Expression and significance of Her2 and Ki-67 in gastric adenocarcinoma without distant metastasis: a cohort study

A total of 210 GaC patients undergoing surgical resection between October 2013 and December 2014 in the First Affiliated Hospital of Anhui Medical University where yearly the greatest number of GaC patients receive resection [15] were potential candidates for this study. The inclusion criteria were: (1) the patient had not received any pre-surgical chemo-, radio-, or bio-therapy; (2) diagnosis of GaC was confirmed by postsurgical pathological examination of resected specimens by at least two experienced pathologists, which was consistent with preoperational pathology; (3) the patient underwent open radical total or partial D2 gastrectomy as appropriate; and (4) all cancers were adenocarcinomas. The exclusion criteria included: (1) the patient had serious comorbidities; (2) the patient had other malignancies simultaneously; (3) the pathological diagnosis was controversial; (4) the patient had previous gastric malignancies; and (5) the patient had received chemotherapy or radiotherapy before resection. In our institution in China, it is not the clinical routine that patients with gastric or cardia cancers receive preoperative treatment. Finally 195 GaC patients were eligible for investigation (Table 1). The resected GaC specimens were collected after resection. Basic patient characteristics were recorded. The clinicopatholgic data were obtained from the surgery and pathology records. Disease stage was classified or recoded according to the American Joint Committee on Cancer/Union for International Cancer Control TNM staging system, seventh edition [16]. Written informed consent was obtained from each participating individual, and our study was approved by the local Institutional Review Board and carried out following the Declaration of Helsinki [17] and Good Clinical Practice guidelines [18].

Anti-Her2 monoclonal antibody was purchased from the Fuzhou New Biotechnology Development company. The other primary antibodies including the mouse-anti-human Ki-67 monoclonal antibody working solution (MAB-0129), the ready-to-use non-biotin immunohistochemistry streptavidin-peroxidase detection kit (KIT-9902), the diaminobenzidine chromogenic reagent (DAB-2301), the citrate tissue-antigen recovery solution (MVS-0100), and the neutral gum were all obtained from the Maixin Biotechnology Co. Ltd., Fuzhou. The reagents were all stored at 4 °C before use.

The obtained tissues were first hematoxylin–eosin-stained to be confirmed as GaC tissues. The protein marker expression in GaC tissues was detected using immunohistochemistry (streptavidin-peroxidase). As standard, automated platform for immunohistochemistry was used. The staining method has been previously described [19], and the major procedures were: (1) the slides were immersed in 10% silver nitrate solution for 24 h, and then dehydrated in 95% ethanol, followed by water rinsing and air drying. Then they were processed by 2% APES acetone for 30 s, followed by wash with pure acetone and airing; (2) the specimens were fixed in 10% formaldehyde, embedded in paraffin, serially sectioned into 3 μm-thick slices, attached to slides, dewaxed by xylene, and hydrated by gradient ethanol, followed by phosphate-buffered saline (PBS) wash and airing; (3) antigen recovery was conducted using citrate with high temperature and pressure, followed by cooling and water and PBS wash. Then the sections were incubated in 3% hydrogen peroxide deionized water for 5–10 min to inactivate the endogenous peroxidase, followed by water and PBS wash; (4) the Her2 or Ki-67 (working solution) monoclonal antibodies (50 μL) was added to the specimens respectively, followed by incubation at 4° C overnight or at room temperature for 1 h and then PBS wash; (5) the polymer enhancer (50 μL) was added followed by incubation at room temperature for 20 min and then PBS wash. Then the general-type Immunoglobulin G antibody-Horseradish Peroxidase polymer (anti-mouse/rabbit, 50 μL) was added followed by incubation at room temperature for 15–30 min and then PBS wash; (6) the sections underwent color-developing in diaminobenzidine solution (100 μL/section) for 2–8 min with microscopic observation, followed by distilled water rinse for 10 min to stop the reaction and then hematoxylin counterstain for 3 min plus water wash; and (7) the sections were acidized using 1% hydrochloric acid and processed by saturated lithium carbonate solution, followed by rinse. Then the sections were gradient alcohol-hydrated, xylene-hyalinized, and neutral gum-sealed, followed by microscopic observation. The positive tissue section in the pre-experiment set was selected as the positive control, and the negative control was obtained by replacing the primary antibody with PBS.

The stained sections were evaluated by 2 experienced pathologists in a blind way. Observed in high power field (× 400), randomly 5 fields were selected and examined for each section, and the number and intensity of positively-stained cells were recorded and averaged, with 100 cells in each field counted respectively. In case of a discrepancy, a third senior investigator was consulted and agreement was reached by consensus. The positivity level of Her2 expression follows the National Comprehensive Cancer Network (NCCN) Guidelines (https://​www.​nccn.​org): − means “no reactivity or membranous reactivity in < 10% of cancer cells”; + means “faint or barely perceptible membranous reactivity in ≥ 10% of cancer cells; cells are reactive only in part of their membrane”; ++ means “weak to moderate complete, basolateral or lateral membranous reactivity in ≥ 10% of cancer cells”; +++ means “strong complete, basolateral, or lateral membranous reactivity in ≥ 10% of cancer cells”. – or + expression indicates a negative result, and +++ expression suggests a positive finding. In case of ++ expression by immunohistochemistry, in situ hybridization was further performed, and cases with an average Her2 copy number ≥ 6.0 signals/cell were considered positive. For Ki-67, the results were assessed according to the percentage of positive cells: − means very low proliferation activity with proportion of Ki-67-positive cells < 25%; + means low proliferation activity with proportion of Ki-67-positive cells 25–50%; ++ means moderate proliferation activity with proportion of Ki-67-positive cells 50–75%; +++ means high proliferation activity with proportion of Ki-67-positive cells > 75%. A section was regarded negative in Ki-67 expression with < 50% positive cells, and positive with ≥ 50% positive cells following relevant reports published recently [20‐24]. Cancers were further categorized into 4 groups according to combined expression of both markers: double negative, Ki-67 positive and Her2 negative, Her2 positive and Ki-67 negative, and double positive.

Continuous variables were presented as mean ± standard deviation; median (interquartile range), and categorical variables as count (percentage [%]). Clinicopathologic factors in different subgroups by the combined expression of Her2 and Ki-67 were compared using the χ² or analysis of variance test according to data type. Logistic regression was used to determine the association of marker expression with other clinicopathologic factors. Kaplan–Meier survival curves stratified by marker expression were plotted, and prognostic differences across groups were examined using the log-rank test. The association of marker expression with survival was further examined using univariable and multivariable Cox proportional hazards regression. Data were analyzed using the R (v. 3.4.0, Vienna, Austria) statistical software (https://​www.​r-project.​org/​). A finding was statistically significant with a 2-sided P value < 0.05.

For the 195 analyzed patients (Table 1), the mean age was 64 years, and males took up 76%. Most of the tumors were located at gastric cardia and/or fundus (52%), and were poorly-differentiated or undifferentiated (75%). The mean tumor length was 4.8 cm. The mean metastatic lymph node number was 4 based on an average harvest of 19 lymph nodes per patient, and the mean positive-harvested lymph node ratio was 0.20.

Both Her2 and Ki-67 were presented as brown or yellow granules in GaC. Her2 was majorly expressed in cell membrane and the positive expression proportion was 28% (Fig. 1). Ki-67 was located at cell nucleus, and the positive proportion was 64% (Fig. 2). Stronger Her2 expression was associated with poorer tumor differentiation, neurovascular invasion, earlier pT stage, and more advanced pN stage; however, expression of Ki-67 was not significantly associated with the investigated clinicopathologic variables (Table 2). Expression of Her2 or Ki-67 was not significantly associated with overall pTNM stage (Table 3).

Based on the combined expression of both markers, cancers were categorized into four groups (Table 4): double negative (n = 49, 25%), Ki-67 positive and Her2 negative (n = 91, 47%), Her2 positive and Ki-67 negative (n = 21, 11%), and double positive (n = 34, 17%). While no significant differences in patient age, sex, tumor location, size, metastatic lymph node number, lymph node ratio, neurovascular invasion, cancer embolus, or pN stage were observed between the four groups, there existed significant inter-group differences in tumor differentiation (P = 0.001), pT stage (P = 0.009), and pTNM stage (P = 0.022). Patients with negative expression of both markers had highest proportions of poorly-differentiated/undifferentiated tumors (86%), pT stage 3–4 (94%), and pTNM stage III (69%).

The above findings appeared contradictory to the well-known knowledge that strong expressions of Her2 and Ki-67 are both predictors of poor survival, and we further explored the association of the combined expression with pTNM stage in overall, node-negative, and node-positive GaCs and with pT and pN stages using logistic regression. Interestingly, in node-negative disease, negative expression of both markers was significantly associated with the most advanced pTNM stage; however, in overall or node-positive GaC, no significant association of combined expression of the two markers with pTNM stage was observed (Table 5). We further investigated the association of the combined expression of both markers with individual pT and pN stages (Table 6), and performed univariable analysis and 2 sets of multivariable analyses (one with pN and pT stage excluded from covariates, and the other with pN or pT stage included). All models revealed that cancers with negative expression of both markers had the most advanced pT stage; while the combined expression was not significantly associated with pN stage.

Kaplan–Meier analysis showed no significantly different survival across the four groups by Ki-67 (P = 0.17), Her2 (P = 0.62), or combined expression of Her2 and Ki-67 (P = 0.24; Fig. 3). Using Cox proportional hazards regression (Table 7), neither univariable nor multivariable-adjusted analysis showed a significant association of Ki-67 expression with survival; a strongly positive (+++) Her2 expression was significantly associated with poorer survival compared to − expression in multivariable analysis (hazard ratio = 2.17, 95% confidence interval = 1.01–4.68). Using the Cox model, neither univariable nor multivariable-adjusted analysis showed a significant association of combined Her2 and Ki-67 expression with survival (Table 7).