Expression Level of Valosin Containing Protein is Associated with Prognosis of Primary Orbital MALT Lymphoma

Non-Hodgkin’s lymphoma (NHL) constitutes one half of malignancies arising in the orbit and the ocular adnexae. Mucosa-associated lymphoid tissue (MALT) lymphoma, a kind of B cell lymphoma, is the most common histological category in this anatomic region (Eckardt et al., 2013; Vollmer, 2013). A substantial percentage of NHL arise from tissues other than lymph nodes and even from sites which normally contain no lymphoid tissue (Padhi et al., 2012). In recent years, the incidence of orbital and ocular adnexal MALT lymphoma is increasing worldwide (Sjö et al., 2008; Moslehi et al., 2011). According to the ILSG classification, MALT lymphoma irrespective of origin is thought to be an indolent disease as reflected by the good general prognosis of patients (Smiljanic et al., 2013). However, it has been reported that the recurrence rate of MALT lymphoma is up to 26.1% (Charlotte et al., 2006). Moreover it is remarkable that as a part of systemic disease, primary orbital MALT lymphoma has the possibility of recurrence in the distant organs after initial therapy (Raderer et al., 2005; Sjö et al., 2009; Goda et al.,


Introduction
Non-Hodgkin's lymphoma (NHL) constitutes one half of malignancies arising in the orbit and the ocular adnexae.Mucosa-associated lymphoid tissue (MALT) lymphoma, a kind of B cell lymphoma, is the most common histological category in this anatomic region (Eckardt et al., 2013;Vollmer, 2013).A substantial percentage of NHL arise from tissues other than lymph nodes and even from sites which normally contain no lymphoid tissue (Padhi et al., 2012).In recent years, the incidence of orbital and ocular adnexal MALT lymphoma is increasing worldwide (Sjö et al., 2008;Moslehi et al., 2011).According to the ILSG classification, MALT lymphoma irrespective of origin is thought to be an indolent disease as reflected by the good general prognosis of patients (Smiljanic et al., 2013).However, it has been reported that the recurrence rate of MALT lymphoma is up to 26.1% (Charlotte et al., 2006).
Moreover it is remarkable that as a part of systemic disease, primary orbital MALT lymphoma has the possibility of recurrence in the distant organs after initial therapy (Raderer et al., 2005;Sjö et al., 2009;Goda et al., 1 Medical College of Qingdao University, Qingdao, 2 Institute of Orbital Disease, General Hospital of Chinese People's Armed Police Forces, 3 Beijing Institute of Basic Medical Sciences, Beijing, China *For correspondence: yanggg@hotmail.com, xiaolihuawj@ sina.com
Valosin-Containing Protein (VCP), a member of the ATPases associated with various cellular activities (AAA) superfamily, acts as a molecular chaperone in many cellular activities.Dai et al have demonstrated the involvement of VCP in the signaling pathway of IκB degradation in human B cell lines (Dai et al., 1998).As we known, IκB is a specific inhibitor of the NFκB transcription factor (Baeuerle et al., 1988).It has become clear that aberrant deregulated NFκB activation is a hallmark of several lymphoid malignancies, particularly MALT lymphoma (Coupland et al., 2013).Moreover, it has been reported that VCP can inhibit apoptosis and facilitate metastasis of the Osteosarcoma Cell Line (Asai et al., 2002).Several studies have revealed VCP is associated with the prognosis of gastric cancer (Yamamoto et al., 2003), colorectal cancer (Yamamoto et al., 2004), and liver cancer (Yamamoto et al., 2003).
Here, we detected the expression level of VCP in 58 samples from primary orbital MALT lymphoma.Combining with the clinical data, the correlation between VCP expression level and the prognosis of the primary orbital MALT lymphoma was analyzed.

Patients
A total of 62 patients underwent curative resections for primary orbital MALT lymphoma at the Institute of  In the present study, the patients were diagnosed exactly with primary orbital MALT lymphoma by the senior pathologists.Before surgery, the patients received comprehensive examinations including physical examination, computerized tomographic (CT) scans of orbit, thorax, abdomen and pelvic, laboratory examinations and bone marrow biopsy.And there was no tumor metastasis in the distant organ.The surgeon removed the tumor as much as possible intraoperatively.
After the incision healed up, all of the patients received the radiation therapy postoperatively.The patients received follow-up visits at 3-month intervals.Through the examination, the recurrence in the orbit and the distant organ could be detected timely.The follow-up period ranged from 24 to 60 months (median, 60 months).

Immunohistochemistry
Samples obtained from the curative resections were fixed in 10% formalin and routinely processed for paraffin embedding.Histologic sections cut at 4 μm were stained immunohistochemically for monoclonal antibody of VCP (abcam, Cambridge, UK).The staining in vascular endothelial cell was used as internal positive control.Stained sections were evaluated in a blinded manner without prior knowledge of the clinicopathologic parameters.The percentage of positive cells was calculated by counting positive cells per 5×100 tumor cells.

Statistical analysis
Time to recurrence was calculated as time to any event related to the same cancer.Overall survival was defined as the time elapsed from the date of surgery to the date of the last follow-up examination or death of any cause.Disease-free survival was defined as the time elapsed from the date of surgery to the date of local or distant recurrence or death of any cause.Statistical analyses were performed using SPSS 17.0 software.The X 2 and Fisher exact test were performed for analyzing the association between VCP expression level and clinicopathologic features of primary orbital MALT lymphoma.Overall survival and disease-free survival curves were calculated by using the Kaplan-Meier method.Different groups were compared with the log-rank test.P values lower than 0.05 were considered statistically significant.

Patients outcome
According to the follow-up visit of all patients in this study, we found that 13/58 (22.4%) patients suffered from tumor recurrence.The mean value of recurrence time was 39.5 months (range from 10 to 56 months).8/58 (13.8%) patients died of tumor, 4/58 (6.9%) patients died of other causes unrelated to the tumor.As shown in Table 1, the 5-year disease-free and overall survival rates were 70.7% and 79.3%, respectively.The mean value of 5-year disease-free survival was 54.7 months (range from 10 to 60 months).The mean value of overall survival in the series was 57.2 months (range from 24 to 60 months).

VCP expression in the tissue samples from patients with primary orbital MALT lymphoma
All the fifty-eight sections of patients that were diagnosed primary orbital MALT lymphoma could be stained by specific monoantibody of VCP.All of the samples were stained by monoclonal antibodies against CD20 (Figure 1B, D).Although the difference of the staining strength was not observed, it was found that the percentage of the VCP stained cells varied from 32% to 95% in these samples.The median value was calculated as 45%.The samples were divided into two groups (level 1 and level 2) according to the median value.The percentage of VCP positive cells lower than 45% was named as level 1, higher than 45% was named as level 2. Thirty sections (51.7%) were regarded as level 1 (Figure 1A).Twentyeight sections (48.3%) were regarded as level 2 (Figure 1C).

The correlation between VCP level and clinical features of patients with primary orbital MALT
We investigated the correlation between VCP expression level and series of clinical features, including age, sex, tumor size, LDH level, erythrocyte sedimentation rate (ESR) level, serum albumin level, B symptoms, and recurrence (Table 2).Interestingly, it was found that patient's tumor size of level 2 is larger than that of level 1 (P=0.008).Meanwhile, 11/28 (39.3%) patients in level 2 recurred, while, only 2/30 (6.7%) patients in level 1 recurred in the follow-up visit period.The recurrence rate of patients of level 1 was significantly lower than that of level 2 (P=0.003).However, there was no correlation between VCP expression level and the other clinical factors.

Survival analysis
It was shown that the 5-year disease-free and overall survival rates of patients are significantly correlated with VCP expression level.Patients with VCP level 1 had better 5-year survival rates than that with VCP level 2 (5-year disease-free survival, 90.0% v 50.0%,P=0.001; 5-year overall survival, 90.0% v 67.9%, P=0.032; Table 3; Figure 2).However, there was no correlation between the other factors and the survival rates (Table 3).

Discussion
In the present study, it has been demonstrated that the expression of VCP varies in different patients with primary orbital MALT lymphoma.The expression level of VCP is significantly correlated with the tumor size, the recurrence rate and 5-year survival rate, which suggests that VCP may be a potential marker for the prediction of prognosis of patients with primary orbital MALT lymphoma.
VCP might play a crucial role in tumor proliferation and metastasis through the activation of NFκB.So it is reasonable that the expression level of VCP is associated with tumor size, recurrence rate and overall survival rate in these patients.
In our cohort of patients, 22.4% of the patients recurred in 5 years.The recurrence rate was similar to the previous study (Charlotte et al., 2006).Moreover, we found that all of these recurred in the distant organ, which suggests that orbital MALT lymphoma, as a part of a systemic disease, may be easy to recur in other organ besides orbit.
The mechanism of lymphoma recurrence has not been clarified.In our study, we surprisingly found that the percentage of VCP positive cells was associated with the prognosis of patients with primary orbital MALT

Figure 1 .
Figure 1.Representative Immunohistochemical Staining for VCP of primary orbital MALT lymphoma samples (×200).A, VCP expression level 1 of primary orbital MALT lymphoma.B, Positive expression of CD 20 in the serial section of (A) section.C, VCP expression level 2 of primary orbital MALT lymphoma.D, Positive expression of CD 20 in the serial section of (C) section

Figure 2 . 5 -
Figure 2. 5-Year Disease-free (A) and Overall (B) Survival of Patients with VCP Level 1 and 2 in Primary Orbital MALT Lymphoma.A significant difference can be