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Abstract. – OBJECTIVE: To explore the relation-ship between IgH gene rearrangement and orbital MALT (mucose-associated lymphoid tissue) lym-phoma removal operation prognosis, and to quanti-fy the effect of IgH gene rearrangement on primary orbital MALT lymphoma prognosis.

PATIENTS AND METHODS: Fifty-eight patient cases with primary orbital MALT lymphoma were included in this study. Orbital lymphoma speci-mens were embedded in paraffin for sectioning. IgH gene rearrangement was detected using PCR. The correlation between IgH gene rearrangement and the patient recurrence and survival rates were determined using statistical analysis. The afore-mentioned rates were calculated and a survival curve was determined. p-values lower than 0.05 was considered statistically significant.

RESULTS: We found that the 5-year disease-free survival rate was 90.8% in patients with orbital MALT lymphoma (mean value 56.7 months, range 52-60 months). The use of IgH gene rearrangement detec-tion methods found that the non-recurrence rate of primary orbital MALT lymphoma cases was 79.3%. Survival analysis revealed that IgH gene rearrange-ment was significantly correlated with recurrence of orbital MALT lymphoma (p<0.001).

CONCLUSIONS: IgH gene rearrangement de-tection can be improved by the combined usage of multiple primer pairs, especially family specific primers. In the future, detection of IgH gene rear-rangement may be used as a novel marker to pre-dict the prognosis of patients with primary orbital MALT lymphoma.

Key WordsIgH gene rearrangement, PCR technology, Orbital

MALT lymphoma.

Introduction

The most common type of non-Hodgkin’s lymphoma is mucosa-associated lymphoid tis-sue (MALT) lymphoma, which mainly develops from low-grade malignant B-cell lymphoma of the lymph nodes, although the orbit and gastro-intestinal tract are also usually involved1. Reports have indicated that orbital MALT lymphoma is a

common lymphoma and a common type of solid tumor. Furthermore, orbital MALT lymphoma is very prone to deterioration even after treatment, which greatly affects patient quality of life and survival time2,3. Therefore, it is important to judge the therapeutic effect on the tumor and determine treatment and follow-up plans to monitor tumor recurrence4. Clinical difficulties in the treatment of this type of tumor are mainly due to the diagnosis method employed. Diagnosis is mainly performed using immunohistochemistry of tumor tissue bi-opsies, which facilitates the examination of cell morphology and clinical manifestations. However, this method cannot determine whether the tumor is benign or malignant, which deleteriously im-pacts the formulation of a treatment plan. In recent years, advances in molecular biology technology have started a boom in the use of cell proliferation monoclonality to assist in the diagnosis of tumors throughout the world5,6.

In this work, the pathological examination of tumor tissues in patients undergoing orbit-al tumor resection was carried out, and fol-low-up treatment was established for patients with MALT lymphoma. Immunoglobulin heavy locus gene (IgH) rearrangement in tumor tissues was detected to explore the correlation between gene rearrangement and prognosis.

Patients and Methods

Materials and InstrumentsPrimers were synthesized by Sangon Biotech

Co., Ltd. (Shanghai, China). Immunohistochemical staining kit PV6000 and gum sealing agent were obtained from Beijing Zhongshan Life-form Tech-nology Co., Ltd. (Beijing, China) RNA extraction kit for paraffin embedded tissues, cDNA synthesis kit, and DEPC treated water were obtained from TaKaRa Co. (Otsu, Shiga, Japan). PCR kit was obtained from Sangon Biotech (Shanghai, China)

European Review for Medical and Pharmacological Sciences 2017; 21: 2561-2566

B. DING1, Z.-C. JU2, J.-M. LIU1

1Department of Anesthesiology, Weifang People’s Hospital, Weifang, Shandong, China2Department of Ophthalmology, Weifang People’s Hospital, Weifang, Shandong, China

Corresponding Author: Jiang-mei Liu, MD; e-mail: oipaie3949@163.com

A correlation study between IgH generearrangement and orbital lymphoma removal operation prognosis

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Co., Ltd. Normal saline was obtained from CR Double-Crane Pharmaceuticals Co., Ltd. (Beijing, China) dimethyl sulfoxide (DMSO) was obtained from UNI-CHEM Company (Greenville, SC, USA). A LEICARM-2145 ultra-thin semiautomat-ic microtome (Wetzlar, Germany), LEICADMLB optical microscope (Wetzlar, Germany), PCR am-plification instrument (Thermo Fisher Scientific, Waltham, MA, USA), high-speed tabletop centri-fuge (Shanghai Anting Scientific Instrument Fac-tory, China), pure water system (Merck Millipore, Billerica, MA, USA), ultrasonic cleaner (Southern China Ultrasonic Equipment Factory, China), and electronic balance (Sartorius AG) were used. The study was approved by the Ethics Committee of the WeifangPeople’s Hospital.

All cases were selected from patients undergo-ing eye surgery at the Weifang People’s Hospital between January 2006 and December 2015. Pri-mary orbital MALT lymphoma patients were di-agnosed by pathological diagnosis7. Samples from a total of 68 patients with primary orbital MALT lymphoma were collected, of which 10 were lost during follow-up, resulting in 58 patients in the fi-nal analysis (male:female ratio of 0.61:1). Extended details of the selected cases are shown in Table I.

Methods

DNA Extraction from Embedded Tissue

Fifteen 10 μm tissue sections were excised un-der surgical microscopy (weighing no more than 25 mg). The normal tissue surrounding the tumor was avoided. To remove the residual wax present in tissue samples after embedding, xylene solu-tion and ethanol were used. DNA was extracted from samples after drying. Spectrophotometric determination of optical density (OD) was used to calculate sample purity and concentration. The remaining samples were stored at -30°C.

Primer Design The core fragment sequence of the IgH gene

in the Gene Bank was used in order to design and synthesize primers. Primer sequences were as fol-lows: FR1: VH1, CCTCAGTGATGGTCTCGT-GCAAGG; VH2, TCCTGCGCTGGTGATAGG-CACACA; VH3, GGTCCCTGAGACTCTC

CTGTGCA; FR2A: TGG(A/G)TCGG(C/A)CTG(G/C)C(T/C)(T/C)CNGG; FR3A: ACAC-GGC(C/T)GT(G/A)TATTACTGT; JHa: ACCT-GAGGAGACGCTGACC; JHb: GTGACGAGG-

GTNCGTTGGCCCCAG; JHc: TGAGGAGACG-GTGACCTGGATCCCTTGGCGCCAG. The re-arrangement of IgH gene was detected by PCR. A two-step amplification which amplified the CDR I region first and then subsequently amplified the CDR II and III regions improved the accuracy of the results and the sensitivity of the method7-9.

Table I. Summary of primary orbital MALT lymphoma patient clinical data.

Age Recurrence IgH gene (years) Sex (month) rearrangement

46 Male No No 61 Female No No 53 Male 40 No 56 Male No No 72 Male No No 67 Male No No 68 Female No No 65 Female 50 No 82 Female No No 50 Male No No 49 Female 18 Yes 47 Male 56 Yes 67 Male No No 56 Male No No 87 Female No No 65 Female No No 45 Male No No 43 Female No No 45 Male 55 Yes 82 Male No No 78 Female 36 Yes 76 Female No No 67 Male No No 69 Female No No 89 Male No No 76 Female No No 65 Female 60 Yes 58 Female 30 Yes 52 Female No No 55 Male No No 41 Female No No 66 Female 10 Yes 76 Male 56 Yes 46 Female No No 71 Female No No 46 Male No No 74 Female 32 Yes 53 Male No No 70 Female No No 35 Female No No 82 Female 36 Yes 60 Male No No 83 Female No No 76 Female No No 81 Male No No 49 Female No No 71 Female No No 66 Male 47 Yes

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PCR Amplification PCR was conducted to detect gene rearrange-

ment using primers targeting the J region (JH a and JH b) or V region (FR3A or FR2A), the fol-lowing reaction protocol was used (Tables II, III).

The products from the first round of PCR served as the template for the second round of PCR, which used the identical reaction conditions and system. The primers for the first round of PCR were FR3A (FR2A) and JH a, and the primers for the second round were FR3A (FR2A) and JH b. The CDR I region of the IgH gene was amplified by PCR using the aforementioned conditions and system. The primers for the first round of PCR were FR1 (VH1, VH2, VH3) and JH a, and the primers for the second round of PCR were FR1 (VH1, VH2, VH3) and JH b and JH c10,11.

Detection of PCR Products After the PCR reaction, PCR products and

sample buffer were added together in a 1:5 ratio. The appropriate amount of ethidium bromide was added to a 1% agarose gel, and samples were loaded after gel solidification. After electropho-resis for 30 min under 120 V current, the results were observed under a UV lamp and analyzed with a gel imaging system.

Statistical Analysis To calculate the recurrence rate, postoperative

survival rate, and to investigate the relationship be-tween IgH rearrangement and survival rate, fol-low-up data were collected. Intergroup comparisons were made by paired t-test. Multi-group comparisons were made by ANOVA. χ2-test was applied to test the

qualitative data. Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA), and survival curves of patients were drawn12. p <0.05 was considered statistically significant.

Results

PCR Product Identification After electrophoresis of PCR products, we

found that IgH FR2A, FR3A, and FR1 fragment lengths were 230-270 bp, 80-120 bp, and 80-90 bp, respectively. Positive results presented 1-2 specific clear bands, whereas negative results showed diffuse and non-clear bands (e.g. Figure 1, lane 1). Detailed results are shown in Figure 1.

IgH Gene Detection ResultsUsing the FR3A and FR2A primers, we em-

ployed hemi-nested PCR to detect the presence of IgH gene rearrangement in the tissue samples of 58 patients with MALT lymphoma. IgH rear-rangement bands were observed in a total of 12 cases, suggesting the presence of a clonally rear-ranged IgH gene. Using FR1 (VH1, VH2, VH3) specific primers, as well as JHb and JHc primers, the mixed PCR method was used to perform the second round of amplification. Again, IgH gene rearrangement was found in 12 cases. A positive result was the presence of a single band (Figure 2).

Prognosis After SurgicalTumor Removal

Through the follow-up of 58 patients in this study, we found tumor recurrence in 13 patients (22.4%). The recurrence time ranged from 18

Table II. PCR reaction system.

Component V (μl)

Template DNA 1dNTP 210×PCR buffer 5Primer 2ddH2O 15Total 25

Table III. Two-step PCR reaction conditions.

Reaction Phase Part 1 Part 2

Denaturation 94°C 60 s 95°C 60 sAnnealing 56°C 60 s 62°C 90 sExtension 72°C 90 s 72°C 60 sCycles 35 40Extension 7 min 7 min

Figure 1. Identification of IgH gene rearrangement using FR3A and FR2A primers. 1: control; 2-5: FR3A primers; M: marker; 6-9: FR2A primers.

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to 60 months, with an average of 42.5 months. The 5-year disease-free survival rate was 90.8%. Survival time when no recurrence was observed within 5 years ranged from 52 to 60 months, with an average of 56.7 months. Results are shown in Figure 3 and Table III.

Relationship BetweenTumor Recurrence and IgH Rearrangement

In the 58 patients, the 5-year disease-free survival rate and overall survival rate were both significantly related to IgH gene rearrangement. The 5-year disease-free survival rate in patients with IgH gene rearrangement was significantly lower than in patients without rearrangement (p=0.0023). Results were shown in Table IV and Figure 4.

Discussion

In recent years, ocular adnexal MALT lym-phoma has been examined in a large number of studies, which indicated that MALT lymphomas are solid tumors with high incidence in the orbit and increasing global incidence rates13. The un-derlying mechanisms behind tumor recurrence are complex and unclear. To determine the effect of treatment on the patient as well as the fol-low-up program, Ann Arbor staging and the IPI index are usually used as the gold standard for evaluation14. The clinical effect of these indices is also recognized, but they facilitate the possibility of subjective judgment error when the score co-efficient is low, thus affecting the result. Clinical data has shown that the clinical evaluation coef-ficient of for this tumor type is generally low15.

Figure 2. Detection of IgH gene rearrange-ment using FR1 primers. 1-6: FR1 (JHb) primers; M: marker; 7-10: FR1 (JHc) primers.

Figure 3. 5-year surviv-al rate for patients with (solid line) and without tumor recurrence (dotted line).

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As such, there is a significant deficiency re-garding the use of Ann Arbor staging and the IPI index for evaluating MALT lymphomas16.

Molecular biology technology has rapidly de-veloped, and can presently be applied for the diagnosis of ocular adnexal MALT lymphoma17. Studies have indicated that recombination in the V, D, J, C regions within the immunoglobu-lin heavy chain gene varies with the degree of cell differentiation – that is, IgH gene rear-rangement occurs in the process of lymphocyte differentiation. Lymphocytes exhibit a specific rearrangement pattern that is different from gene rearrangement, indicating the complexity of clon-ality18. Thus, normal PCR amplification products will present differences in product length, lead-ing to a diffuse result19. Since IgH gene rear-rangement can accurately predict the prognosis of patients with primary orbital MALT lymphoma, it is an independent and effective prognostic fac-tor in cases of primary orbital MALT lymphoma, and provides a basis for treatment and follow-up plan development. In this study, we found that the incidence of IgH gene rearrangement was not uniform among 58 patients with primary orbital MALT lymphoma and that presence of IgH gene rearrangement was significantly correlated with tumor recurrence rate. Therefore, IgH gene rear-rangement can be used as an effective index to predict patient prognosis.

Most MALT tumors can be detected using specific primers designed specifically for FR1, but because the experiments are more complex, they are not suitable for clinical application20. By improving the original experimental method, IgH gene rearrangement can be detected using hemi-nested PCR, and FR1 family specific prim-ers and conservative primers were used for ampli-fication. In order to increase the rate of positives, reduce false negatives, and improve the detection rate of IgH gene rearrangement, we improved the PCR method currently available. We first detected specific primers FR3A and FR2A, and amplified FR1 mixed primers (VH1, VH2, VH3) and JH b and JH c primers according to the detection re-

sults. This method is simple and sensitive and can be applied to the clinic. In this study, we found that the positive rearrangement bands of two or more primers could be detected simultaneously in the same patient, possibly because primer amplified bands merely reflect the oligoclonal or subclonal proliferation of infiltrating lymphocytes, and the positive rearrangement bands of amplified by one primer reflects the monoclonal proliferation of tumor cells21,22. There was one case without IgH gene rearrangement, and the possible reason is that DNA has different degrees of decomposition in the embedding, resulting in false negatives. Therefore, a clinical diagnosis should be based on pathological morphology in combination with clin-ical manifestations and immunological phenotype. These parameters should be analyzed from multi-ple perspectives to avoid subjectivity influencing the final judgment23.

Conclusions

In summary, IgH gene rearrangement effec-tively and specifically predicts the prognosis of orbital MALT lymphoma, and can be used as an index to judge orbital MALT lymphoma progno-sis, with great significance in the determination of the treatment and follow-up plans of the patient. However, the relationship between IgH gene re-arrangement and the prognosis of other tumors

Table IV. Tumor recurrence and IgH rearrangement.

Incidence IgH gene number rearrangement rate (%)

Denaturation 94°C 60 s 95°C 60 sAnnealing 56°C 60 s 62°C 90 sExtension 72°C 90 s 72°C 60 s

Figure 4. Survival time after tumor recurrence. **p<0.05.

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remains to be further studied. Whether ocular ad-nexal lymphoid reactive hyperplasia and atypical hyperplasia presents a similar trend as we have found regarding MALT lymphoma is a potential research focus and direction.

Conflict of interestThe authors declare no conflict of interest.

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