Follicular lymphoma (FL) is one of the most frequent indolent B-cell lymphomas. Most of them arise from the lymph nodes and are at advanced clinical stages at the time of diagnosis.1^–3 They sometimes show high-grade transformation of diffuse large B-cell lymphomas.4 We previously described five duodenal cases; they are more frequently found in the duodenum than in other areas of the gastrointestinal tract.5 They are often localised tumours with multiple polyps around the ampulla of Vater, although five of eight surgically resected cases had adjacent lymph nodal metastasis. Recently there have been several reports of duodenal follicular lymphomas,6^–13 and most reported cases are confined to this region. Localised indolent lymphomas are rare except for MALT lymphomas, which are extranodal lymphomas. Therefore, duodenal follicular lymphomas might have characteristics similar to those of MALT lymphomas. Though we described some characteristics of duodenal follicular lymphomas previously, the number of examined cases was small, and clinicopathological features need to be examined in more cases. The majority of previous reports were single case reports, except for Shia et al.7 We have studied 40 cases of duodenal follicular lymphomas, including the reported cases. In the present paper, we attempted to examine a IgH/bcl-2 hybrid gene and VH gene occurrence in order to study the difference between duodenal follicular lymphomas and nodal and MALT lymphomas.

MATERIALS AND METHODS

All cases were taken from our surgical pathology files for the past 12 years. Diagnostic criteria of the duodenal follicular lymphomas are detailed in a previous report.5

Immunohistology

In order to determine the cellular characters, immunohistological examination was performed with the indirect immunoperoxidase method using dextran polymer-conjugated secondary antibody labelled with peroxidase (Envision+ immunostaining system, DAKO, Glostrup, Denmark). In brief, approximately 3 μm-thick paraffin sections were made and deparaffinised. The sections were pretreated by microwave heating with citrate solution (pH 6.0) for antigen-retrieval before the immunohistochemical procedure. After the blocking of endogenous peroxidase and treatment with normal bovine serum to reduce non-specific staining, the primary antibodies were applied. The primary antibodies included monoclonal antibodies (MAbs) to CD10 and CD5 (Novocastra, Newcastle upon Tyne, UK), MAbs to BCL-2, CD20, CD79a and CD45RO and polyclonal antibody to CD3 epsiron (DAKO, Glostrup, Denmark) and monoclonal antibody to cyclin D1 (Zymed, San Francisco, California, USA). Further steps were carried out according to the manufacturer’s instructions using the Envision+ immunostaining system.

PCR for detecting immunoglobulin gene rearrangement and for detecting t(14;18)

DNA was extracted from paraffin sections according to the method described by Isaacson et al.14

The variable regions (CDR2 and FW3) and VDJ region (CDR3) of the immunoglobulin heavy chain (IgH) gene were amplified by two-step PCR methods according to a previously described method.15 16 If neither template concentration amplified the β-globin product, the case was considered unsuitable PCR. As a negative control, each PCR experiment contained a sample lacking the DNA template. A sample with DNA extract from the lymph node of a diffuse large B-cell lymphoma was used as a positive control.

DNA was extracted from rearranged bands, and oligonucleotide sequences of CDR2 and FW3 were analysed by directly sequence method using an ABI sequencer (ABI PRISM 377) with dye terminators (Perkin Elmer, Warrington, UK), and compared with germline sequences recorded in the GenBank database.

DNA extracted from paraffin-embedded tissues was also assessed for the presence of t(14;18) chromosomes involving the BCL-2 major breakpoint region (mbr) using the protocol of Crescenzi et al17 and the primers MBR5: 5′-TTA GAG AGT TGC TTA CGT G and SKJH: 5′-ACC TGA GGA GAC GGT GCC AGG GT. The products were then subjected to electrophoresis in 2% agarose, transferred to nylon membranes, and probed with an end-labelled BCL-2 mbr oligonucleotide (5-GCC TGT TCA ACA CAG AC). DNA from the t(14;18)/mbr cell lines SU-DHL-6 or RL-7 was used as a positive control.

The products were analysed by Southern blotting with a 32P-end labelled BCL-2 mcr oligonucleotide probe (5-GAC TCC TTT ACG TGC TGG TAC C). DNA from the mcr t(14;18) containing cell line, SU-DHL-16, served as a positive control. In all PCR experiments, DNA from the myeloid leukaemia cell line HL60 served as a negative control. All experiments included a no-template DNA control.

RESULTS

In our department, the incidence of nodal and extranodal lymphomas was approximately equal, and gastrointestinal tract lymphomas comprised 38.9% of the extranodal ones. FLs accounted for 10.5% of all lymphomas and were mostly nodal. Marginal zone lymphomas accounted for 16.0% and were mostly extranodal. Table 1 summarises the clinicopathological features of duodenal FLs.

The patients (17 women and 23 men) were aged 37–80 years. Endoscopy showed that in most cases the main lesions were located around the ampulla of Vater: 38 cases (95%) showed a nodular growth pattern of the involved mucosa surface described as multiple whitish small polyps, protrusions or irregular mucosa on the surface. Two cases had a submucosal lesion, and one of these had a stenosis lesion. Clinical staging according to the Ann Arbor classification was as follows: 30 patients had stage I, 7 patients stage II, and 3 patients stage III. One stage III case was associated with the ileum, and another with stomach and ileum involvement other than the duodenal lesion. The third stage III vase (patient 21) was originally diagnosed with follicular hyperplasia approximately 10 years ago without any other outside lesions; when the patient consulted a doctor because of abdominal discomfort, duodenal and inguinal lymph node involvement of the follicular lymphoma was diagnosed. We re-examined the original duodenal sample and diagnosed follicular lymphoma.

The examined cases were evaluated according to the WHO classification, and histological grading was determined. Thirty-six cases were grade 1 and four were grade 2; none was grade 3a or 3b. All cases were positive for CD10 and BCL-2 protein.

Follow-up clinical data were available in 32 patients. Initial treatment for the 19 patients was surgery alone in 8 cases, chemotherapy only in 2 cases, chemotherapy plus radiation in 1 case, chemotherapy with rituximab in 2 cases, chemotherapy plus eradication in 1 case, and eradication only in 5 cases. Of the 19 patients who received therapy, 11 achieved a complete initial response and 2 achieved a partial response. Recurrence of disease did not occur, including the eight cases who underwent surgical resection. No patient died of disease.

The juxtaposition of the IgH/bcl-2 gene at the major break point was detected in 4/15 cases (27%). Immunoglobulin heavy chain gene rearrangement was detected in 8/15 (53.3%) examined cases. Examination of VH occurrence showed that three cases were VH4: two of them were VH4-34, the other was VH4-21. The other four were VH3-21, VH3-15, VH3-74 and VH3-11. The other case was VH5-51. Sequencing of these genes indicated that homology to the germ line gene was 90.8–95.2% (table 2).

DISCUSSION

In our surgical pathology file, the number of follicular lymphomas was 550, which comprised 10.5% of all lymphomas. A total of 498 cases were lymph nodal and the other 52 were extranodal origin. Therefore, it was confirmed that the follicular lymphomas frequently involved the duodenum in extranodal lymphomas. Of 40 in the present series, 37 were in clinical stage I (n = 30) or stage II (n = 7). These results differ from those of Federico et al3 on nodal follicular lymphomas (p<0.001). Shia et al7 reported 26 primary follicular lymphomas of the gastrointestinal tract; 10 cases were duodenal, which also confirms the distinct specific localisation. In the previous report, 5 cases were diagnosed in almost seven years; an additional 35 cases were diagnosed in the past five years. It is notable that clinicians and gastrologists have more frequently experienced such cases after they began to examine the descending portion of the duodenum as well as the stomach and duodenal bulb. All five cases in the previous report were female, but this gender difference disappeared with larger numbers of cases. In the present series, however, specific localisation of the region, the second portion of the duodenum, macroscopic characteristics, the majority with multiple whitish small polyps, were confirmed.

Histological examination revealed that all cases were positive for CD10 and BCL-2 protein, and were classified as grade 1 or 2. Shia et al7 reported that out of 26 gastrointestinal FLs, only 3 were grade 3. Differential diagnosis of examined cases was MALT lymphomas and mantle cell lymphomas. As gastrointestinal mantle cell lymphomas are almost all CD5-positive with cyclin D1 overexpression, differentiation to mantle cell lymphoma was not difficult. We had few cases that were composed of small to medium sized lymphoma cells, positive for B-cell markers and negative for CD5 and CD10. In these cases without follicular structures, diagnosis as MALT lymphoma or follicular lymphoma is difficult because there is no specific marker for MALT lymphoma. Tzankov et al18 reported a case of primary gastric follicular lymphoma which mimics MALT lymphoma. This also indicates the difficulty of differential diagnosis. We diagnosed such cases as low-grade B-cell lymphoma. Even if these few cases were follicular lymphomas, the positive rate of CD10 of duodenum follicular lymphomas seemed to be higher than that of lymph nodal ones, which was approximately 70%. In the Shia series, 24/26 were CD10 positive.7

Follicular lymphoma cells of this site were uniform, and we had no malignant transformed cases. Shia et al7 described four mixed follicular and large cell cases. In their three cases, two cases were confined to the mucosa and submucosa, and the other one showed deep invasion beyond the submucosa. These findings coincided with their slow clinical course. In our series, 8 cases were surgically resected, 6 were treated with Helicobacter pylori eradication, and 5 were treated with combination chemotherapy. One case was treated with chemotherapy with irradiation, and 13 cases were followed up without treatment. Cases treated by surgery, and chemotherapy with irradiation were in complete remission. The other cases were alive with disease for 1–60 months. One stage III case showed dissemination for 10 years. Therefore, this tumour is quite indolent but has malignant potential like the MALT lymphomas. As our patients have been followed up for rather a short period, the conclusive long term survival rate with special references to their treatment should be checked in the future. Gastric MALT lymphomas which are resistant to eradication are likely to have their clinical course improved by irradiation19; indeed one cases of our series responded to irradiation, including therapy. A recent report showed successfully radiation therapy for a duodenal follicular lymphoma.20 The best therapeutic options should be determined in the future.

With PCR analysis, immunoglobulin gene rearrangement was detected in 53.3% of examined cases, and rearrangement of IgH/bcl-2 gene at the major break point was detected in 27% of cases. The former ratio was slightly low, mainly due to the small biopsy sample, and extranodal lymphoma was less frequently detected by PCR analysis.15 The latter ratio resembled that of Japanese nodal FLs.21 Nomura et al9 also detected t(14;18) by FISH analysis, and Bende et al, Matsuzawa et al and Shia et al also reported such cases.7 10 11 These findings strongly indicate that duodenal FLs have the same chromosomal abnormality as nodal FLs.

Three of 8 examined cases were VH4, and 2 were VH4-34. The biased VH gene type is suggested to be related to specific antigen selection. VH occurrence in peripheral blood lymphocytes shows VH3 predominance; only 5% are VH4.22 Nodal follicular lymphoma also shows VH3 predominance.23 In contrast, MALT lymphoma cells of various organs are VH4 dominant (table 3).15 24^–32 These data might suggest that duodenal follicular lymphomas have intermediate characters of nodal FL and MALT lymphomas, although the number of examined cases did not cover all cases.

It is interesting that Toyoda et al reported a case of duodenal FL which regressed following eradication of H pylori.33 This case indicates that some duodenal FLs are dependent on antigenic stimulation, such as MALT lymphomas. Bende et al reported a case of duodenal follicular lymphoma which expressed α-4,β-7-integrin, which also is a characteristic of MALT lymphoma of the gastrointestinal tract.10

In conclusion, duodenal FLs are distinct and careful clinical checking seems increase the number of cases which are diagnosed. The chromosomal abnormality deviation of VH occurrence as well as their clinicopathological features might reflect that they are composed of intermediate feature of nodal FLs and MALT lymphomas.