(+) and eb virus (-)/tp53

Int J Clin Exp Pathol 2015;8(9):11766-11771www.ijcep.com /ISSN:1936-2625/IJCEP0012638

Case ReportA case of gastric cancer with heterogeneous components of EB virus (+)/TP53 (+) and EB virus (-)/TP53 (-)

Ikuo Matsuda1, Kazuomi Kan2, Sadayuki Doi2, Yoshiyuki Motoki2, Masayuki Onodera3, Seiichi Hirota1

1Department of Surgical Pathology, Hyogo, Japan; 2Department of Surgery, Kawanishi City Hospital, Hyogo, Japan; 3Department of Pathology, Kawanishi City Hospital, Hyogo, Japan

Received July 9, 2015; Accepted August 21, 2015; Epub September 1, 2015; Published September 15, 2015

Abstract: Epstein-Barr virus (EBV)-associated gastric adenocarcinoma is a histological subtype of gastric adeno-carcinoma, in which all of the carcinoma cells are basically positive for EBV-encoded small RNA (EBER) by in situ hybridization. Although its typical histology has some overlap with gastric carcinoma with lymphoid stroma, absence of massive lymphoid infiltrate is sometimes observed either in whole or in part. EBV-associated adenocarcinoma is one of the four representative molecular pathological subtypes recently identified by comprehensive genomic analysis of gastric adenocarcinomas. According to the analysis, typical EBV-associated gastric adenocarcinoma constitutes an independent molecular pathological subgroup, which is mutually exclusive to TP53-mutated ad-enocarcinoma with chromosomal instability, another molecular pathological subtype in gastric adenocarcinomas. Here, we report a rare case of gastric cancer heterogeneously composed of EBER (+)/TP53 (+) and EBER (-)/TP53 (-) portions. The EBER (+)/TP53 (+) component with massive lymphoid infiltrate surrounded the EBER (-)/TP53 (-) component showing well to moderately differentiated tubular adenocarcinoma. Although collision of two indepen-dent gastric cancers could be the simplest and most possible explanation for this situation, we discussed another possibility. In the case of gastric collision tumors, concurrent development of EBER (+) gastric adenocarcinomas and EBER (-) gastric adenocarcinomas in a single stomach is a rare incident. Since presence of the EBER (+)/TP53 (+) tumor component is atypical in itself, we also discussed the mechanism of development of the clone.

Keywords: Gastric adenocarcinoma, Epstein-Barr virus, adenocarcinoma with lymphoid stroma, TP53, collision tumor

Introduction

Epstein-Barr virus (EBV)-associated gastric adenocarcinoma is a histological subtype of gastric adenocarcinoma, in which all of the car-cinoma cells are basically positive for EBV-encoded small RNA (EBER) by in situ hybridiza-tion [1]. Therefore, it is reasonable to assume that the EBV infection occurs at the early stage of the tumorigenesis and may be related to its initiation. Although its typical histology has some overlap with gastric carcinoma with lym-phoid stroma, absence of massive lymphoid infiltrate is sometimes observed either in whole or in part. More than 80% of cases of gastric carcinoma with lymphoid stroma are associat-ed with Epstein-Barr virus (EBV) infection [2].

A recent comprehensive genomic analysis of gastric adenocarcinoma identified at least four

molecular pathological subtypes [3]. The first subtype is EBV-associated adenocarcinoma, the second one is associated with microsatel-lite instability characterized by loss of expres-sion of MLH1 protein, the third one is diffuse-proliferating and poorly-differentiated histological subtype characterized by mutations in RhoA signaling pathway, and the last one is intestinal subtype often associated with chro-mosomal instability and TP53 mutation [3]. The subtype with MLH1 loss is often associated with CpG island methylator phenotype [3]. It was reported that these four subtypes are mutually exclusive in general [3]. Therefore, EBER mRNA positivity concurrent with diffuse TP53 protein positivity within a tumor is consid-ered to be an exception rather than a rule.

Histologically, it is common that mixture of dif-ferent patterns composes a tumor. At cellular

Gastric cancer with EBV (+)/TP53 (+) and EBV (-)/TP53 (-) components

11767 Int J Clin Exp Pathol 2015;8(9):11766-11771

level, moreover, tumors are composed of as- sembly of multiple heterogeneous clones. The so-called intratumor heterogeneity has been recently refined as genomic heterogeneity among the tumor cells, revealed by analysis using next generation sequencing [4-6]. The case of gastric adenocarcinoma is no excep-tion. In the routine diagnostic histopathology, the correlation between morphological hetero-geneity and genomic one in the tumor is of clini-cal significance. Morphological heterogeneity of the tumor may sometimes provide an impor-tant clue to genomic heterogeneity of the tumor, which is often associated with responses to particular molecular-targeted therapy. A colli-sion tumor, in which more than two indepen-dent tumors collide to constitute a tumor, might be one of the extreme presentations of intratu-mor heterogeneity.

Here we report a rare case of gastric cancer heterogeneously composed of EBER (+)/TP53 (+) and EBER (-)/TP53 (-) portions. EBER (+)/

TP53 (+) component in the peripheral zone sur-rounded EBER (-)/TP53 (-) component in the central zone. Although we considered that colli-sion of two independent gastric cancers could be the most possible explanation for this situa-tion, we discussed another possibility. Since EBER (+)/TP53 (+) tumor component in this tumor is atypical in itself, we discussed mecha-nism for this phenomenon.

Case report

A 70-year-old Japanese man was admitted to Kawanishi City Hospital for examination of pro-gressive anemia. At the age of 58, he had suf-fered from acute myocardial infarction, for which coronary stenting was performed, and intrahepatic cholangiocellular carcinoma, which was completely resected by partial hepatec-tomy. He had also been followed for type 2 dia-betes mellitus and chronic hepatitis C. Blood test showed that his hemoglobin had decreased from 13.1 to 11.1 g/dL (normal range: 13.2-17.2) during 4 months. Gastrointestinal bleed-ing was suspected. Endoscopic examination of the gastrointestinal tract revealed Borrmann type 2 tumor of around 2.5 cm in diameter in the posterior wall of the gastric corpus (data not shown). The biopsy of the tumor showed the proliferation of tubular adenocarcinoma (data not shown). Pylorus-preserving partial gastrec-tomy was performed.

The HE image of the resected specimen showed that the tumor was saucer-shaped at low power view (Figure 1A), which was composed of the central portion and the peripheral one. The deepest portion of the invasion reached to the superficial layer of the muscularis propria (Figure 1A). The central portion of the tumor mainly consisted of tubular proliferation of atypical epithelial cells, which was diagnosed as tubular adenocarcinoma of well to moder-ately differentiated type (Figure 2A). In con-trast, peripheral portion consisted of tumor cells proliferating diffusely and sparsely, form-ing small aggregates or nests, which was diag-nosed as poorly differentiated adenocarcinoma (Figure 2C). The morphological distinction between the central and peripheral compo-nents of the tumor was clearly highlighted with immunohistochemistry of pankeratin AE1/AE3 (Figures 2B, 2D, 3B). In addition to the morpho-logical differences, the peripheral portion of

Figure 1. Loupe view of the resected tumor recon-structed by two virtual slides. (A) Hematoxylin-eosin (HE) stain. (B) In situ hybridization of Epstein-Barr virus-encoded small RNAs (EBER). (C) TP53 immuno-histochemistry. The area of the whole tumor is out-lined by a dotted curve in (A). In (B) and (C), positive cells are stained brown. The intratumor heterogene-ity was illustrated by in situ hybridization of EBER (B) and immunohistochemistry for TP53 (C). That is, the tumor cells in the peripheral zone, demarcated by a solid curve in each figure, were positive for EBER (B) as well as TP53 (C). In contrast, the tumor cells in the central zone, which is the residual portion of the tumor and is demarcated by dotted and solid lines, were basically negative for EBER (B) and TP53 (C). Both of the two zones were homogeneously positive for MLH1 immunohistochemistry (data not shown). Bar: 5 mm.



the tumor was intermingled with heavy lym-phoid infiltrate, which obscured the distinction between the tumor foci and inflammatory cells (Figure 2C). This so-called lymphoepithelial lesion was reminiscent of EBV-associated car-cinoma such as nasopharyngeal carcinoma. In order to examine a possibility of EBV-associated gastric adenocarcinoma, in situ hybridization of EBER was performed. Unexpectedly, the tumor cells were heterogeneously positive for EBER; that is, the central and tubular portion of the tumor was negative for EBER, while the periph-eral and poorly differentiated portion of the tumor was positive (Figures 1B and 3C).

Further immunohistochemical examination of the tumor showed that the central component of the tumor was almost negative for TP53 except for sparsely distributed positive cells in the mucosal surface, while the peripheral com-ponent was diffusely positive for TP53 (Figures 1C and 3D). Both the central and peripheral

portions of the tumor were positively stained for MLH1 immunohistochemistry (Figure 3E). Thus, the central zone of the tumor was com-posed of EBER (-)/TP53 (-)/MLH1 (+) cells, while the peripheral zone of the tumor was composed of EBER (+)/TP53 (+)/MLH1 (+) cells (Figure 3C-E).

Discussion

In this paper, we report a case of gastric adeno-carcinoma composed of two heterogeneous portions with clear borderline formation: the central core zone and the peripheral zone. The central zone was composed of EBER (-)/TP53 (-)/MLH1 (+) tumor cells. In contrast, the periph-eral zone was composed of EBER (+)/TP53 (+)/MLH1 (+) tumor cells, surrounding the central core zone.

A recent comprehensive genomic analysis revealed that gastric adenocarcinomas are com-

Figure 2. Representative histology of the resected tumor in the central and peripheral zones. (A, B) Central zone. (C, D) Peripheral zone. (A, C) HE stain. (B, D) Immunohistochemistry for pankeratin AE1/AE3. The tumor cells formed tu-bular structure in the central zone (A, B), while they proliferated diffusely in nested or isolated clusters intermingled with lymphoid infiltrate in the peripheral zone (C, D). Original magnification: × 200. Bar: 100 μm.



posed of at least four molecular pathological subtypes, each characterized by association with either EBV, loss of MLH1 protein, TP53 mutation, or mutations in RhoA pathway [3]. It was reported that these four subtypes are mutually exclusive in general [3]. Consistent with this, there were several reports suggesting that EBV-associated gastric adenocarcinomas do not harbor TP53 mutations in most cases [7-11], although some discrepant results were also reported [12-14]. In this regard, the EBER (+)/TP53 (+) tumor component in the peripheral zone appears to be atypical for EBV-associated gastric adenocarcinoma.

The most straightforward explanation for the present case might be a collision tumor com-posed of two independent gastric adenocarci-nomas, one of which is EBER (+)/TP53 (+) while the other is EBER (-)/TP53 (-). EBV-associated gastric adenocarcinomas are reported to be associated with synchronous or metachronous independent adenocarcnomas in the stomach, and Matsunou et al. [15] reported that 22 out of 26 cases (86.4%) of gastric adenocarcino-mas with lymphoid stroma which developed synchronously or metachronously in patients

with gastric adenocarcinomas with lymphoid stroma were EBER-positive. They also described that all of 4 gastric adenocarcinomas without lymphoid stroma which developed synchro-nously or metachronously in patients with gas-tric adenocarcinomas with lymphoid stroma were EBER-positive as well [15]. Since these data indicate that concurrent development of EBER (-) gastric adenocarcinomas is a rare inci-dent in the stomach with EBER (+) gastric ade-nocarcinomas, collision of EBER (+) adenocarci-noma with EBER (-) adenocarcinoma in our case might be an extraordinary event.

As another possibility for the present case, one of the two zones may be a metastasis of other primary tumor to the original gastric cancer. Since the patient had undergone partial hepa-tectomy for cholangiocellular carcinoma 12 years before, the EBER (-)/TP53 (-)/MLH1 (+) zone could be the metastatic recurrence of the cholangiocellular carcinoma. Although the chol-angiocellular carcinoma was also shown to be EBER (-)/TP53 (-)/MLH1 (+) (data not shown), CK7-positivity of the cholangiocellular carcino-ma was not consistent with CK7-negativity of the present gastric cancer (data not shown).

Figure 3. High power view of the resected tumor at the borderline between the central and peripheral zones de-scribed in Figure 1. (A) HE stain. (B, D, E) Immunohistochemistry. (C) In situ hybridization. (B) Pankeratin AE1/AE3. (C) EBER. (D) TP53. (E) MLH1. In each figure, the central zone is in the left half side of the solid line, while the peripheral zone is in the right half side. In (B) to (E), positive cells are stained brown. All of the tumor cells were highlighted by AE1/AE3 immunohistochemistry (B). The central zone of the tumor in the left half of each figure was negative for EBER (C) and TP53 (D), and positive for MLH1 (E). On the other hand, the peripheral zone of the tumor in the right half of each figure was positive for EBER (C) and TP53 (D), as well as MLH1 (E). Original magnification: × 200. Bar: 100 μm.



How did TP53-immunopositivity evolved in EBER (+) tumor cells in the peripheral zone? A candidate cell-autonomous link, if any, between EBV infection and TP53 mutation may be the expression of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family proteins in the tumor cells [5, 16]. As a mechanism of host immunity to virus, APOBEC family restricts replication of viral genomes (for example, EBV genome) by enzymatically intro-ducing cytidine to thymine conversion in the DNA [17]. This may not be achieved without the cost of potential introduction of mutations into the host genome, presumably including TP53 mutation. A recent study by next generation sequencing showed that intratumor heteroge-neity is partly generated by an APOBEC-mediated mechanism [5]. Expression of activa-tion-induced cytidine deaminase (AID), another APOBEC family, in gastric adenocarcinoma was also reported [18]. AID may be an APOBEC fam-ily involved in TP53 mutation in Helicobacter pylori-mediated tumorigenesis of the stomach [18].

The borderline formation between the two com-ponents in the present case can be easily explained by a cell competition mechanism at the junction of the two. A typical example of cell competition may be observed in senescence-associated secretory phenotype (SASP) in aging, where senescent “loser” subclones cea- se to proliferate while the surrounding “winner” cells proliferate in response to cytokines secreted by the senescent “loser” subclones. It is tempting to speculate that cytokine secre-tion, presumably associated with inflammatory infiltration in EBV-positive cancer, may play a role in this borderline formation, just as in SASP. The importance of non-cell-autonomous driving of tumor growth and clonal heterogeneity has recently been experimentally substantiated [19].

In conclusion, we report a rare case of gastric cancer, in which EBER (-)/TP53 (-) component in the central zone and EBER (+)/TP53 (+) one in the peripheral zone are coexistent. A collision tumor of two independent gastric cancers could be the most possible explanation for this situation. Concurrent development of EBER (+) gastric adenocarcinoma and EBER (-) gastric adenocarcinoma in a single stomach is a rare incident. Moreover, EBER (+)/TP53 (+) tumor

component in this tumor is considered to be atypical in itself for EBV-associated gastric ade-nocarcinoma. We speculate that the sharp bor-derline formation of two components in the present case may be promoted by cell-autono-mous as well as non-cell-autonomous mecha-nisms, both of which may be related to EBV infection.

Acknowledgements

We thank all the colleagues in the Department of Surgical Pathology, Hyogo College of Me- dicine, and Department of Pathology, Kawanishi City Hospital, for preparation of pathological specimen. This work was partly supported by Grant-in-Aid for Researchers, Hyogo College of Medicine, 2014.

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Seiichi Hirota, De- partment of Surgical Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 6638501, Japan. Tel: +81798456666; Fax: +8179- 8456671; E-mail: [email protected]

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