Gastric Cancer With Primitive Enterocyte Phenotype: An Aggressive Subgroup of Intestinal-type Adenocarcinoma : The American Journal of Surgical Pathology

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Gastric Cancer With Primitive Enterocyte Phenotype

An Aggressive Subgroup of Intestinal-type Adenocarcinoma

Yamazawa, Sho MD*; Ushiku, Tetsuo MD, PhD*; Shinozaki-Ushiku, Aya MD, PhD*; Hayashi, Akimasa MD, PhD*; Iwasaki, Akiko MD*; Abe, Hiroyuki MD, PhD*; Tagashira, Amane MD*,†; Yamashita, Hiroharu MD, PhD; Seto, Yasuyuki MD, PhD; Aburatani, Hiroyuki MD, PhD; Fukayama, Masashi MD, PhD*

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The American Journal of Surgical Pathology 41(7):p 989-997, July 2017. | DOI: 10.1097/PAS.0000000000000869
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Some cancers may develop through the reactivation of embryonic developmental programs. This notion has been postulated since the 19th century. On the basis of histologic similarities between tumors and embryonic tissues, Rudolph Virchow and Julius Cohnheim proposed “embryonal-rest hypothesis,” which suggested that cancer arises from the activation of dormant, embryonic-like cells present in mature tissue.1,2 In recent studies, primitive cell-like gene expression signatures have been recognized and reported to be associated with tumor aggressiveness in various cancers including lung adenocarcinoma, breast cancer, bladder cancer, and glioma.3–7 Because the hallmark traits of primitive cells—self-renewal, multipotency, and unlimited proliferation capacity—parallel the high proliferative capacity and phenotypic plasticity of cancer cells, upregulation of embryonic developmental programs may be associated with the highly aggressive nature.

In gastric cancer, adenocarcinoma with enteroblastic differentiation may be a representative example of “primitive phenotype” cancer because of its morphologic resemblance to early fetal-gut epithelium as well as frequent upregulation of oncofetal proteins such as alpha-fetoprotein (AFP) and glypican-3 (GPC3).8,9 In addition, previous studies have reported that several embryonic stem cell marker genes, such as spalt-like transcription factor 4 (SALL4), LIN28, and claudin-6 (CLDN6), were frequently expressed in a subset of gastric cancers including carcinoma with enteroblastic differentiation and AFP-producing gastric adenocarcinoma.9–12 Importantly, upregulation of these primitive phenotypic markers is associated with a highly aggressive nature in gastric cancer.8–10,12–14 Although previous studies focused on individual markers in isolation, expression profiles of a panel of genes associated with the primitive phenotype and its correlation with clinicopathologic significance have not been systemically analyzed in gastric cancer.

This study aims to better characterize the clinicopathologic and molecular features of gastric carcinoma with primitive phenotype. First, we performed an immunohistochemical analysis of a panel of primitive phenotypic markers in a series of gastric cancer cases (n=386), and subsequently, performed hierarchical clustering analysis on the basis of the identified expression profiles. Primitive phenotypic markers analyzed in this study included embryonic stem cell markers (OCT4, NANOG, SALL4, CLDN6, and LIN28) and known oncofetal markers in the stomach, AFP and GPC3, both of which are expressed in non-neoplastic gastrointestinal epithelium only during early fetal development.15–17 Second, we assessed the correlation of primitive phenotype with clinicopathologic parameters as well as molecular phenotyping based on the status of mismatch repair (MMR) proteins, Epstein-Barr virus (EBV), and TP53. Finally, we performed in silico analysis using the Cancer Genome Atlas (TCGA) database to validate our observations and to further characterize the molecular basis of gastric cancer with primitive enterocyte phenotype (PEP).


Case Selection

This study included 386 consecutive cases of gastric adenocarcinoma surgically resected between January 2007 and December 2010 at Tokyo University Hospital, Tokyo, Japan. We excluded cases undergoing neoadjuvant chemotherapy, palliative surgery, or second surgery for recurrent disease.

The study was approved by the Institutional Review Boards of Tokyo University Hospital.

Clinical Data

Demographic and clinical follow-up data were obtained by reviewing medical records. Tumor staging was performed according to the tumor-node-metastasis classification system.18 Early cancer was defined as a T1 tumor and advanced cancer as a tumor with T2 or higher stage.

Histologic Assessment

The following histologic features were recorded: histologic type, tumor size, depth of tumor invasion (T-stage), lymphovascular invasion, and nodal metastasis. The histologic type was determined according to the criteria of Lauren19 classification. Two distinct histologic types, fetal gut-like adenocarcinoma and hepatoid adenocarcinoma, were also recorded when present.

Immunohistochemical Analysis

Tissue microarrays were constructed for immunostaining and in situ hybridization by using a manual tissue arrayer (Beecher Instruments Inc., Sun Prairie, WI). We punched and retrieved duplicate 2-mm-diameter tissue cores from each donor’s tissue block and arrayed them in recipient blocks. Each array block contained 48 tissue cores from 24 tumors. Immunohistochemical staining was performed on tissue microarray samples using the Ventana Benchmark automated immunostainer (Ventana Benchmark; Ventana Medical Systems Inc., Tucson, AZ), according to the manufacturer’s instructions. The primary antibody used in this study is listed in Table 1. The presence of EBV in tumor cells was confirmed by in situ hybridization targeting an EBV-encoded small RNA (EBER-ISH) with an FITC-labeled peptide nucleic acid probe (Y5200; Dako, Glostrup, Denmark) and a polyclonal anti-FITC antibody (dilution 1:25; Thermo Fisher Scientific, Waltham, MA).

List of Antibodies Used for Immunohistochemistry

All samples were evaluated by at least 1 junior pathologist (S.Y. or A.I.) and 1 senior pathologist (A.S.-U. or T.U.). Discrepant cases were discussed by consensus review. As for the evaluation of primitive phenotypic markers, nuclear staining for OCT4, NANOG, and SALL4, cytoplasmic staining for AFP and LIN28, membranous staining for CLDN6, and membranous or cytoplasmic staining for GPC3 were evaluated. The scoring system was 3-tiered according to the percentage of positive tumor cells: negative, no staining in any tumor cell; focal, positive staining between 1% and 49% tumor cells; and diffuse, positive staining in 50% or more tumor cells.

MMR deficiency was defined by the complete loss of nuclear staining in tumor cells for at least one of the 4 proteins (MLH1, PMS2, MSH2, or MSH6). TP53 overexpression was defined as positive staining in 50% or more tumor cells. Tumors with diffuse EBER-ISH labeling were considered EBV-positive gastric cancers.

In Silico Analysis of TCGA Database

The clinicopathologic information and molecular data including the gene expression profile and mutated gene list of each tumor were obtained from the TCGA database.20 We analyzed the correlations of primitive phenotypic gene expression at the mRNA level with molecular phenotype and clinicopathologic features, including patient prognosis.

Statistical Analyses

All statistical analyses were conducted with JMP Pro 11 software (SAS Institute Inc., Cavy, NC). We performed hierarchical clustering analysis by the Wald clustering linkage method. The Fischer exact tests in analyzing correlation between each group and clinicopathologic variables was used. The Kaplan-Meier methods and log-rank test for survival analyses and Cox proportional hazard regression models for multivariate survival analyses were used. Differences were considered significant when the P-value from the 2-tailed test was <0.05.


Expression of Primitive Phenotypic Markers

By immunohistochemical analysis, 149 of 386 (38.6%) tumors were positive for at least 1 primitive phenotypic marker, including SALL4 (32.3%, focal in 23.0% and diffuse in 9.3%), CLDN6 (7.4%, focal in 4.1% and diffuse in 3.3%), GPC3 (18.6%, focal in 16.3% and diffuse in 2.3%), and AFP (2.6%, focal in 2.3% and diffuse in 0.3%), whereas none of the cases were positive for OCT4, NANOG, and LIN28 (Fig. 1). Of note, expression patterns of SALL4, CLDN6, GPC3, and AFP were significantly correlated to one another (P<0.001 in any combination of the 4 proteins, Fig. 2). Ten AFP positive tumors, that is, AFP-producing adenocarcinomas, were invariably positive for both GPC3 and SALL4 and 8 (80%) of them were also CLDN6-positive.

Immunohistochemistry and in situ hybridization. Representative examples displaying positive staining for AFP (cytoplasmic) (A), CLDN6 (membranous) (B), GPC3 (membranous & cytoplasmic) (C), and SALL4 (nuclear) (D). An example with loss of MLH1 nuclear expression (E). Diffuse nuclear labeling with EBER-ISH in an EBV-positive carcinoma (F).
Hierarchical clustering analysis by expression profiling of primitive phenotypic markers and correlation with other features. Gastric cancers (n=386) were clustered into 3 subgroups by immunohistochemical evaluation of primitive phenotypic markers including SALL4, CLDN6, GPC3, AFP, NANOG, LIN28, and OCT4. Intestinal-type histology was predominant in groups 1 and 2. Fetal gut-like histology was exclusively seen in group 1. Most tumors with EBV or MMR deficiency were clustered into group 3.

On the basis of the expression profile of each tumor, a hierarchical clustering analysis classified the 386 tumors into 3 groups: group 1 (primitive phenotype, n=93), characterized by AFP, CLDN6, GPC3, or diffuse-SALL4 positivity; group 2 (SALL4-focal, n=56), which shows only focal SALL4 expression; and group 3 (negative, n=237), which comprises all negative markers (Fig. 2). The sensitivity of each marker for identifying group 1 tumor is summarized in Table 2. Combinations of GPC3 plus SALL4-diffuse or CLDN6 were highly sensitive for identifying group 1 tumors with sensitivity of 87% and 94%, respectively. These triple markers in combination detected all of the group 1 tumors.

The Sensitivity of Each Marker for Identifying Tumors With Primitive Enterocyte Phenotype

Correlation With Clinicopathologic Features

The clinicopathologic features of the 3 groups are summarized in Table 3. The female-to-male ratio was significantly higher in group 3 (35%) compared with group 1 (18%, P=0.002) and group 2 (16%, P=0.006). There were more patients under 65 years old in group 3 (44%) than the other groups (34%), although this did not reach statistical significance. Group 1 exhibited a significantly higher frequency of advanced staging (T2-T4), presence of vascular invasion, and lymph node metastasis greater than those in group 2 or 3. In terms of histologic type, a majority of group 1 (83%) and group 2 (88%) cases were of the intestinal type, whereas intestinal-type and diffuse-type histologies were almost equally represented in group 3 (Fig. 3A). To better characterize the histologic features of group 1 tumors, intestinal-type tumors were further classified into tubular, papillary, solid, and mixed of them according to the WHO classification,21 but we could not find significant difference in the histologic pattern among the 3 groups. Of note, fetal gut-like pattern (n=13) and hepatoid pattern (n=1) were noted exclusively in group 1 (Fig. 3B).

Clinicopathologic Features of Groups 1, 2, and 3 Gastric Adenocarcinomas
Histology of gastric carcinoma with primitive phenotype. Most tumors comprised intestinal-type adenocarcinoma with high-grade nuclear atypia (A). Thirteen tumors show characteristic histology of fetal gut-like adenocarcinoma composed of columnar neoplastic cells with clear cytoplasm (B).

Association With Molecular Features

In our cohort, 26 (6.7%) tumors were EBV positive and 27 (7.0%) were MMR deficient (26 cases with loss of MLH1 and PMS2 expression and a single case with loss of MSH2 and MSH6 expression). These 2 subtypes were mutually exclusive and mostly classified into group 3: 20 of 26 (76.9%) EBV-positive tumors and 22 of 27 (81.5%) MMR-deficient tumors were clustered into group 3 (Fig. 2). TP53 overexpression was more frequently seen in group 1 (51%) than in group 2 (39%, P=0.182) or group 3 (27%, P<0.001).

Patient Outcome and Prognostic Factors

The Kaplan-Meier survival plots demonstrated that group 1 was significantly associated with poor disease-specific and disease-free survival when compared with groups 2 and 3 (Fig. 4). Tumor size, T-stage, lymphovascular invasion, and TP53 overexpression were also significantly associated with disease-specific and disease-free survival, while other features, including EBV infection, MMR deficiency, and histologic type, were not associated with differences in disease-free and disease-specific survival (Table 4). In multivariate analysis, group 1, in addition to tumor size and T-stage, was independently associated with reduced disease-free survival, although there was no significant impact on disease-specific survival (Table 4).

Survival analysis. The Kaplan-Meier curves demonstrated that patients in group 1 experienced significantly worse prognosis than those in the other 2 groups in terms of disease-specific survival (A) and disease-free survival (B). P-values less than 0.05 are indicated in bold.
Univariate and Multivariate Analyses for Disease-specific Survival and Disease-free Survival

Validation in TCGA Database

We analyzed a cohort (n=295) of gastric adenocarcinoma published by the TCGA project for the mRNA expression levels of the 4 genes (SALL4, CLDN6, GPC3, and AFP) characterizing the primitive phenotype in our cohort.20 After excluding cases with no available expression data for any of the 4 genes, or cases with preoperative neoadjuvant therapy or palliative surgery, 210 cases were included for further analysis. On the basis of the z-scores for reads per kilobase per million mapped reads, expression levels of each gene were classified into 3 tiered categories: negative (z-score <1.0), low expression (z-score ≤1.0 to <10.0), and high expression (z-score ≥10.0), which correspond to the scoring system in the immunohistochemical analysis of our cohort (ie, negative, focal, and diffuse). On the basis of this, the 210 tumors were clustered into 3 groups: group 1 (primitive phenotype, n=25), low or high expression of at least one of the 3 genes (AFP, CLDN6, or GPC3) or high expression of SALL4; group 2 (SALL4 low, n=55), low expression of SALL4 but negative for the other 3 genes; group 3 (negative, n=130), negative expression of all 4 genes (Fig. 5A).

Hierarchical clustering analysis in TCGA cohort by primitive gene expression profiles. Gastric cancers of TCGA cohort were clustered into 3 groups by mRNA expression profiles of SALL4, CLDN6, GPC3, and AFP (A). In terms of molecular subclassification, group 1 and 2 cases were predominantly composed of CIN tumors. A majority of group 1 tumors exhibited intestinal-type histology. Mutations of TP53 were enriched in groups 1 and 2. By survival analysis, group 1 patients showed significantly poorer disease-free survival than group 3 patients, although the difference in overall survival was not significant (B and C). RPKM indicates reads per kilobase per million mapped reads. P-values less than 0.05 are indicated in bold.

In terms of molecular subtypes defined by the TCGA project, namely chromosomal instability (CIN), genomically stable (GS), EBV-positive (EBV), and microsatellite instability (MSI), the majority of groups 1 (23/25=92%) and 2 (44/55=80%) were classified as CIN, whereas group 3 consisted of 4 types at nearly the same proportions. Frequent TP53 mutation was significant in groups 1 (17/25=68%, P=0.002) and 2 (37/53=70%, P<0.001) in comparison with group 3 (45/128=35%). Further, in an enrichment analysis employing the cBioPortal for Cancer Genomics, only TP53 mutation was identified as significantly enriched in tumors with SALL4, CLDN6, GPC3, or AFP expression (z-score≥1.0) compared with the other tumors (64.3% vs. 34.4%, q-value=0.0361 when calculated using the Benjamini-Hochberg procedure).20,22,23 Regarding histologic type, group 1 consisted essentially of intestinal-type tumors (23/25=92%), except for 2 tumors with mixed histology. Group 2 was also enriched with intestinal-type tumors (41/54=76%). The frequency of intestinal-type histology was significantly lower in group 3 (80/124=65%) compared with group 1 (P=0.008). Finally, group 1 patients showed significantly worse disease-free survival than group 3 patients (P=0.049), although the difference was not significant in terms of overall survival (Figs. 5B, C).


We identified gastric cancer with PEP on the basis of protein expression profiles of a panel of primitive phenotypic markers. This subgroup (group 1) was characterized by male predominance, intestinal-type histology including fetal gut-like pattern, poor patient prognosis, frequent TP53 overexpression, and little association with EBV and MMR-deficiency. AFP-producing gastric adenocarcinoma—a well-known aggressive tumor entity—was included in this subset. These observations in our cohort were well reproduced in TCGA datasets by analysis based on mRNA expression profiles. We could successfully identify a subset of tumors with primitive phenotypes in TCGA’s cohort, and this subset was also characterized by intestinal-type histology as well as poorer disease-free survival. Frequent TP53 mutation was also a feature of this subset, a finding consistent with our observation of TP53 overexpression in immunohistochemistry.

In terms of molecular subclassification proposed by the TCGA project,20 CIN was the predominant type of gastric cancer with PEP. Group 2 was also enriched with CIN tumors and had relatively similar clinicopathologic features to group 1, but tumors were less aggressive. The majority of EBV, MSI, and genomically stable tumors were not associated with up-regulation of primitive phenotype genes and were included in group 3. The CIN subgroup may be still heterogeneous because this is the largest subgroup comprising half of all gastric cancers, and is defined by the exclusion of EBV and MSI subtypes and based on the presence of extensive somatic copy-number aberrations.20 Therefore, it would be important to further identify distinct subsets within CIN tumors in the quest toward precision medicine. Our observations suggest that PEP gastric cancer is a distinct subset of CIN tumors with aggressive behavior. A recent study by Ahn et al24 demonstrated that EBV-ISH as well as immunohistochemistry of MLH1, E-cadherin, and TP53 could successfully classify gastric cancers into subtypes corresponding to molecular classifications previously reported. Simple methods like this as well as our study would be highly valuable to spur further research on this area or for clinical application in the future, because molecular classifications require complicated and expensive analyses, preventing widespread use.

In comparison with ontogenesis, the expression patterns of primitive phenotype genes in group 1 tumors were comparable with those of gastrointestinal epithelium in early gestation.10,11,15–17 When compared with primitive germ cell tumors, which parallel embryonal development,25 expression patterns of group 1 tumors—positive for SALL4, CLDN6, GPC3, and AFP but negative for OCT4, NANOG, and LIN28—were close to those of yolk sac tumor or immature teratomas.11,26,27 Expression of OCT4 and NANOG is limited to the very early stages of embryogenesis, and is present only in the most primitive form of germ cell tumors, embryonal carcinoma and seminoma.26 On the basis of these findings, PEP gastric cancer in our study is likely to recapitulate fetal gut epithelium in early gestation, but is not as primitive as embryonic stem cells, which corresponds to embryonal carcinoma.25

The mechanism of primitive phenotypic gene activation remains an important question for future analysis. Recent studies have demonstrated that an epigenetic mechanism similar to cellular reprograming might activate embryonic stem cell-like gene expression signature in several tumors.7,28 For example, SALL4 re-expression induced by DNA demethylation was reported in hepatocellular carcinoma and hematopoietic malignancies.29–31 In line with these findings, a recent comprehensive analysis demonstrated that SALL4 was hypomethylated and overexpressed in 34% of 98% gastric cancers, with significant negative methylation–expression correlation (Pearson correlation coefficient=−0.655).32 In addition, the absence of TP53 function has been shown to enhance the efficiency of cellular reprogramming.33,34 Given these observations, we speculate that group 1 tumors in this study develop through primitive phenotypic transformation by an epigenetic mechanism, which is most likely to occur in CIN tumors with TP53 alterations.

The National Institutes of Health TCGA project has highlighted drug-targetable pathways in each molecular subgroup. Relevant targetable pathways identified in CIN tumors were related to receptor tyrosine kinase gene amplifications, including HER2, EGFR, MET, FGFR, and VEGFA.20 Novel agents for these targets have been examined in clinical trials, but the results were largely disappointing, with the notable exception of trastuzumab targeting HER2.35 Because the primitive markers analyzed in this study are not expressed in normal adult tissues, they are ideal therapeutic targets, and in fact, several novel agents are under evaluation. Anti-CLDN6 monoclonal antibodies such as IMAB027 or 6PHU3 have been developed for cancer treatment, and the early phase of clinical trials of IMAB027 has been conducted involving patients with ovarian cancer.36,37 GPC3 is also expected to be a target for immunotherapy as well as monoclonal antibody therapy (Codrituzumab), currently under clinical trials.38–40 As for SALL4, a peptide that inhibits interaction of SALL4 with its downstream target was developed, which successfully reduced tumorigenesis of xenograft hepatocellular carcinoma in the NOD/SCID mouse.41,42 Given the aggressive nature of PEP gastric cancer, development of these novel agents is greatly anticipated.

In summary, we identified PEP gastric cancer as an aggressive subgroup of intestinal-type adenocarcinoma on the basis of expression profiles of a panel of primitive phenotypic markers. Molecular characteristics included frequent TP53 mutation and weak association with EBV and MSI. In terms of TCGA molecular subclassification, this group comprised a distinct subset of CIN tumors. Developing therapeutic strategies targeting primitive phenotypic markers, such as GPC3, CLDN6, or SALL4, for this aggressive tumor is promising in relation to precision medicine approaches.


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gastric cancer; SALL4; GPC3; CLDN6; AFP

Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc.