Keywords
gastric cancer - gastroesophageal adenocarcinoma - Her2-Neu - targeted therapy - trastuzumab
Introduction
Gastric/gastroesophageal (GE) carcinoma is one of the common causes for cancer-related
mortality in India and worldwide. The Global Burden of Disease Study 1990–2016 observed
that gastric cancer is one of the top 10 common cancers in India and accounts for
9% of the total cancers disability-adjusted life years.[1]
[2]
[3]
[4] The incidence of gastric cancer is higher in South India[5] and often presents at advanced stages. The mainstay of treatment is surgery with
perioperative chemotherapy.
The role of various cell surface antigens/transmembrane proteins/growth factor receptors
like CDH1, MCT1, and MCT4[6]
[7] have been studied in gastric cancer. Her2-Neu oncogene is a member of the erbB-like
oncogene family which are physiologically involved in signal transmission and are
constituted by family of four cell surface receptors—HER1, HER2, HER3, and HER4, which
exist as monomers and heterodimerize with her2 on ligand binding, in turn generating
strong intracellular signals controlling normal cell proliferation.[8]
[9] Hence, Her2 overexpression in neoplastic cells causes uncontrolled cancer cell proliferation.[8]
The role of expression of Her2-Neu in gastric/GE junction (GEJ) cancers have gained
interest after witnessing improved outcome on targeted therapy with trastuzumab (recombinant
human anti-her2 monoclonal antibody).[9]
[10]
[11]
Despite being a poor prognosticator, it has been proved promising in early gastric
cancers when used in combination with other chemotherapeutic agents.[10]
[12]
[13]
[14]
[15]
[16]
The immunohistochemical expression of Her2-Neu in gastric/GEJ cancers displays heterogeneity
and also varies geographically.[17]
[18]
As there is paucity of Indian data on this, we conducted this study to evaluate the
role of expression of Her2-Neu in gastric/GE cancer as a prognostic marker.
Materials and Methods
This is a 4-year (January 2011–December 2014) retrospective study followed by 7 years
of follow-up (i.e., 2015–2021) of patients who underwent surgical resection for primary
gastric or GEJ adenocarcinoma, at a tertiary center in South India. Institutional
ethics committee approval was obtained for the study.
Details regarding patient demographics, tumor site and stage, and treatment including
neoadjuvant/adjuvant therapy were obtained from the hospital information system. All
cases were histologically categorized as per Lauren's classification into intestinal
or diffuse and staged as per TNM 8th Edition. Immunohistochemistry (IHC) with Her2-Neu
monoclonal primary antibody (polyclonal rabbit anti-human c-erbB-2 oncoprotein [DAKO]
with a dilution of 1:600) was performed on 3-μm thick deparaffinized sections of tumor
tissue using the two-step Super Sensitive Polymer HRP IHC detection kit including
positive and negative controls in each run. Her2-Neu expression in the neoplastic
cells was evaluated using previously validated scoring system adapted from Hofmann
et al[19] and Rüschoff et al.[14] Score of 0 and 1+ was considered as negative, while 3+ was considered as positive.
Score 2+ was considered as equivocal/borderline and further assessed by fluorescent
in situ hybridization (FISH) for definitive categorization.
Follow-up period: The median follow-up period was 2 years (range 1–7 years). Information regarding
disease progression/overall survival was obtained from hospital case notes and/or
telephonic contact/cancer registry data.
Statistical analysis was performed using IBM SPSS version 20.0 software program (SPSS
Inc., Chicago, Illinois, United States). Pearson's chi-square test was used to analyze
the association of Her2-Neu expression with clinical details, type, and differentiation
of adenocarcinoma and pathological stage of the tumor. To find the probability of
overall survival and disease-free survival, Kaplan–Meier analysis was done and followed
by log rank test for the comparison. A p-value of < 0.05 was considered to be statistically significant.
Results
A total 114 patients of histologically proven gastric/GE adenocarcinoma was considered
in the study with a mean age of 61.75 ± 12.07. There were 90 males (78.95%) and 24
females (21.05%); male-to-female ratio was 3.75:1. The median age of the patients
was 64.5 years (range 22–80 years). Eighty-two percent of gastric cancer patients
were older than 50 years of age. The most common location of the tumor was the gastric
cardia and GEJ (42.9%). The predominant histological type of adenocarcinoma was intestinal
subtype (83.3%). Fifty-two percent of the tumor was of poor histological differentiation
(grade 3). Majority (79%) of the patients had advanced stage of disease with metastases
([Table 1]).
Table 1
Site, histological characteristics, and staging of tumors in the study population
(total n = 114)
|
Characteristics
|
n
|
Percentage (%)
|
|
Site
|
|
Gastric cardia and gastroesophageal junction
|
49
|
42.9
|
|
Gastric body/fundus
|
37
|
32.45
|
|
Distal gastric tumor (antrum/pylorus)
|
28
|
24.56
|
|
Histological type (Lauren's classification)
|
|
Intestinal type
|
95
|
83.33
|
|
Diffuse type
|
19
|
16.66
|
|
Grade/histological differentiation
|
|
Grade I (well differentiation)
|
16
|
14.03
|
|
Grade II (moderate differentiation)
|
39
|
34.2
|
|
Grade III (poor differentiation)
|
59
|
51.75
|
|
Tumor staging (as per AJCC TNM 8th edition)
|
|
Lower tumor stage (pT1 and pT2)
|
24
|
21.06
|
|
Advanced tumor stage (pT3 and pT4)
|
90
|
78.94
|
|
Metastatic disease Including nodal metastasis and distant metastasis
|
|
Present
|
82
|
71.93
|
|
Absent
|
32
|
28.07
|
Abbreviations: AJCC, American Joint Committee on Cancer; TNM, tumor, nodes, and metastases.
Her2-Neu expression profile: Only 13 patients were found to have positive staining for Her2-Neu (Score 3 + )
([Fig. 1]). Four patients with equivocal IHC results (2 + ) were confirmed to be Her2 overexpressed
by FISH ([Table 2]).
Fig. 1 Immunohistochemistry (IHC) for Her2-Neu positive staining (3 + ).
Table 2
Expression profile of Her2-Neu (N = 114)
|
IHC scoring for Her2-Neu
|
N (frequency)
|
Interpretation/result
|
|
0
|
89
|
Negative
|
|
1+
|
3
|
Negative
|
|
2+
|
9
|
Negative
|
|
2+ FISH negative
|
5
|
Negative
|
|
2+ FISH positive
|
4
|
Positive
|
|
3+
|
13
|
Positive
|
Abbreviations: FISH, fluorescent in situ hybridization; IHC, immunohistochemistry.
-
1. Tumors with Her2-Neu positivity (n = 17)
The tumors with positive Her2-Neu expression were more frequently located in the proximal
stomach (cardia) and GEJ (64.7%), with a predominant intestinal morphology (94.1%)
and well/moderate differentiation (grade 1 and 2; 82.35%). The comparison of histological
differentiation with Her2-Neu expression was found to be statistically significant
(p-value 0.005) ([Fig. 2]). Diffuse morphology was seen in only one patient. Seventy-five percent of patients
had advanced stage of disease with distant/nodal metastases.
Fig. 2 Intestinal type adenocarcinoma morphology.
-
2. Tumors showing Her2-Neu negative (n = 97) had no specific site location, histology, grade, or stage bias. In univariate
analysis there were found to be no statistically significant association for tumor
attributes with Her2-Neu expression status ([Table 3]).
Table 3
Multivariate table showing comparison of tumor attributes/characteristics with Her2-Neu
expression status
|
Variable
|
|
Her2-Neu positive (total 17)
|
Her2-Neu negative (total 97)
|
p-Value
|
|
N
|
%
|
N
|
%
|
|
Age at diagnosis years
|
> 50
|
15
|
88.24
|
79
|
81.44
|
0.57
|
|
< 50
|
2
|
11.76
|
18
|
18.56
|
|
Gender
|
Male
|
14
|
82.35
|
76
|
78.35
|
0.95
|
|
Female
|
3
|
17.65
|
21
|
21.65
|
|
|
Site of tumor
|
GEJ
|
11
|
64.71
|
38
|
39.18
|
0.08
|
|
Gastric body/fundus
|
2
|
11.76
|
35
|
36.08
|
|
Distal stomach
|
4
|
23.53
|
24
|
24.74
|
|
Histological subtype
|
Intestinal type
|
16
|
94.12
|
79
|
81.44
|
0.34
|
|
Diffuse type
|
1
|
5.88
|
18
|
18.56
|
|
Differentiation
|
Well/moderate
|
14
|
82.35
|
41
|
42.27
|
0.005
|
|
Poor
|
3
|
17.65
|
56
|
57.73
|
|
Tumor stage
|
T1/T2
|
4
|
23.53
|
20
|
20.62
|
1.00
|
|
T3/T4 (advanced stage)
|
13
|
76.47
|
77
|
79.38
|
|
Node/distant metastasis
|
Present
|
13
|
76.47
|
69
|
71.13
|
0.8
|
|
Absent
|
4
|
23.53
|
28
|
28.87
|
Abbreviation: GEJ, gastroesophageal junction.
Follow-Up
The mean and median follow-up period was 2.6 and 3 years, respectively. Eight patients
with Her2-Neu positive (2) and Her2-Neu negative (6) were lost to follow-up. Fifty-six
patients among 106 (52.8%) in our study population had disease recurrence and 51 (51%)
died of the disease.
Eleven Her2-Neu positive patients (73.3%) had disease recurrence and 9 (69.23%) died
of the disease. In comparison, with negative expression for Her2-Neu protein, 45 (49.5%)
had disease recurrence and 42 (48.28%) died of the disease ([Fig. 3]). The comparison of mortality and recurrence status was not found to be statistically
significant (p-value 0.086 and 0.159, respectively).
Fig. 3 Frequency of disease recurrence in relation to Her2-Neu expression.
The overall mean disease-free survival for our study population was 21.15 months with
standard error of 1.498 (95% confidence interval [CI]: 18.216–24.087). The overall
mean disease-free survival for the patients showing positive Her2-Neu expression was
15.4 months with standard error of 3.55 and for patients with negative Her2-Neu expression
was 22.07 months with standard error of 1.364 (p-value 0.08) ([Fig. 4]).
Fig. 4 Kaplan–Meyer curve depicting comparison of overall disease-free survival between
Her2-Neu positive and negative groups.
The mean overall survival for our study population was 23.51 months with standard
error of 1.364 (95% CI: 20.841–26.189). The mean overall survival for the patients
showing positive Her2-Neu expression was 21.14 months with standard error of 3.702
and for patients with negative Her2-Neu expression was 23.91 months with standard
error of 1.474 (p-value 0.245) ([Fig. 5]). As the number of patients with Her2 positive disease were low, multivariate analysis
was not feasible.
Fig. 5 Kaplan–Meyer curve depicting comparison of overall survival between Her2-Neu positive
and negative groups.
Discussion
The incidence of gastric cancer in India is relatively low compared with other countries.
However, a regional variation has been observed in India with the highest incidence
recorded from Southern and Eastern India.[20] There is paucity of data from these regions and hence this study was undertaken
to understand the clinicopathological characteristics and survival outcome of patients
with gastric and GEJ adenocarcinoma in relation to the expression of Her2-Neu, a potential
oncological target. Concordant with other Indian studies,[21]
[22] this study found a male gender preponderance for gastric cancer with male-to-female
ratio of 2:1, 2.3:1, and 3.75:1, respectively. Other studies including a meta-analysis[15]
[23]
[24] have also reported similarly.
Gastric antrum has been observed to be the most common site of involvement as seen
in studies from India and other Asian countries like Japan and Korea.[24]
[25] However, Devesa et al,[26] Harikumar et al,[27] and Abdi-Rad et al[24] have mentioned a paradigm shift of gastric cancer to cardia/proximal stomach in
the West and in an Indian state Kerala. This was observed in this present study also
and can be attributed to changing colonization of Helicobacter
pylori with atrophic gastritis, dietary habits, lifestyle, obesity, and GE reflux.[28]
Frequency of positivity for Her2-Neu in gastric/GEJ cancers has been found variable
across continents. Lei et al[15] in their meta-analysis have stated a slightly higher rate of positivity in Asian
countries, that is, 19.52% in comparison to the European countries which is 16.91%.
This heterogeneous expression has been observed between Asian countries and even within
the Indian subcontinent. Shan et al[29] in a Chinese study demonstrated a frequency of 9.8%, while Matsusaka et al[30] in a Japanese multicenter observational study demonstrated 15.6% frequency of high
Her2-Neu expression. Among the Indian studies, a geographic variation has been observed
with Sukanya et al[22] from Tamil Nadu reporting a 12% frequency, Patil et al[31] in their multicentric clinical trial showed 7% frequency and a systematic review
of 41 studies reported a median of 15%,[32] Gupta et al[21] from Delhi showed 24.5%frequency, Panda et al[33] from the state of Orissa showed 18.7% frequency, and the frequency in the present
study done in a tertiary care institute of Kerala was 15%.
Significant association between Her2-Neu expression and proximal gastric/GEJ tumor
site has been observed in studies done by Roy et al,[23] Shan et al,[29] and Lei et al[15] and in the present study. Although Sukanya et al[22] demonstrated higher expression of Her2-Neu in distal gastric cancers, the number
of GEJ tumors was low in their comparatively smaller study population (4 out of total
70). A significant association of Her2-Neu expression with well/moderately differentiated
(grade 1 and 2) intestinal type adenocarcinoma is there in the present study and in
those conducted by Gupta et al,[21] Panda et al,[33] Lei et al,[15] and Shan et al.[29]
GEJ tumors are more aggressive and with advanced stage of disease.[24] In the present study, both Her2-Neu positive and Her2-Neu negative groups showed
distant/nodal metastasis with only a borderline difference between them, not statistically
significant. Lei et al[15] and Panda et al[33] in their meta-analysis and study, respectively, have demonstrated a statistical
correlation between Her2-Neu positive tumor status and advanced tumor staging.
There is limited Indian data on survival/outcome of patients with Her2-Neu expression
on gastric cancer; hence, this index study seems to be one of its kind. The mean overall
disease-free survival and overall survival for the patients showing positive Her2-Neu
expression in tumor was lower in comparison to those with negative Her2-Neu tumor
expression in the present study; despite administration of neoadjuvant therapy (to
35 and 42% patients with Her2-Neu positive and negative expression in tumor, respectively)
and adjuvant chemotherapy (to 70% patients from each group). Trastuzumab was not given
to any of the patients in the present study at our center, due to the high cost of
the drug in the given period of study. Chua and Merrett[32] in their review of 49 studies comprising 11,337 patients found that in patients
with positive Her2-Neu expression the median 3-year disease-free survival was 58%
(range: 50–88%) and in those with negative Her2-Neu expression the median 3-year disease-free
survival was 86% (range: 62–97%); the median overall survival and 5-year survival
rate was 21 (42%) and 33 months (52%) in Her2-Neu positive and Her2-Neu negative groups,
respectively.
The fact that Her2-Neu positive tumors demonstrate advanced disease with lower survival
rates despite giving neoadjuvant and adjuvant chemotherapy, targeted interventions
are a ray of hope.
Few large-scale studies like the ToGA trial have not only indicated the poor prognostic
value of Her2-Neu expression in gastric/GEJ cancers, but also had underlined the improved
survival by using trastuzumab in combination with chemotherapy as new standard option
for patients with Her2-Neu positive primary/advanced or recurrent gastric and GEJ
cancers.[16] Kim et al in their study conducted in a single institution, investigated the outcomes
of trastuzumab-based chemotherapy administrated to 47 Her2-Neu positive patients with
recurrent or primary metastatic gastric cancer. The outcomes were comparable to that
of the ToGA trial, and also concluded that the consideration of conversion surgery
in fit patients can be done with R0 resection.[34] After observing significantly improved survival on the use of dual-targeted anti-Her2-Neu
therapy in breast cancer, Tabernero et al had designed a study—JACOB—to assess the
efficacy and safety of pertuzumab plus trastuzumab and chemotherapy in patients with
previously untreated Her2-positive metastatic gastric/GEJ cancers. The primary results
showed that the addition of pertuzumab did not significantly improve overall survival
at ≥ 24.4 months' median follow-up, possibly due to increased complexity of gastric
cancer and multifactorial disease progression.[35] However, descriptive end-of-study results showed some but limited evidence of treatment
activity and acceptable toxicity profile for the use of above combination.[36]
Conclusion
Expression of Her2-Neu in gastric/GEJ adenocarcinomas though low in our population,
was associated with lower survival and found to be a poor prognosticator.
