**7.** *Hp***-specific CagA oncoprotein may play an important role in the molecular mechanism of** *Hp***-dependent gastric lymphoma**

Previous studies have demonstrated that one of the factors promoting the proliferation of *Hp*-related gastric MALT lymphoma is dependent on the communication between tumor-infiltrating T cells and tumor B cells [32–35]. In addition, CD40 mediated signaling, T helper-2-type cytokines, chemokines such as interleukin-22, the costimulatory molecule CD86, and regulatory T cells (Foxp3+) have been reported to help and promote the proliferation of MALT lymphoma cells [32–35, 92–94]. Through the eradication of *Hp*, malignant B cells are no longer subjected to antigen stimulation, *Hp*-related T-cell interaction, and immune-related regulations; this leads to their gradual regression and death [93, 94]. These findings may explain why MALT lymphomas are more likely to remain localized, and most of them are cured by HPE (**Figure 5**).

Previous studies have shown that the *Hp* strain associated with inflammatory and virulent processes carries the pathogenicity island (*cag*PAI) which includes the *cagA* gene which encodes the CagA protein [95–97]. The CagA protein contains three distinct domains (Domain I to III) at the N-terminal region, and a 5-amino acid-repetitive tandem motif (EPIYA, glutamic acid-proline-isoleucine-tyrosinealanine) at the C-terminal region [98–100]. The EPIYA motif is characterized by the presence of a tyrosine phosphorylation site; the CagA is phosphorylated at this site by the Src-family kinase (SKFs) and c-Abl [100–102]. The *Hp* CagA-positive strain has been reported to be epidemiologically linked with the development of lymphoid follicles and neoplasms of the stomach [45, 103–105]. For example, Eck et al. reported that the positive rate of serum CagA immunoglobulin G antibodies was higher in *Hp*-positive gastric MALT lymphoma patients than in *Hp*-positive patients with chronic active gastritis (95.5% vs. 67.0%) [45]. In addition to epidemiological studies, other studies have found that CagA promotes the proliferation of B-lymphocytes through CagA-phosphorylation-dependent and -independent signaling pathways [36, 106, 107]. Ohnishi et al. reported that in CagA-transgenic

#### **Figure 5.**

*Schema illustrating the direct and indirect lymphomagenesis of* Helicobacter pylori *(*Hp*)-dependent gastric lymphomas, including mucosa-associated lymphoid tissue (MALT) lymphoma, diffuse large B-cell lymphoma (DLBCL)(MALT), and "pure" DLBCL. (A) After a long-term* Hp *infection,* Hp *can cause inflammation and destroy gastric epithelial cells, and stimulate immune B-lymphocytes to migrate into these lesions and progressively develop MALT or lymphoid follicle; and this phenomenon may allow the* Hp *to directly contact B-lymphocyte. Simultaneously, the* Hp *could stimulate the production of* Hp*-specific intratumoral T-helper cells. (B) When* Hp *directly communicates with B-lymphocyte, cytotoxin-associated gene A (CagA), an*  Hp*-specific oncoprotein, can translocate into the subcellular area of B-lymphocyte and can undergo tyrosine phosphorylation (TP); phosphorylated CagA can interact with the cytoplasmic Src homology region 2 domain-containing phosphatase-2 and further trigger the activation of extracellular signal-regulated kinase, p38 mitogen activated protein kinase, and the production of anti-apoptosis proteins, Bcl-2, Bcl-xL, and Bad. CagA can also promote tumor growth and differentiation by activating TP-independent signaling. (C)* Hp *infection also indirectly produces antigens, which can communicate with B-cell receptors (BCRs) and elicit the BCR-related survival signal. Simultaneously,* Hp *can indirectly foster growth and differentiation of lymphoma B-cells with exhaustive help from* Hp*-stimulating helper T-cells, T helper (Th)2- or Th17-type cytokines-mediated signaling, the interaction with chemokines and their receptors, the co-communication of CD40/CD40 ligand, and stimulatory molecules such as CD86/CD28. (D)* Hp *infection can engender responses of CD4+ CD25+ regulatory T-cells (Tregs) in the gastric microenvironment, and these Tregs could suppress immune-mediated pathogenesis and further cause immune evasion of these lymphoma B-cells. In addition,*  Hp*-producing cytotoxic T-cells exhibit defective functionality of perforin and Fas/Fas-Ligand interaction and, thus, have less roles of cytotoxicity and apoptosis for lymphoma B-cells.* Hp*,* Helicobacter pylori*; Lym, lymphocyte; DLBCL, diffuse large B-cell lymphoma; MALT, mucosa-associated lymphoid tissue; CagA, cytotoxin-associated gene; TP, tyrosine phosphorylation; SHP-2, Src homology region 2 domain-containing phosphatase-2; ERK, extracellular signal-regulated kinase; MAPK, mitogen activated protein kinase; BCR, B-cell receptor; Th, T helper; Tregs: CD4+ CD25+ regulatory T-cells.*

mice, CagA caused the occurrence of myeloid leukemia and malignant B-cell neoplasms (histologically similar to DLBCL) through SHP-2 phosphorylation-dependent signaling [37]. Other investigators showed that the deregulation of SHP-2 was associated with the proto-oncogenic functions associated with the creation of lymphoid and hematopoietic progenitor cells [108, 109].

Lin et al. revealed that CagA that is injected into B cells via type IV secretion systems (T4SS) can activate ERK, p38 MAPK, Bcl-2, and Bcl-xL molecules through SHP-2 phosphorylation-dependent signaling [38]. Kuo et al. found that in tumor samples of 64 gastric MALT lymphoma patients who received first-line HPE, the expression of CagA was significantly associated with *Hp*-dependence (*Hp*dependence vs. *Hp*-independence: 68.4% [26/38] vs. 19.2% [5/26], P < 0.001) [39]. Among *Hp*-dependent patients, those with tumors expressing CagA responded more rapidly to HPE when compared to patients with tumors without CagA expression (median time to CR after completing HPE, 3.0 vs. 6.5 months, P = 0.025) [39]. In addition, in gastric MALT lymphoma patients with known tumor invasion depth, CagA expression was closely associated with less depth of tumor invasion (tumors

#### *Revisiting the Full Spectrum of* Helicobacter pylori*-Related Gastric Lymphoma DOI: http://dx.doi.org/10.5772/intechopen.97424*

limited to mucosa/submucosa vs. tumor involved muscularis propria or beyond: 58.5% vs. 22.2%, P = 0.010) [39]. Furthermore, Kuo et al. found that CagA expression significantly correlated with p-SHP-2, p-ERK, p-38 MAPK, Bcl-2, and Bcl-xL expression, and the expression of the aforementioned molecules correlated with the *Hp* dependence of these tumors [40]. When compared with CagA expression alone, the co-expression of CagA, p-SHP-2, and p-ERK provided an augmented positive predictive value (93.3% vs. 81.8%) and better specificity (95.5% vs. 81.8%) for *Hp* dependence in gastric MALT lymphoma patients [40]. Based on the prospective T3206 trial, Tsai et al. reported that CagA expression correlated significantly with *Hp* dependence in gastric MALT lymphoma and DLBCL(MALT) (85.3% [29/34] vs. 33.3% [4/12], P = 0.001) [25]. Furthermore, downregulation or absence of CagA expression was documented in the residual tumor cells of *Hp*-dependent cases after HPE [25]. Ben Younes et al. also showed that CagA correlated with p-PAKT expression in the tumor cells of *Hp*-positive gastric MALT lymphoma and DLBCL patients, although this study did not show an association between CagA and *Hp* dependence [110]. In addition to CagA expression in tumor cells, Sumida et al. found that among patients with t(11;18)(q21;q21)-negative gastric MALT lymphoma, the serum titer of the CagA antibody was significantly higher in patients with *Hp*-dependent tumors than in those with *Hp*-independent tumors [111]. Taken together, these findings imply that the *Hp* CagA oncogenic protein may participate directly in the molecular mechanisms of *Hp*-dependent gastric MALT lymphoma (**Figure 5**).

In addition to gastric MALT lymphoma, the *Hp* CagA strain may be associated with the development of *Hp*-related gastric DLBCL [112, 113]. Peng first showed that the rate of detection of the CagA gene in gastric biopsy samples was significantly higher in gastric DLBCL(MALT) patients (76.7% [23/30]) than in gastric MALT lymphoma patients (37.8% [14/37]) or in patients with gastritis (30.3% [17/56]) [112]. Delchier et al. also revealed that the *Hp*-seropositive rate was greater in gastric DLBCL patients (100% [16/16]) than in those with gastric MALT lymphoma (78% [29/37]), whereas the CagA-seropositive rate was also higher in DLBCL patients than in MALT lymphoma patients (75% [12/16] vs. 44.8% [13/29], P < 0.05) [113]. Considering that a certain proportion of gastric DLBCLs, including DLBCL(MALT) and "pure" DLBCL, as well as gastric MALT lymphoma are responsive to antibiotics eradicating *Hp*, the clues from *Hp*-specific intratumor T cells and the interacting co-stimulatory molecules, and *Hp* CagA-triggering signaling in MALT lymphoma may also participate in the lymphomagenesis of gastric DLBCL [32–35, 92–94]. Regarding the interaction between *Hp*-specific T cells and co-stimulator molecule CD86 expressed in lymphoma B cells [94], Kuo et al. first showed that CD86 expression in tumor cells was significantly associated with *Hp* dependence in gastric DLBCL(MALT) (68.8% vs. 0%, P = 0.001) [114]. This finding is in line with the findings that *Hp*-dependent gastric MALT lymphoma exhibited a higher expression of CD86 than *Hp*-independent gastric MALT lymphoma [115]. Furthermore, Kuo et al. showed that among gastric "pure" DLBCL patients receiving first-line HPE, CD86 expression was more frequently found in *Hp*-dependent tumors than in *Hp*-independent tumors (61.5% [8/13] vs. 25% [2/8], P = 0.023) [41]. Lin et al. also revealed that in cocultures of *Hp* and B-lymphoma cells, *Hp* CagA upregulated CD86 expression in B cells [38], indicating that a proportion of gastric DLBCLs are still dependent on the triggering of T cells by *Hp* CagA for promoting proliferation (**Figure 5**).

When exploring the possible role of CagA in the lymphomagenesis of gastric DLBCLs, Kuo et al. revealed that CagA expression significantly correlated with p-SHP-2 expression and limited stages (I-IIE1, 82% vs. 47%, P = 0.017) in *Hp*-positive gastric "pure" DLBCL patients receiving systemic chemotherapy [29]. Kuo et al. also showed that CagA expression was associated with significantly better CR (CagA[+]

vs. CagA[−]: 89% vs. 59%, P = 0.030), 5-year EFS (CagA[+] vs. CagA[−]: 85.2% vs. 46.3%, P = 0.002), and OS (CagA[+] vs. CagA[−]: 88.9% vs. 52.9%, P = 0.003) [29]. These findings suggest that *Hp* CagA and its regulated signaling participate in the lymphomagenesis of *Hp*-related gastric "pure" DLBCL. Furthermore, Kuo et al. showed a close association between CagA expression and *Hp* dependence of patients with gastric DLBCL (including DLBCL(MALT) and "pure' DLBCL) who received first-line HPE (*Hp*-dependence vs. *Hp*-independence: 74.3% [26/ 35] vs. 25.0% [7/28], P < 0.001) [41]. Furthermore, CagA expression significantly correlated with the expression of p-SHP-2 and p-ERK, and the expression of these molecules was significantly associated with *Hp* dependence of gastric DLBCL [41]. Among *Hp*-dependent gastric DLBCL patients, the median time to CR after completing HPE was quicker in tumors with CagA expression than in tumors without CagA expression (4.0 months vs. 5.0 months, P = 0.050) [41]. Kuo et al. also observed that CagA and CagA signaling molecules were diminished or absent in a series of biopsy samples after HPE [41]. These results indicate that CagA and its regulated signaling molecules may be involved in the pathogenesis of *Hp*-dependent gastric DLBCL (**Figure 5**).

Epidemiological studies have reported that the incidence of gastric MALT lymphoma in East Asia (including Taiwan, Korea, and Japan) is higher than that in Western countries (Netherlands, Italy, and USA) [70, 72, 116–119]. In addition to the distinct prevalence of gastric lymphoma, the rate of CagA-positivity in *Hp* strains was higher in East Asian populations (at nearly 90%), when compared with that in Western populations (at approximately 60%) [120–125]. In contrast to CagA from Western *Hp* isolates containing EPIYA-A, EPIYA-B, and EPIYA-C segments, the EPIYA motifs in East Asian *Hp* strains (including those from Taiwan), mainly consist of EPIYA-A, EPIYA-B, and EPIYA-D segments [36, 97, 100, 126–128]. CagA activates ERK/MAPK signaling in gastric epithelial cells or lymphoma B-cells mainly by interacting with SHP-2; the CagA-SHP-2 complex is characterized by an interaction between the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment of CagA with the SH2 domain of SHP-2 [97, 128, 129]. In addition, the *Hp* CagA strains bearing the EPIYA-D motif had a greater affinity for binding SHP-2, a capacity for phosphorylating tyrosine, and conferred a risk for developing gastric cancer [97, 126, 129, 130]. Chuang et al. assessed the intensity of tyrosine-phosphorylated CagA (p-CagA) in *Hp* strains isolated from Taiwanese patients with a distinct disease status, including gastric cancer, gastric ulcer, duodenal ulcer, and gastritis; the authors reported that the p-CagA intensity was higher in patients with gastric cancer or gastritis accompanied by intestinal metaplasia than in patients with gastritis but without intestinal metaplasia [131], indicating that a higher tyrosine phosphorylation activity of CagA may be associated with a risk of developing precancerous lesions and subsequent gastric cancer [131]. In *Hp* strains isolated from *Hp*-dependent cases of gastric lymphoma including five DLBCLs and six MALT lymphomas, Kuo et al. showed that all cases were CagA-positive strains [41]. In their studies, the positive CagA *Hp* strains were significantly associated with a rapid time to CR after completing HPE in *Hp*-dependent gastric lymphoma patients, including MALT lymphoma and DLBCLs [39, 41]. In addition, CagA expression correlated significantly with p-SHP-2 expression and the expression of tyrosine phosphorylation-dependent molecules such as ERK and p38 MAPK, in the lymphoma cells [39–41]. These findings further support the findings of a systematic review of HPE for treating gastric MALT lymphoma from Zullo et al. the authors investigated the reason for the significantly higher CR rate of tumors in Asian populations (84.1%) than that of tumors in Western populations (73.8%) (P < 0.0001) [62]. Although the association between tumors expressing CagA and the *Hp* dependence of gastric lymphoma may explain why most CagA-positive gastric MALT lymphomas (even for gastric DLBCLs) remain localized and show a quick response to HPE, approximately 30–50% of *Hp*-dependent gastric

lymphoma patients lack CagA expression in their tumors [39, 41]; this suggests that other underlying molecular mechanisms responsible for antibiotic responsiveness may exist, and need to be explored.
