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CH 1302003702
change or only that infrequently was an increase seen (20, 29, 37– 41). of the IFN-␥ gene was produced using the following primers: sense, TCTTT-
Therefore, the current study was designed to examine whether TCTTTCCCGATAGGT; antisense, CAGGGATGCTCTTCGACCTC. Both
HER-2/neu gene amplification and/or overexpression occurred in fragments were amplified in the same vial using the following conditions:
gastrinomas, which are the most common functional malignant 94°C for 10 min and 35 cycles of 94°C, 50°C, and 70°C for 1 min each in a
PET (9, 42). Sufficient numbers of cases were included, which had thermocycler (9700; Perkin-Elmer Corp.). Bands were separated on 10%
polyacrylamide gels, stained with ethidium bromide, and band intensities were
been followed long term to allow correlations with tumor growth
measured with an image analysis program (NIH-IMAGE).4
patterns. Such correlations allowed us to assess whether levels of
RNA Quantitation. Tumor RNA was extracted from 8-m cryosections of
expression of this proto-oncogene might be useful either prognos- the specimens using a commercial kit (RNeasy Mini kit; Qiagen Inc.) after
tically for identifying patients with aggressive disease or possibly analyzing an adjacent slide with H&E staining to determine that the slides
for identifying a subset that possibly might benefit from trastu- contained ⬎80% tumor tissue. Purified pancreas RNA was purchased from
zumab treatment. Clontech (Clontech, Palo Alto, CA), and RNA was isolated from four breast
cancer cell lines SK-BR-3, BT474, MDAMB453, and BT 483 (American Type
Culture Collection) using a commercial kit (Trizol; Life Technologies, Inc.
MATERIALS AND METHODS
Grand Island, NY). Random hexamer-primed first-strand cDNA was prepared
Patients with reverse transcription (RNA PCR kit; Perkin-Elmer, Foster City, CA). The
integrity of the cDNA was assessed by detecting a diffuse smear from 0.6 to
Briefly, 8-m thick paraffin sections were mounted on plain glass slides. After
deparaffinization and rehydration the slides were placed in a microwave
pressure cooker in 0.01 M citrate buffer (pH 6.0; Biogenex, San Ramon, CA.)
containing 0.1% Tween 20 and heated in a microwave oven at maximum
power (900 W) for 40 min. Sections were immediately cooled in Tris-buffered
saline (0.05 M; pH 7.6). Thereafter, all of the sections were washed in
Tris-buffered saline (pH 7.6) containing 5% goat serum (Life Technologies,
Inc.) for 30 min. Slides were incubated with the primary polyclonal antibody
A0485 (Hercept; Dako Corp.) in a dilution of 1:500 for 12 h at room
temperature. The rest of the procedure (secondary antibody, avidin-biotin
complex, color development, and counterstain) was performed on the Ventana
immunostainer.
RESULTS
Table 1 Clinical and laboratory characteristics of patients studied a primary tumor localized. During the postoperative follow-up of
Characteristic Number (%) 3.1 ⫾ 0.4, one-half the patients remained disease free (Table 1). Of the 22
Patients 43 patients that were not disease-free after the resection almost two-thirds of
Male gender 27 (62%) the tumors did not show any growth during the postoperative follow-up.
Age (yrs)a
Mean ⫾ SE 48.2 ⫾ 1.5
In 40% of the patients not cured the tumors displayed growth, and the
[Range] [15–62] tumor in 3 of these patients demonstrated aggressive growth with the
Duration of diseaseb development of liver metastases during the postresection follow-up time
Mean ⫾ SE 8.9 ⫾ 1.1
[Range] [0.2–35] (Table 1).
Preoperative fasting serum gastrin (pg/ml) DNA was extracted from all 43 of the tumors (the primary or a
Mean ⫾ SE 5151 ⫾ 260 regional lymph node metastasis), leukocytes from 17 of these patients,
[Range] [87–110000]
Median 746 and from the breast cancer cell line SK-BR-3, which is reported to
Preoperative BAO (mEq/h)c have amplification of the HER-2/neu gene copy number (54 –57) for
Mean ⫾ SE 44 ⫾ 3.6
[Range] [10–92]
differential PCR of the HER-2/neu and IFN-␥ genes. Fig. 1 (left
Preoperative MAO (mEq/h)c panel) shows an example of the differential PCR result from 4 patients
Mean ⫾ SE 69 ⫾ 5.4 and from the SK-BR-3 breast cancer cells. Differential PCR demon-
[Range] [23–135]
Primary tumor locationd strated a 3-fold amplification of the HER-2/neu oncogene over the
Duodenum 18 (42%) IFN-␥ gene in the breast cancer cell line SK-BR-3 (Fig. 1, top left
Pancreas 8 (19%) panel). However, in the 4 gastrinomas (Fig. 1, middle left panel) the
Lymph noded 10 (23%)
Othere 7 (16%) intensity of the HER-2/neu band was less than that of the IFN-␥ band;
Surgical outcome therefore, the HER-2:IFN-␥ ratio was ⬍1, indicating no amplification
Disease-free 21 (49%)
Not disease-free 22 (51%)
of the HER-2/neu oncogene relative to the IFN-␥ gene in any of the
Tumor growth patternf gastrinomas if this criteria of overamplification is used (58 – 61). In
No growth 13 (60%) the WBCs from the same 4 patients (Fig. 1, bottom left panel),
Growth 6 (27%)
New liver metastases 3 (14%) similarly no amplification of the HER-2/neu oncogene over the IFN-␥
a
Age at time of surgery. gene was seen using a ratio ⬎1. In the first 17 patients both the
b
Duration of disease is defined as time from onset of persistent symptoms compatible gastrinoma and WBC HER-2:IFN-␥ ratio were determined using
with gastric acid hypersecretion to surgery (7). differential PCR, and the presence of the two genes was not signifi-
c
BAO and MAO are from the 36 patients without previous acid-reducing surgery.
d
Primary tumor location was determined during surgery (46). Lymph node primary cantly different in both tissues (tumor versus WBC: 0.60 ⫾ 0.03
indicates a gastrinoma resected in lymph node only and a disease-free status postopera- versus 0.62 ⫾ 0.02; Fig. 1, right panel). Using the SD of the
tively (92).
e
Other primary tumor locations include: heart (n ⫽ 1), bile ducts (n ⫽ 1), liver (n ⫽ 3),
HER-2:IFN-␥ ratio for the leukocytes in these 17 patients a 97.5%
and unknown (n ⫽ 2). confidence interval was calculated (Fig. 1, right panel). Gastrinomas
from 6 patients had a significantly higher HER-2:IFN-␥ ratio than
f
Tumor growth pattern was defined by annual evaluations with imaging studies (17)
(computed tomography, magnetic resonance imaging, and SRS (Somatostatin receptor
scintigraphy). Growth was defined as an increase in size of lesion or number of imaging the ⬎ mean ⫹3 SD of the leukocytes (i.e., ⬎0.86; P ⬍ 0.025; Fig. 1,
lesions as defined in “Materials and Methods” (17). right panel). In none of the tumor specimens was the ratio ⬎1.5-times
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HER-2/NEU EXPRESSION IN GASTRINOMA
the ratio in nontumor cells, ⬎1, and in no patient gastrinoma did the
ratio approach the 3.2-fold amplification seen in the SK-BR-3 breast
cancer cell line (Fig. 1, right panel).
RNA of sufficient quality and quantity for amplification of the
HER-2/neu and -actin transcripts was available from gastrinomas
from 33 patients. Fig. 2 shows an example of the competitive PCR for
the determination of the HER-2/neu transcript amount from 2 patients.
Where the intensity of the ethidium bromide staining of the upper
band is calculated to equal that of the lower band is the amount of
molecules in the unknown sample. A similar competitive PCR was
performed to determine the amount of -actin in each sample. Using
the dilution of the sample added, the number of molecules of
HER-2/neu and -actin were determined in all of the samples and
expressed as a ratio (i.e., neu:-actin ratio; Fig. 3). The amount
detected in the gastrinomas varied over a 700-fold range from 0.00029
to 0.19 with a mean of 0.0193 ⫾ 0.007 neu molecules per -actin
molecule (Fig. 3). In tissue from 4 normal pancreas the ratio for
HER-2/neu over -actin expression varied over a 4.5-fold range from
0.0275 to 0.125 with a mean of 0.075 ⫾ 0.032 HER-2/neu molecule
per -actin molecule (Fig. 3, middle column). In contrast, in the four
breast cancer cell lines known to overexpress HER-2/neu (SK-BR-3,
BT474, MDA-MB453, and BT483; Refs. 54, 56, 62) significantly
more HER-2/neu molecules per -actin molecule were found com-
pared with two cell lines not overexpressing HER-2/neu (BT20 and
MDA-MB468; Refs. 54, 62; Fig. 3, right column). When a 95%
coefficient interval for the relative expression of HER-2/neu com-
pared with -actin was calculated from normal pancreas only 1of 33
(3%) gastrinomas exceeded this range (Fig. 3, left panel).
HER-2/neu overexpression is known to correlate with tumor ag-
gressiveness, prognosis, and invasiveness in some endocrine (thyroid Fig. 3. Distribution of the amount of HER-2/neu mRNA present in gastrinomas, normal
cancer and pheochromocytomas) and nonendocrine (breast, gastric, pancreas, and positive controls. Quantitative PCR results were obtained from gastrinomas
from 33 patients (F; left column), four normal pancreas (E), and six breast cancer cell
pancreatic, and esophageal) cancers (23, 25, 26, 30, 31, 33). There- lines (BT 20, MDAMB468, SK-BR-3, BT474, MDAMB453, and BT 483; right column).
fore, we compared the amount of HER-2/neu expression with the Four cell lines (SK-BR-3, BT474, MDAMB453, and BT 483; half-filled squares) are
presence or absence of various clinical, laboratory, and tumoral fea- reported to overexpress HER-2/neu and two cell lines (䡺) not to overexpress the
HER-2/neu gene (54, 56, 62). The horizontal line with vertical bars represents the mean
tures reported to correlate with malignant behavior of gastrinomas for the neu:-actin ratio determined by quantitative PCR in the gastrinomas; bars, ⫾ SE.
and/or PETs (Refs. 1, 6, 7; Fig. 4). No clinical (gender, curability, and The 䡠䡠䡠 is the 95% confidence interval (mean ⫹2 SD) for the normal pancreas (i.e., 0.185).
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HER-2/NEU EXPRESSION IN GASTRINOMA
breast cancer cells (Fig. 5, top left). In the normal pancreas removed in other cancers with HER-2/neu amplification/overexpression (25,
during surgery from one of the patients with a gastrinoma, a few 35, 65).
normal islet cells stained with the antibody (Fig. 5, bottom left). This Our results demonstrate that HER-2/neu gene amplification is un-
indicated that our tissue samples still contained the HER-2/neu anti- common in gastrinomas, and when it occurs it is only a minimal
gen and therefore any lack of immunostaining in gastrinomas was not increase compared with the magnitude of the overexpression in other
likely because of damage to the tissue, which can occur during storage tumors. Differential PCR was used, which is one of the established
(37, 39, 64). In the normal pancreas some of the islet cells and entire methods to detect amplification of the HER-2/neu oncogene (47, 58,
exocrine pancreas were devoid of any positive HER-2/neu staining 66). Using this method, none of the 43 gastrinomas in the present
(Fig. 5, bottom left). Similarly, in each of the 10 gastrinomas exam- study demonstrated amplification of the HER-2/neu oncogene to a
ined no immunohistochemical staining with the HER-2/neu antibody level greater than the single copy IFN-␥ gene in any of the tumors.
could be detected in the tumor. Examples of two of the gastrinomas However, in 14% of the tumors (i.e., 6 of 43) the ratio of HER-2/neu
with negative staining are shown in Fig. 5 (right column). Two oncogene to IFN-␥ exceeded by 3 SDs the mean of the ratio for the
gastrinomas that had slightly elevated HER-2/neu:IFN-␥ ratios leukocyte DNA of the patients (P ⬍ 0.025), suggesting mild ampli-
(Patients 38 and 40) were included in those studied by immunocyto-
fication of the HER-2/neu oncogene was present in these gastrinomas.
chemistry and each was negative for HER-2/neu staining in the tumor.
This level of HER-2/neu amplification is much less than the 2 to
⬎30-fold reported in gastric cancer (67), breast cancer (54, 58 – 60,
DISCUSSION 68), urinary bladder cancer (57, 61), and ovarian cancer (69). Fur-
The purpose of the present study was to assess the frequency of thermore, using the criterion of at least a 2-fold amplification, which
amplification of HER-2/neu gene and possible overexpression of its is used in many other studies for a positive result for HER-2/neu gene
products in gastrinomas. The identification of a subset of patients with amplification (58 – 61, 69), our findings in gastrinomas would be
gastrinomas overexpressing either the HER-2/neu gene and/or its interpreted as none of the gastrinomas showing significant HER-2/neu
product might be clinically useful for management of these patients. gene amplification.
HER-2/neu amplification/overexpression might prove to be an Our results differ from one study (40) that reported all 12 of the
independent predictor of the growth behavior of the gastrinoma as it gastrinomas studied demonstrated HER-2/neu gene amplification (2–
has for a number of nonendocrine tumors (23, 25, 26). Secondly, 12-fold). However, they are in agreement with three other studies
HER-2/neu amplification/overexpression could provide the basis to (70 –72) involving small numbers of gastrinomas. In each of these
consider treatment of this subset of patients with the HER-2/neu latter three studies (70 –72) comparative genomic hybridization was
humanized monoclonal antibody, trastuzumab. Trastuzumab, either used and identified no overexpression on chromosome 17q in gastri-
alone or in combination with other antitumor agents, is currently being nomas, the location of the HER-2/neu gene. Similarly, in a study of 18
used in patients with breast cancer (23, 34) and considered for use midgut carcinoids, which have many similarities to PETs, by com-
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HER-2/NEU EXPRESSION IN GASTRINOMA
parative genomic hybridization, 0% demonstrated overexpression on other endocrine cancers [papillary thyroid, 52–100% (33, 75, 84 – 86);
17q (73). Our results in gastrinomas are similar to results with other and pheochromocytomas, 48 –79% (29, 87)] and common nonendo-
endocrine tumors, which demonstrate no HER-2/neu gene amplifica- crine tumors [esophageal, 23% (25); gastric, 33% (25); breast, 20%
tion [pituitary, 0% (74); and thyroid cancer, 0% (28, 30, 75)] and (29, 39); and colon, 33% (25)]. To investigate the possibility that
various nonendocrine tumors where HER-2/neu gene amplification HER-2/neu mRNA and/or protein overexpression could be occurring
was uncommon [non-small cell lung cancer, 3% (76); prostate, 0% in gastrinomas, as well as to determine whether the mild amplification
(27, 77); and urinary bladder cancer, 4 – 8% (78, 79)]. However, our of the HER-2/neu gene in a subset of the gastrinomas resulted in
results in gastrinomas differ from that reported in other PETs and in increased expression, the HER-2/neu mRNA levels were determined
some carcinoids. In these studies gains were seen on chromosome 17q by qualitative PCR in 33 gastrinomas in which frozen tissue existed
by comparative genomic hybridization in 55% of the tumors (n ⫽ 20) and HER-2/neu protein expression was examined by immunocyto-
in one study (71), 57% in a second study (n ⫽ 26; 70), and 41% in a chemistry in a proportion of the tumors. Quantitative PCR was used
third study (n ⫽ 44) with one of two nongastrinoma PETs showing because studies demonstrate that immunocytochemical methods may
overexpression of HER-2/neu gene by fluorescence in situ hybridiza- not be reliable for quantitative assessment of low levels of increased
tion analysis (71). These results suggest that different PETs/carcinoids HER-2/neu protein expression (23, 88). Furthermore, in both endo-
may differ in the frequency with which HER-2/neu gene amplification crine tumors including PETs and carcinoids (29), as well as some
occurs and its role in their molecular pathogenesis. Our results with nonendocrine cancers [i.e., gastric (89)] there was a close correlation
gastrinomas also differ from findings in a number of nonendocrine between HER-2/neu mRNA expression and protein expression. To
cancers where HER-2/neu gene amplification is not infrequent [breast, correct for small variations in both sample input and amount detected,
10 –33% (60); gastric, 10 – 43% (25); esophageal, 35% (25); and in our study, qualitative PCR was used for both determining the
ovarian, 19 –59% (80)]. amount of HER-2/neu mRNA and -actin present, and the result
In some tumors such as breast cancer there is a close correlation normalized to the -actin amount. The relative amount of HER-2/neu
between HER-2/neu gene amplification, and overexpression of and -actin molecules expressed as the neu:-actin ratio varied
HER-2/neu mRNA and/or protein (23, 56). However, with other widely over a 700-fold range in gastrinomas. However, in only 3%
tumors [thyroid cancer (28), gastric cancer (67), prostate cancer (27), (1 of 33 gastrinomas) did the ratio exceed the upper limit of normal
and urinary bladder cancer (79)] overexpression of HER-2/neu for pancreatic tissue. Similarly none of the 10 gastrinomas, which all
mRNA and/or protein occurs without HER-2/neu gene amplification. had normal mRNA levels, overexpressed HER-2/neu protein on im-
Using assessments of HER-2/neu mRNA and/or protein levels, studies munocytochemical analysis including 2 patients who had tumors with
suggest HER-2/neu overexpression is not important in some endo- slightly increased HER-2/neu gene amplification. These results are
crine cancers [adrenocortical tumors, 0% (81); medullary thyroid similar to two other studies involving small numbers of gastrinomas,
cancer, 0% (29, 82); and follicular thyroid cancer, 0% (83, 84)]. which reported 1 of 6 tumors (17%; Ref. 37) and 0 of 20 (0%; Ref.
However, overexpression of HER-2/neu protein occurs frequently in 20), demonstrated HER-2/neu protein overexpression. These results
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HER-2/NEU EXPRESSION IN GASTRINOMA
are also similar to some studies involving other PETs and/or carci- ses, or decreased survival. In the present study we found no correla-
noids, which reported 0 –9% (20, 38, 39, 72) but not others, which tion between the expression level of HER-2/neu in gastrinomas and
reported 17–100% of the tumors (29, 37, 41, 90), demonstrated the occurrence of abnormalities in either of these two tumor suppres-
HER-2/neu overexpression with increased membrane staining using sor genes, providing no support for a cooperative interaction of these
immunohistochemical methods. The findings in some of these studies abnormalities in increasing gastrinoma aggressiveness.
on various PETs/carcinoids at first appear to conflict with results from In conclusion, our results do not provide evidence for a general role
a study involving 36 PETs examined by comparative genomic hy- for overexpression of either the HER-2/neu gene or its product in the
bridization (72) in which 41% had amplification of chromosome 17q, molecular pathogenesis of gastrinomas, in contrast to the suggestion
the location of the HER-2/neu gene. However, in that study (72) only from a previous study (40) involving a few cases. However, our
9% of the PETs with HER-2/neu gene amplification had overexpres- results do show that minimal HER-2/neu gene amplification occurs in
sion of HER-2/neu protein by immunohistochemical studies. The a small subset (14%) of gastrinomas. Furthermore, significantly
above results on HER-2/neu mRNA and/or protein expression in higher HER-2/neu mRNA levels are found in more aggressive gas-
gastrinomas are consistent with the conclusion that amplification of trinomas. Because the current treatments of advanced malignant
the HER-2/neu gene or overexpression of its product do not seem to PETs/carcinoids are generally inadequate and because tumor progres-
be involved in the molecular pathogenesis of most gastrinomas. sion is now the main determinant of survival in these patients (1, 6, 7),
In a number of both endocrine [thyroid cancer (30, 33) and pheo- the finding of higher levels of HER-2/neu mRNA in more aggressive
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