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European Journal of Cancer 53 (2016) 42e50
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Original Research
Extra-gain of HER2-positive cases through HER2
reassessment in primary and metastatic sites in advanced
gastric cancer with initially HER2-negative primary
tumours: Results of GASTric cancer HER2 reassessment
study 1 (GASTHER1)
Sook Ryun Park a,1, Young Soo Park b,1, Min-Hee Ryu a,1,
Baek-Yeol Ryoo a, Chang Gok Woo b, Hwoon-Yong Jung c,
Jeong Hoon Lee c, Gin Hyug Lee c, Yoon-Koo Kang a,*
a
Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
c
Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
b
Received 5 May 2015; received in revised form 9 September 2015; accepted 19 September 2015
Available online 13 December 2015
KEYWORDS
HER2;
Gastric cancer;
Intratumoural
heterogeneity;
Primary lesion;
Metastatic lesion;
Conversion;
Reassessment
Abstract Purpose: The intratumoural heterogeneity of human epidermal growth factor receptor 2 (HER2) expression in gastric cancer is a major challenge when identifying patients
who might benefit from HER2-targeting therapy. We investigated the significance of reevaluation of HER2 status in primary sites and metastatic or recurrent sites in advanced
gastric cancer patients whose primary tumours were initially HER2-negative.
Patients and methods: In part I of this study, we evaluated the significance of repeat endoscopic
biopsy in unresectable or metastatic gastric cancer patients whose tumours were initially HER2negative. In part II, we examined the HER2 positivity rate in metastatic or recurrent sites in patients whose primary tumours were HER2-negative in biopsy or surgical specimens.
Results: In part I (n Z 183), we identified patients with HER2-positive tumours for a rescued
HER2 positivity rate of 8.7% (95% confidence interval [CI], 4.6e12.8%) that was associated with
tumour location (diffuse stomach versus other Z 0% versus 11.7%, P Z 0.013), Bormann type (IV
versus others Z 0% versus 11.7%, P Z 0.013), and initial biopsy HER2 immunohistochemistry
score (0 versus 1 versus 2 Z 6.7% versus 15.4% versus 25.0%, P Z 0.028). Part II (n Z 175)
* Corresponding author: Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil,
Songpa-gu, Seoul, 138-736, Republic of Korea. Tel.: þ82 2 3010 3230; fax: þ82 2 3010 8772.
E-mail address: [email protected] (Y.-K. Kang).
1
The first three authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejca.2015.09.018
0959-8049/ª 2015 Elsevier Ltd. All rights reserved.
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
43
resulted in HER2 positivity of 5.7% (95% CI 2.3e9.1%) that was significantly associated with metastatic site (liver versus others Z 17.2% versus 3.4%, P Z 0.012). When compared with a historical
control that showed HER2 positivity on initial assessment, patients who had rescued HER2 positivity had similar treatment benefits from trastuzumab-containing first-line chemotherapy.
Conclusion: Repeat HER2 assessment in primary and metastatic or recurrent sites is recommended in patients with advanced gastric cancer whose primary tumour is initially HER2negative.
ª 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Nowadays, new agents that target critical oncogenic
pathways can improve treatment outcomes, and management based on the molecular characteristics of tumours is becoming routine. Human epidermal growth
factor receptor 2 (HER2) is a transmembrane tyrosine
kinase receptor and a well-established therapeutic target
in breast and gastric cancer [1e3]. A randomised phase
III study (Trastuzumab for GAstric Cancer [ToGA])
demonstrated the clinical benefit of combining trastuzumab, an anti-HER2 monoclonal antibody, with
chemotherapy in patients with unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) cancer [3]. The survival benefit compared
with chemotherapy alone was more prominent in patients with immunohistochemistry (IHC) scores of 3þ or
2þ with gene amplification as evidence by fluorescence
in situ hybridization (FISHþ) [3]. Based on these results,
trastuzumab was integrated into the standard chemotherapy for HER2-positive gastric cancer [4].
Because of its therapeutic implications, the correct
identification of HER2 positivity on tissue specimens is
critical. Unlike breast cancers, gastric cancers show
frequent incomplete membrane staining, and heterogeneity of HER2 status is common, which makes the
diagnosis of HER2 overexpression difficult. Therefore,
the ToGA study employed a new set of IHC scoring
criteria that considered as positive basolateral or bilateral membranous reactivity as well, and used a 10%
cutoff instead of the 30% used in breast cancers which
was also modified to the 10% in the 2013 HER2 testing
guideline for breast cancer to reduce false-negative results [5,6]. Nonetheless, intratumoural heterogeneity of
HER2 positivity may still result in inaccurate assessment
of HER2 status, which presents a major challenge in
identifying patients who could benefit from HER2targeting therapy. Also important is that HER2 status
is usually assessed in primary gastric tumours that is
used to guide therapy in the metastatic or recurrent
setting, but discordance between HER2 status in primary and metastatic tumours that develop synchronously or metachronously may be misleading.
The purpose of this prospective study was to determine how many additional gastric cancer patients with
HER2-positive tumours could be found through reassessment of HER2 in 1) primary tumour sites by repeat
endoscopic biopsy and 2) metastatic or recurrent
tumour sites in patients whose tumours were HER2negative in the initial evaluation for primary tumours,
and to identify possible baseline factors associated with
rescued HER2 positivity through reassessment.
2. Methods
2.1. Study design
The study consisted of two parts, each with a separate
patient population. In part I, we evaluated the significance of a repeat endoscopic biopsy in unresectable or
metastatic gastric cancer patients whose tumours were
HER2-negative in the initial endoscopic biopsy, and in
part II we examined the HER2 positivity rate in metastatic or recurrent sites in patients whose primary tumours were HER2-negative in biopsy or surgical
specimens.
Patients were eligible for this study if they were over
18 years old with histologically proven unresectable,
metastatic, or recurrent (recurrent disease was included
only in part II) gastric or GEJ adenocarcinoma and no
prior chemotherapy in the palliative setting. Initial
HER2 assessment of the primary gastric tumour had to
be negative in endoscopic biopsy in part I and in the
endoscopic biopsy or surgical specimen in part II. HER2
positivity was defined as IHC 3þ or IHC 2þ plus
FISHþ [5,7]. In part I, immediately after obtaining
HER2-negative results in an initial biopsy, a repeat
gastroendoscopic biopsy was performed at six
different sites of the primary tumour lesions.
To compare outcomes of trastuzumab-containing
first-line chemotherapy between patients with HER2
positivity detected on the initial assessment in primary
tumours and those with rescued HER2 positivity
detected on the repeat assessment in primary or metastatic tumours, we used historical control data of unresectable, metastatic, or recurrent gastric cancer patients
who had HER2 positivity on the initial endoscopic biopsy or gastrectomy specimen and were treated with
trastuzumab-containing first-line chemotherapy between April 2009 and March 2013 at Asan Medical
44
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
Center. The Center’s Institutional Review Board
approved the study protocol, and all patients provided
written informed consent.
2.2. Immunohistochemistry staining and fluorescence in
situ hybridization
HER2 IHC was performed on formalin fixed, paraffin
embedded tissue using Pathway (monoclonal antibody,
4B5; Ventana Medical System, Tucson, AZ) with an
automated staining device according to the manufacturer’s US Food and Drug Administration-approved
procedures (see Appendix for more detail). HER2 protein expression was scored on a scale of 0 to 3 using the
gastric cancer consensus panel recommendations [5,7].
FISH was performed only in cases with IHC 2þ
using the Abbott PathVysion HER2 DNA Probe Kit
protocol (Abbott Laboratories, Abbott Park, Des
Plaines, IL) according to the manufacturer’s instructions
(see Appendix for more detail). HER2 gene amplification status was evaluated by counting signals in 20 nonoverlapping tumour cells with the highest gene count.
The interpretation followed the criteria of the American
Society of Clinical Oncology (ASCO)/College of
American Pathologists (CAP) guidelines [8]; negative for
gene amplification if the HER2/chromosome enumeration probe (CEP)17 ratio was <1.8, positive if the
HER2/CEP17 ratio was >2.2, and equivocal if the
HER2/CEP17 ratio was 1.8e2.2 where we counted 20
additional tumour cells and considered the case positive
if the HER2/CEP17 ratio was 2.0.
2.3. Statistical analysis
The primary end-point was the rate of HER2 positivity
that was converted through reassessment following
initial HER2 negativity. Assuming that the rate of
HER2 positivity meaningful in reassessment was 5%
with a 95% confidence interval (CI) of 0.023e0.097, we
needed a sample size of 179 patients in each study part.
We used univariate logistic regression analysis to
evaluate the effects of clinicopathologic factors on
rescued HER2 positivity. For independent variables, we
used the Wilcoxon-Mann-Whitney test for quantitative
variables and the Pearson c2 test or the Fisher exact test
for categorical variables. We used the CochranArmitage trend test to assess statistical significance of
changes in the relationship between rescued HER2
positivity and HER2 IHC score on the initial biopsy.
We assessed objective tumour response according to
Response Evaluation Criteria in Solid Tumours v1.1 [9].
We defined progression-free survival as the time between
the initiation of treatment and documented disease
progression or death from any cause. We used the
Kaplan-Meier method and the log-rank test to estimate
and compare survival distribution. All tests were twosided, and we considered P 0.05 significant.
Table 1
Baseline characteristics of patients at the time of initial HER2
assessment for primary tumour sites.
Characteristic
Part I
Part II
Number %
Number %
Eligible patients
183
100 175
100
Sex, male
121
66.1 96
54.9
Median age, years (range)
55 (24e78)
56 (30e79)
Disease status
Locally advanced unresectable
13
7.1 1
0.6
Initially metastatic
170
92.9 60
34.3
Recurrent
0
0
114
65.1
Primary tumour location
GEJ/cardia/fundus
22
12.0 15
8.6
Body
47
25.7 92
52.6
Antrum
68
37.2 56
32.0
Diffuse stomach
46
25.1 12
6.9
Bormann type
1
3
1.6 3
1.7
2
34
18.6 19
10.9
3
98
53.6 105
60.0
4
46
25.1 25
14.3
EGC
2
1.1 23
13.1
Histology
W/D adenocarcinoma
5
2.7 7
4.0
M/D adenocarcinoma
55
30.1 41
23.4
P/D adenocarcinoma
106
57.9 96
54.9
Signet ring cell carcinoma
12
6.6 25
14.3
Mucinous carcinoma
5
2.7 6
3.4
Lauren classification
Intestinal
53
29.0 57
32.6
Diffuse
111
60.7 101
57.7
Mixed
19
10.4 17
9.7
HER2 IHC score
0
149
81.4 144
82.3
1
26
14.2 18
10.3
2
8
4.4 13
7.4
Tumour tissues for HER2 assessment in primary tumour
Endoscopic biopsy
183
100 52
29.7
Surgical resection
0
0
123
70.3
Tumour tissues for HER2 assessment in metastatic or recurrent
tumour
Biopsy
NA
NA 119
68.0
Surgical resection
NA
NA 55
31.4
or wide excision
Malignant ascites
NA
NA 1
0.6
Metastatic or recurrent sites where HER2 was assessed
Peritoneum
NA
NA 37
21.2
Liver
NA
NA 29
16.6
Ovary
NA
NA 29
16.6
Lymph node
NA
NA 22
12.6
Anastomosis/remnant
NA
NA 24
13.7
stomach/afferent loop
Others
NA
NA 34
19.4
Time relationship between HER2 assessments in primary and
metastatic/recurrent sites
Synchronous
NA
NA 61
34.9
Metachronous (recurrent disease) NA
NA 114
65.1
Distant metastatic
NA
NA 91
52.0
Locoregional
NA
NA 23
13.1
GEJ, gastroesophageal junction; EGC, early gastric cancer; W/D, well
differentiated; M/D, moderately differentiated; P/D, poorly differentiated; HER2, human epidermal growth factor receptor 2; IHC,
immunohistochemistry; NA, not applicable.
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
3. Results
3.1. Study population
3.1.1. Part I
From May 2011 to April 2013, 186 patients were
enrolled in part I of the study; 183 met eligibility requirements. Table 1 shows their baseline characteristics.
The median interval between the initial and repeat biopsies was 13 d (range, 5e147 d).
3.1.2. Part II
From May 2011 to February 2014, 179 patients were
enrolled in part II of the study; 175 met eligibility requirements (Table 1). The median time interval of
HER2 assessment between primary and metastatic/
recurrent lesions was: 0.2 months (range, 0e4.0 months)
in initially metastatic/locally advanced disease and 28.8
months (range, 3.7e158.2 months) in recurrent disease.
3.2. Rescued HER2 positivity through repeat biopsy in
primary tumours and associated clinical factors in part I
Table 2 shows the HER2 status of initial and repeat
endoscopic biopsy of primary gastric tumours. The
repeat biopsy identified 16 patients with HER2-positive
tumour, resulting in a rescued HER2 positivity rate of
8.7% (16 of 183) (95% CI, 4.6e12.8%). Fig. 1A and B
show representative photomicrographs of a case with
rescued HER2 positivity.
In univariate analysis, rescued HER2 positivity was
significantly associated with primary tumour location
(diffuse stomach versus others Z 0% versus 11.7%,
P Z 0.013), Bormann type (IV versus others Z 0%
versus 11.7%, P Z 0.013), and the initial biopsy HER2
IHC score (0 versus 1 versus 2 Z 6.7% versus 15.4%
versus 25.0%, P Z 0.065 (trend test, P Z 0.028);
0 versus 1/2 Z 6.7% versus 18.2%, P Z 0.045) (Table 3).
In logistic regression analysis, patients who had HER2
IHC 1 or 2þ primary tumours on initial biopsy were 3.1
times (95% CI, 1.04e9.28) more likely than those with
HER2 IHC 0 to show HER2 positivity on repeat biopsy.
45
The median number of biopsy pieces per patient was
five (range, 1e15) on the initial biopsy and 10 (range,
1e15) on the repeat biopsy (P < 0.0001). At least six
pieces per patient were obtained in 64 patients (35.0%)
on the initial biopsy and 177 patients (96.7%) on the
repeat biopsy. There was no difference in the median
ratio of number of cancer-containing biopsy pieces/total
pieces; 0.86 (range, 0.13e1) on the initial biopsy
versus 0.89 (range, 0.10e1) on the repeat biopsy
(P Z 0.679). The median number of biopsy pieces per
patient was five for the HER2-negative tumours and
four for the HER2-positive tumours on the initial biopsy (P Z 0.420), and 10 for both on the repeat biopsy
(P Z 0.083). The rescued HER2 positivity rate according to the number of biopsy pieces on the initial
biopsy was 9.2% for <six pieces versus 7.8% for six
(P Z 0.744) and 11.1% for <three versus 8.5% for
three (P Z 0.661).
3.3. Rescued HER2 positivity through reassessment in
recurrent or metastatic tumours and associated clinical
factors in part II
Reassessment of recurrent or metastatic sites identified
10 patients with HER2-positive tumour for a conversion
rate of 5.7% (10 of 175) (95% CI, 2.3e9.1%) (Table 4).
Fig. 1C and D show representative photomicrographs of
such a case.
In univariate analysis, this converted HER2 positivity
was significantly associated only with metastatic sites
where HER2 was reassessed (liver versus others Z
17.2% versus 3.4%, P Z 0.012) (Table 3). In logistic
regression analysis, patients who had HER2 reassessment in liver metastasis had a 5.88 (95% CI, 1.58e21.84)
times higher probability of HER2 positivity on repeat
assessment than those who had HER2 reassessment in
other recurrent or metastatic sites. Time relationship
between primary and metastatic/recurrent sites (synchronous versus metachronous) was not associated with
HER2 positivity at metastatic/recurrent sites in the
entire population (3.3% versus 7.0%; P Z 0.497) or the
subgroup with liver metastasis (21.1% [4/19] versus 10%
[1/10]; P Z 0.633).
3.4. Outcomes of trastuzumab-treated patients with
rescued HER2 positivity
Table 2
Comparison of HER2 status between initial endoscopic biopsy and
repeat biopsy in primary gastric cancer sites.
HER2 IHC score in
the initial biopsy
HER2 IHC score in the repeat biopsy
0
1
2
3
0
1
2
98
8
3
32
12
1
11 (2)
3 (1)
2
8
3
2
HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry.
The number indicates number of patients. Numbers in parenthesis
indicate patients who had a HER2 IHC score of 2þ and FISHþ on the
repeat biopsy.
Twelve of the 16 patients who had rescued HER2 positivity by repeat endoscopic biopsy in primary tumours,
and 7 of the 10 who had HER2 positivity at metastatic
or recurrent sites, had received trastuzumab-containing
first-line chemotherapy. We compared treatment outcomes between these 19 rescued HER2þ patients with
94 patients who had HER2 positivity on the initial
assessment for primary tumours in biopsy or surgical
specimens and also received trastuzumab-containing
first-line chemotherapy as a historical control. In
46
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
Fig. 1. Representative photomicrographs of converted human epidermal growth factor receptor 2 (HER2) positivity. Initial endoscopic
biopsy in primary tumour shows HER2 negativity in immunohistochemistry (IHC) staining, score 0 (A, original magnification 200), but
the tumour cells in repeat biopsy express strong HER2 protein, IHC score 3 (B, original magnification 200). While initial gastrectomy
specimen shows HER2 negativity in IHC staining, score 0 (C, original magnification 40), the metastatic tumour cells express strong
HER2 protein, IHC score 3 in liver biopsy (D, original magnification 200).
patients with measurable lesion(s), the overall tumour
response rate was 90.9% (95% CI, 73.9e100%) in patients with rescued HER2 positivity and 76.5% (95% CI,
67.5e85.5%) in patients with HER2 positivity in the
initial assessment (P Z 0.446). With a median follow-up
duration of 11.9 months (range, 1.5e25.4 months) and
14.2 months (range, 1.4e51.7 months), respectively, the
median progression-free survival was 7.6 months (95%
CI, 6.5e8.7 months) in patients with rescued HER2
positivity and 8.3 months (95% CI, 6.9e9.7 months) in
patients with HER2 positivity in the initial assessment
(P Z 0.444) (Fig. 2).
4. Discussion
On the basis of therapeutic value of trastuzumab, the
National Comprehensive Cancer Network (NCCN)
Guidelines recommend that patients with inoperable
locally advanced, recurrent, or metastatic adenocarcinoma of the stomach or GEJ be tested for HER2 status
[10]. Although intratumoural heterogeneity of HER2
status reportedly occurs in breast cancer [11,12], it seems
to occur more commonly in gastric cancer, causing
sampling errors in determining patient eligibility for
anti-HER2 therapy to be more of an issue [5,7,13e16].
In a study comparing whole tissue sections and corresponding multiple tissue micro-arrays (TMAs) of gastrectomy specimens that served as biopsies procedure,
TMAs showed a false-negative HER2 status rate of
24%, providing strong evidence of the risk of biopsy
sampling errors [17].
In the present study, we determined the clinical usefulness of the repeat endoscopic biopsy in patients with
previous negative HER2 biopsies who would not usually
be considered for anti-HER2 therapy for advanced
gastric cancer. With repeat biopsy, 8.7% of patients were
shown to have HER2-positive gastric cancer. In addition to primary tumour sites, metastatic and/or recurrent sites could be also considered for re-evaluation in
cases of initially negative HER2 status. By doing that,
5.7% of patients turned out to have HER2-positive
disease. Since the rate of HER2 positivity defined as
HER2 IHC 3þ or IHC 2þ/FISHþ in advanced gastric
cancer is already low (<20%) [3,18,19], the additional
identification in this study of 8.7% (primary sites) and
5.7% (metastatic or recurrent sites) HER2-positive
gastric cancers that were considered HER2-negative on
initial assessment shows the need for repeat assessment
so as not to deny patients the potential benefit of
receiving anti-HER2 therapy for HER2-positive gastric
cancer.
According to our prospective database, HER2 status
was evaluated in 875 patients before first-line chemotherapy during the same period as this study, and among
them, 110 patients had HER2-positive tumours on
initial endoscopic biopsy or gastrectomy assessment,
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
47
Table 3
Univariate analysis for HER2 positivity in reassessment according to clinicopathologic characteristics.
Characteristic
HER2 positivity
Part I
Sex
Male
Female
Age
<65 years
65 years
Disease status
Locally advanced unresectable
Metastatic
Recurrent
Primary tumour location
GEJ/cardia/fundus
Body
Antrum
Diffuse stomach
Bormann type
1
2
3
4
EGC
Histology
W/D adenocarcinoma
M/D adenocarcinoma
P/D adenocarcinoma
Signet ring cell carcinoma
Mucinous carcinoma
Lauren classification
Intestinal
Diffuse
Mixed
HER2 IHC score at primary tumour
0
1
2
Tumour tissues in primary gastric HER2 assessment
Endoscopic biopsy
Surgical resection
No. of biopsy pieces obtained at primary tumourd
<6
6
Time relationship between HER2 assessment at
primary and metastatic/recurrent sites
Synchronous metastatic site
Recurrent metastatic site
Recurrent locoregional site
Sites of metastasis/recurrence where HER2 was assessed
Peritoneum
Liver
Ovary
Lymph node
Anastomosis/remnant stomach
Others
Chemotherapy between HER2 assessment at primary
and metastatic/recurrent sites
No chemotherapy
Adjuvant chemotherapy
Part II
Number
%
10/121
6/62
8.3
9.7
9/134
7/49
6.7
14.3
3/13
13/170
NA
23.1
7.6
NA
2/22
6/47
8/68
0/46
9.1
12.8
11.8
0
P
Number
%
0.749
0.756
5/96
5/79
5.2
6.3
6/132
4/43
4.5
9.3
0/1
2/60
8/114
0
3.3
7.0
2/15
7/92
1/56
0/12
13.3
7.6
1.8
0
0/3
1/19
5/105
1/25
3/23
0
5.3
4.8
4.0
13.0
0/7
5/41
5/96
0/25
0/6
0
12.2
5.2
0
0
4/57
3/101
3/17
7.0
3.0
17.6
0.138
0.263
0.091
0.526
0.013a
0.194
0.013b
0/3
3/34
13/98
0/46
0/2
0
8.8
13.3
0
0
0/5
4/55
10/106
2/12
0/5
0
7.3
9.4
16.7
0
4/53
9/111
3/19
7.5
8.1
15.8
P
0.513
0.799
0.352
0.530
0.055
0.045c
0.821
10/149
4/26
2/8
6.7
15.4
25.0
8/144
1/18
1/13
5.6
5.6
7.7
NA
NA
NA
NA
2/52
8/123
3.8
6.5
11/119
5/64
9.2
7.8
1/28
1/24
3.6
4.2
0.725
0.744
1.000
0.485
NA
NA
NA
NA
NA
NA
2/61
6/91
2/23
3.3
6.6
8.7
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0/37
5/29
2/29
1/22
2/24
0/34
0
17.2
6.9
4.5
8.3
0
0.019
0.749
NA
NA
NA
NA
4/84
6/91
4.8
6.6
HER2, human epidermal growth factor receptor 2; GEJ, gastroesophageal junction; EGC, early gastric cancer; W/D, well differentiated; M/D,
moderately differentiated; P/D, poorly differentiated; IHC, immunohistochemistry; NA, not applicable.
a
P value for diffuse stomach versus others.
b
P value for type 4 versus others.
c
P value for IHC 0 versus 1/2.
d
Patients only who had HER2 assessment for primary tumour in endoscopic biopsy.
48
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
Table 4
HER2 status of primary tumour sites versus metastatic or recurrent
tumour sites.
HER2 IHC score
at primary
tumour sites
HER2 IHC score at the metastatic
or recurrent sites
0
1
2
3
0
1
2
107
9
8
26
7
1
5 (2)
2 (1)
4 (1)
6
0
0
The number indicates number of patients. Numbers in parenthesis
indicate patients who had a HER2 IHC score of 2þ and FISHþ at
metastatic or recurrent sites.
HER2, human epidermal growth factor receptor 2; IHC,
immunohistochemistry.
resulting in a 12.6% HER2-positive rate. If the remaining 765 patients with HER2-negative primary tumour
had been reassessed both at primary and metastatic
tumour sites in cases with initially metastatic/locally
advanced disease (N Z 413) and at recurrent tumour
sites in cases with recurrent disease (N Z 352), 60 cases
(413 8.7% þ 413 5.7%) and 20 cases (352 5.7%),
respectively, would have had rescued HER2 positivity,
and consequently, the rate of HER2 positivity would
have been 21.7% (190/875). This corresponds to a 72.2%
relative increase of HER2 positivity through repeat
assessment. Although the cost and potential harm
associated with repeat assessment requires further evaluation, our study demonstrates that this approach is
Fig. 2. Kaplan-Meier curves of progression-free survival in patients with human epidermal growth factor receptor 2 (HER2)
positivity in the initial assessment in endoscopic biopsy or gastrectomy specimen for primary tumour (initial HER2 positivity)
and in patients with HER2 positivity in the repeat endoscopic
biopsy after initial HER2-negative endoscopic biopsy or in the
reassessment of metastatic or recurrent sites after initial HER2negative biopsy or gastrectomy specimen for primary tumour
(converted HER2 positivity). All patients were treated with
trastuzumab-containing first-line chemotherapy.
feasible and not associated with significant complications while it has major therapeutic implications in that
patients with rescued HER2 positivity can be offered the
same benefits of trastuzumab-containing chemotherapy
as those showing HER2 positivity on initial assessment
(Fig. 2).
Previous studies show that HER2 positivity in
advanced gastric cancer is associated with clinicopathologic characteristics of Lauren’s intestinal type, the
proximal primary tumour region encompassing gastric
cardia/GEJ, and liver metastasis [19,20]. In the present
study, the factors significantly associated with rescued
HER2 positivity on the repeat endoscopic biopsy
included the primary tumour location other than diffuse
stomach, Bormann type other than type four, and the
higher HER2 IHC score on the initial endoscopic biopsy. Of note, since patients who had HER2 IHC 1 or
2þ primary tumours on the initial biopsy were 3.1 times
as likely as those who had HER2 IHC 0 tumours to
show HER2 positivity on repeat biopsy, they, in
particular, should be considered for re-biopsy.
Regarding the number of endoscopic biopsy fragments, although there were significant differences between the initial and repeat biopsy (median, 5 versus 10,
respectively; P < 0.0001), the number of biopsy fragments on the initial biopsy was not associated with
rescued HER2 positivity on the repeat biopsy (9.2% for
<six fragments versus 7.8% for six; P Z 0.744; 11.1%
for <3 versus 8.5% for three, P Z 0.661). While the
NCCN Guidelines recommend multiple (6e8) endoscopic biopsies to provide an adequate-sized material for
histologic interpretation [10], there are controversial
data about optimum numbers of viable biopsies of the
cancer to account for intratumoural heterogeneity of
HER2 expression. Tominaga and colleagues suggested
at least five biopsy fragments based on 100% concordance between the biopsy and resection specimens in
HER2 status in 24 HER2-positive gastric cancers
(defined as HER2 IHC 2þ or 3þ) [21], whereas Warneke and colleagues showed the 24% false-negative
HER2 status rate with five TMAs in each tumour
when compared to corresponding whole tissue sections
[17]. Huang and colleagues showed that there was no
association between the number of biopsy fragments
and HER2 discordance between biopsy and surgical
specimens [22]. Further studies are needed to clarify the
optimal number of endoscopic biopsy for accurate
HER2 evaluation in gastric cancer.
Previous studies reported variable concordance rates
of HER2 status between primary gastric cancer sites and
paired metastatic sites [15,23e25]. Bozzetti and colleagues [23] reported 94.9% concordance between HER2
status by IHC on 39 primary and corresponding metastatic sites, but they allowed equivocal (IHC 2þ) cases in
one site to have either negative (IHC 0/1þ) or equivocal
(IHC 2þ) status in the other site. Kim and colleagues
[15] reported significant discordance (17.2%) between
S.R. Park et al. / European Journal of Cancer 53 (2016) 42e50
250 paired primary and metastatic lesions by IHC.
Kochi and colleagues [25] showed 90.2% concordance
between HER2 status by IHC and FISH on 102 paired
primary gastric cancer and related regional metastatic
lymph nodes. Our study focused only on patients who
had HER2-negative primary tumours on initial assessment and prospectively evaluated how many patients
had positive conversion in recurrent or metastatic sites.
Although the overall positive conversion rate of 5.7%
was not so high, an interesting finding was that the
positive conversion of HER2 status at metastatic sites
occurred about six times more frequently in liver metastases than at other sites (17.2% versus 3.4%;
P Z 0.012). This could be partially explained by preferential liver metastasis of HER2-positive cancer cells
[19,26]. This relatively high conversion rate indicates
that reassessment of HER2 status is particularly warranted in liver metastases even when the primary tumours were HER2-negative.
In conclusion, on the basis of the recognition of intraand inter-tumoural heterogeneity, the rescued HER2
positivity found in our study, and its important therapeutic implications, we recommend that repeat assessment of HER2 status in primary and metastatic and/or
recurrent tumours in patient with unresectable, metastatic, or recurrent gastric cancer even when initial
assessment showed the primary tumour to be HER2negative.
Conflict of interest statement
Yoon-Koo Kang declares receiving research grants
from Sanofi, Bayer, Roche, and Novartis and discloses a
consultant role for Roche, Bayer, Sanofi, Taiho, Ono,
Pfizer, and Novartis. Min-Hee Ryu declares receiving
honoraria from Novartis, Roche, Sanofi, and Taiho,
research grants from Novartis and having a consultant
role with Novartis and Roche. All other authors declare
no conflicts of interest.
Acknowledgements
This work was supported in part by Roche Korea.
Appendix A. Supplementary data
Supplementary data related to this article can be found
at http://dx.doi.org/10.1016/j.ejca.2015.09.018
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