]. This enzyme makes tumor cells
susceptible to ganciclovir. Subsequent administration of
ganciclovir resulted in a reduction of the mass of tumor
deposits.
In an animal model of liver metastases mice were vaccinated
with dendritic cells prior to the intrasplenic injection of
tumor cells [67]. The dendritic cells impeded the
establishment and growth of liver metastases. This occurred
even when the dendritic cells were not pulsed with tumor
cell lysate. However, the effect was greater in mice receiving
dendritic cells that had been pulsed with tumor cell lysates.
A recent clinical study of melanoma patients with metastatic
disease patients were vaccinated with irradiated autologous
melanoma cells engineered to secrete human GM-CSF [68].
Metastatic lesions resected after vaccination were infiltrated
with T lymphocytes and plasma cells, which was not the case
prior to vaccination. In addition, there was extensive tumor
destruction and fibrosis.
Acknowledgements
This study was supported in part by the Veterans Affairs
Medical Research Service and USPHS Grant CA 24321
from the National Cancer Institute.
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Correspondence to:
Jenny J.L. Ho, Ph.D.
Gastrointestinal Research Laboratory (151 M2)
Veterans Affairs Medical Center
San Francisco, CA 94121
U.S.A.