Introduction
Trastuzumab (Herceptin) is a monoclonal antibody that specifically targets human epidermal growth factor receptor 2 (HER2), a receptor associated with gastric cancer (GC) tumorigenesis, by directly binding its extracellular domain [
1]. The Trastuzumab for GAstric Cancer (ToGA) study, an open-label, international, multicenter, phase III, randomized controlled trial, examined the clinical efficacy and safety of trastuzumab combined with standard chemotherapy (capecitabine or intravenously administered 5-fluorouracil and cisplatin) for first-line treatment of HER2-overexpressing advanced gastric or gastroesophageal junction cancers. Addition of trastuzumab therapy to chemotherapy improved median survival (13.8 months) compared with chemotherapy alone (11.1 months) (
P = 0.0046), and showed significant improvements in time to progression and progression-free survival in the trastuzumab-treated group, with a comparable toxicity profile [
2]. As a result, trastuzumab therapy plus chemotherapy has become the standard treatment for HER2-positive advanced GC patients, as determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). In Japan and the USA, trastuzumab is approved for patients with metastatic GC whose tumors are HER2 positive, as defined by a positive FISH result or an IHC score of 3+. In the European Union, however, trastuzumab is recommended only for individuals whose tumors have high HER2 protein expression, as defined by an IHC score of 2+/positive FISH result or an IHC score of 3+ based on the subset analysis of the ToGA study. HER2 evaluation has therefore become an important approach for predicting clinical efficacy of trastuzumab. The variation in the HER2-positivity rate between countries possibly reflects the unstandardized testing modality and other country-specific factors; it was identified as 27 % in Japanese patients in the ToGA study [
3,
4], which was higher than that identified in previous studies in Japan [
5‐
7].
In the ToGA study, the strong effect of trastuzumab was evident in patients with higher HER2 protein expression (IHC score 2+/FISH positive or IHC score 3+), whereas the efficacy was unclear in patients with low HER2 expression (IHC score 0/FISH positive or IHC score 1+/FISH positive). These results were obtained via a subgroup analysis, and may be affected by the smaller number of patients with low HER2 expression than higher HER2 protein expression. Thus, it is premature to conclude that addition of trastuzumab therapy to chemotherapy is not beneficial in patients with low HER2 expression. Additionally, little has been reported about the clinicopathological features of patients with low HER2 expression [
8‐
10].
In unresectable cases, tumor behavior before treatment is evaluated by biopsy specimens. However, because GC is considered a mixture of heterogeneous tumor types, small biopsy specimens may not reflect its overall behavior, and few studies have focused on HER2-positivity concordance between diagnostic biopsy specimens and surgical specimens [
11,
12]. Because of tumor heterogeneity, the accuracy of HER2 testing can be affected by the site of the examined HER2-stained cells; thus, gastric biopsies could yield false-negative results [
13].
We performed a prospective, multicenter, observational cohort study (JFMC44-1101) to evaluate HER2 expression and gene amplification in consecutively registered Japanese patients with metastatic (excluding curatively resected cases) or recurrent GC, and explored the clinicopathological features in relation to HER2 positivity (IHC score 3+ and/or FISH positive) or low HER2 expression (IHC score 0/FISH positive or IHC score 1+/FISH positive). Furthermore, we evaluated the relationship between HER2 protein expression/gene amplification and sampling conditions to ascertain whether HER2 positivity in GC patients can be accurately determined from routinely prepared formalin-fixed, paraffin-embedded tissues.
Discussion
Previous studies reported that the rate of HER2 positivity (IHC score 3+ and/or FISH positive) in Japanese GC patients was approximately 10–20 % [
5‐
7], but testing methods and interpretation criteria were not standardized. In this study, HER2 status was centrally assessed with a standardized method, which was used to prospectively interpret both the IHC data and the FISH data of the ToGA study; The rate of HER2 positivity was 21.2 % in Japanese patients, identical to the ToGA screening population [
3]. The rate of HER2 positivity was reported as 27 % in Japanese patients in the ToGA study [
4], higher than previously reported rates. This might be a result of bias toward patient selection from past reports [
16‐
18], because the primary purpose of the ToGA study was to assess the clinical efficacy and safety of trastuzumab rather than to evaluate HER2 positivity. The incidence of higher HER2 protein expression (IHC score 2+/FISH positive or IHC score 3+; 15.6 %) and the proportions of FISH positivity in IHC score 0 and IHC score 1+ cases (3.2 and 11 %) were comparable with those reported in the ToGA study [
3]. Similarly, the concordance between IHC and FISH in our results is consistent with that reported in the ToGA study.
A high correlation between HER2 positivity and histological subtype was reported by several authors [
19‐
24]. In the ToGA study, HER2 positivity varied significantly according to histological subtype (intestinal type 31.8 %; diffuse type 6.1 %; mixed type 20 %) [
3]; thus, intestinal type was strongly correlated with HER2 expression. Several reports indicated that intestinal type is associated with hematogenous metastasis, particularly to the liver [
25], and with older age [
26], whereas the diffuse type is adversely related to peritoneal dissemination [
27]. In the present study, intestinal type, absence of peritoneal metastasis, and hepatic metastasis were shown to be independent factors related to HER2 positivity in a multivariate logistic regression analysis. This agrees with what is known about the histological type, i.e., intestinal or diffuse, and the association with accompanying hepatic or peritoneal metastasis, respectively.
Moreover, intestinal type, age (65 years or older), and T1–T3 stage were independent factors related to low HER2 expression (IHC score 0/1+ and FISH positive). This result reveals that HER2-related factors are associated with intestinal-type GCs. Diffuse-type GCs are more malignant than their intestinal-type counterparts, demonstrating early invasion into the muscularis propria [
25]. A previous report demonstrated that diffuse-type advanced GC was significantly associated with advanced pathological T stage [
28]. Thus, diffuse type is commoner in T4 tumors, whereas intestinal type is commoner in T1–T3 tumors. As intestinal type is the most robust factor related to HER2 expression, T1–T3 stage may be an independent factor related to low HER2 expression even in intestinal-type IHC score 0/1+ GC cases. However, the current study was limited by the extent and accuracy of the T staging, which was determined by either pathological or clinical diagnosis methods. To resolve these limitations, we performed ad hoc analyses for low HER2 expression (IHC score 0/FISH positive or IHC score 1+/FISH positive) in the surgical specimen group, because the T stage in the surgical samples was accurately determined pathologically. T1–T3 stage was statistically significantly correlated with low HER2 expression in the univariate analysis, but was not significantly correlated in the multivariate analysis. Likewise, intestinal type, sex, hepatic metastasis, and formalin concentration were statistically significantly associated with low HER2 expression in the univariate analysis; however, there were no significant differences in the multivariate analysis. The discrepancies in these analyses may result from the multivariate analysis being performed only in 392 of 569 cases owing to missing data in the remaining cases, thereby conferring a lack of statistical significance. Further studies are required to confirm this result, and considering these limitations, we cannot conclude that depth of tumor invasion is a factor related to low HER2 expression.
There are several factors that are reported to affect HER2 staining results, such as type of fixative, total fixation time, fixative pH, tissue type, and time before fixation. In the present study, we evaluated the relationship between HER2 expression and sampling conditions; however, the number of biopsy samples, formalin concentration, and formalin-fixation time had no significant effect on HER2 positivity and low HER2 expression. Unfortunately, the recommended conditions for fixation could not be adhered to in this study because the biopsy specimens and surgically resected specimens were mixed up and because correlations between formalin concentration and fixation time could not be undertaken. Moreover, the time before fixation (so-called cold ischemia) and the specimen size were unclear. Further prospective studies aiming to comprehensively evaluate the effects of formalin concentration, formalin-fixation time, and cold ischemia on HER2 testing are required.
There was concern that examination of gastric biopsy samples alone might introduce false-positive and/or false-negative data, because HER2 intratumoral heterogeneity in GC is observed in 20–70 % of HER2-positive tumors [
13,
29] and is the major cause of discrepancies between biopsy samples and surgical specimens. In the multivariate analysis of the present study results, HER2-positivity rates in surgically resected tumors and biopsy samples were not significantly different, similar to the findings in the HER-EAGLE study [
24]. However, these studies were limited in that the correlation between surgical specimens and biopsy samples was not paired, although this contrasts with the GERCOR study, where the overall concordance rate between surgical specimens and paired biopsy samples reached 94 % [
12]. We also examined the concordance between predominant histological type and histological type with a HER2-positive component, which was determined as 81.3 % with the Lauren classification (data not shown). Approximately 20 % of cases showed a discrepancy; therefore, gastroenterologists should consider performing multiple biopsy sampling from varied collection sites to overcome tumor heterogeneity in GC.
In conclusion, HER2 expression in a Japanese GC population was similar in distribution to that identified in the ToGA study. Intestinal type was revealed as an independent factor related to both HER2 positivity and low HER2 expression.
Acknowledgments
This trial was supported by the Japanese Foundation for Multidisciplinary Treatment of Cancer. Editorial support was provided by H. Nikki March and was funded by Chugai Pharmaceutical Co. Ltd.
Conflict of interest
S. Matsusaka has received commercial research grants from Taiho Pharmaceutical Co. Ltd. K. Nishikawa has received commercial research grants and personal fees from Yakult Honsha Co. Ltd, Taiho Pharmaceutical Co. Ltd, and Ajinomoto Pharmaceuticals, and personal fees from Chugai Pharmaceutical Co. Ltd. Tsumura Co. Ltd, and Terumo Corporation. T. Yoshikawa received commercial research grants and personal fees from Chugai Pharmaceutical Co. Ltd during the conduct of the study, has received grants and personal fees from Taiho Pharmaceutical Co. Ltd, Novartis Pharma K.K., Ono Pharmaceutical Co. Ltd, Yakult Honsha Co. Ltd, Covidien Japan Co. Ltd, and the nonprofit organization Kanagawa Standard Anti-cancer Therapy Support System, has received personal fees from Eli Lilly Japan K.K., Abbott Japan Co. Ltd, Kaken Pharmaceutical Co. Ltd, Johnson & Johnson K.K., Takeda Pharmaceutical Co. Ltd, Secom Medical System Co. Ltd, Nikkei Business Publications, and the nonprofit organizations Tokyo Cooperative Oncology Group, Clinical Training Support Council, and Cancer Net Japan, and has received grants from Daiichi Sankyo Co. Ltd and Nippon Kayaku Co. Ltd. A. Ochiai has received commercial research grants from Bayer Ltd, Ventana Medical Systems Inc., Eli Lilly Japan K.K., Toray Industries Inc., Takeda Pharmaceutical Co. Ltd, Taiho Pharmaceutical Co. Ltd, Merck Serono Co. Ltd, Janssen Pharmaceutical K.K., Eisai Co. Ltd, Otsuka Pharmaceutical Co. Ltd, and Daiichi Sankyo Co. Ltd, and nonfinancial support from Fujifilm Co. Ltd. S. Morita has received commercial research grants and personal fees from Chugai Pharmaceutical Co. Ltd. T. Sano has received personal fees from Taiho Pharmaceutical Co. Ltd, Sanofi K.K., Yakult Honsha Co. Ltd, and Chugai Pharmaceutical Co. Ltd. Y. Kodera has received commercial research grants and personal fees from Chugai Pharmaceutical Co. Ltd, grants and personal fees from Taiho Pharmaceutical Co. Ltd, Sanofi K.K., Bristol-Myers Squib Ltd, Merck Serono Co. Ltd, Yakult Honsha Co. Ltd, Daiichi Sankyo Co. Ltd, Otsuka Pharmaceutical Co. Ltd, Takeda Pharmaceutical Co. Ltd, Johnson & Johnson K.K., Asahi Kasei Pharma Corporation, Shionogi & Co. Ltd, Eli Lilly Japan K.K., Pfizer Japan Inc., Ajinomoto Pharmaceuticals, Ono Pharmaceutical Co. Ltd, Kaken Pharmaceutical Co. Ltd, Covidien Japan Co. Ltd, Tsumura Co. Ltd, and grants from Eisai Co. Ltd, Abbott Japan Co. Ltd, CSL Behring Ltd, Teijin Pharma Ltd, the Japan Blood Products Organization, Torii Pharmaceutical Co. Ltd, and Mitsubishi Tanabe Pharma Corporation. Y. Kakeji has received personal fees from Chugai Pharmaceutical Co. Ltd. J. Sakamoto has received personal fees from Takeda Pharmaceutical Co. Ltd and Tsumura Co. Ltd. K. Yoshida received commercial research grants from Chugai Pharmaceutical Co. Ltd during the conduct of the study, has received grants and personal fees from Taiho Pharmaceutical Co. Ltd, Pfizer Japan Inc., Chugai Pharmaceutical Co. Ltd, Yakult Honsha Co. Ltd, Bristol-Myers Squibb Ltd, and Kyowa Hakko Kirin Co. Ltd, honoraria from Taiho Pharmaceutical Co. Ltd, Pfizer Japan Inc., Chugai Pharmaceutical Co. Ltd, Kyowa Hakko Kirin Co. Ltd, and Yakult Honsha Co. Ltd, and has acted as a consultant or advisor for Taiho Pharmaceutical Co. Ltd and F. Hoffmann-La Roche Ltd. All remaining authors declare that they have no conflict of interest.