Clonality analysis of lymphoproliferative disorders in patients with Sjögren's syndrome

Abstract

The aim of this study was to clarify the nature of the clonal lymphocyte infiltration in Sjögren's syndrome (SS) patients associated with lymphoproliferative disorders. We examined B cell clonality in lymphoproliferative tissues from six primary SS patients associated with lymphoproliferative disorders or lymphoma by cloning and sequencing of the gene rearrangement of the immunoglobulin heavy chain complementarity determining region 3 (IgVH–CDR3). Three patients with sequential observation showed progressional clonal expansion with the presence of the same subclone in different tissues during the course of disease. Among them, one patient developed mucosa-associated lymphoid tissue (MALT) lymphoma in glandular parotid. The other three SS patients concomitant with malignant B cells lymphomas showed different clonal expansion of B cells between nodal sites and salivary glands. The cloanality analysis indicated that monoclonal B cell population could spread from one glandular site to another site during the course of SS, suggesting that the malignant clone may arise from the general abnormal microenvironment, not restricted to the glandular tissue, in some SS patients.

Introduction

Sjögren's syndrome (SS) is a systemic as well as an organ-specific autoimmune disease with destructive lymphocyte infiltration of salivary and lacrimal glands, which results in dry eyes and dry mouth. Myoepithelial sialadenitis (MESA) or lymphoepithelial lesion (LEL), consisting of T cell and B cell infilitration, are characteristic features of SS that may range from benign to malignant [1,2]. It has been estimated recently by a cohort study that patients with SS have a 16-fold increased risk of developing lymphoma [3]. Lymphomas that occur during the course of SS are mainly B cell lymphomas, the majority being of low-grade marginal zone type, particularly of mucosa-associated lymphoid tissue (MALT) origin, and develop most often from lymphoproliferative lesions in salivary and lacrimal glands [4,5].

Lymphoproliferative malignancy is the most alarming complication of SS [6]. It is suggested that patients with clonal expansion of B cells in their salivary glands are at high risk of developing lymphomas, and hypothesized that chronic exogenous or endogenous stimulation in MESA could play an important role in the lymphoproliferative process during the course of SS through restricted usage of immunoglobulin heavy chain complementarity determining region 3 (IgVH–CDR3) from MESA-associated clones [7,8]. However, predicting and diagnosing lymphoma by clonal analysis alone is unreliable [9,10]. To understand further the relationship between lymphocyte monoclonality and lymphoma development in SS patients, we studied the lymphocyte clonality by cloning and sequencing IgVH-CDR3 in lymphoproliferative tissues at multiple time-points from six SS patients who developed lymphoproliferative disorder (LPD) or lymphoma during the course of disease.

Materials and methods

Patients

Patient profiles are summarized in Table 1. All six patients satisfied the SS classification criteria of the American–European Community Classification Criteria [11]. Patient 1 (61 years old, male) was SS associated with LPD. He suffered initially from abdominal pain, with signs of enlargement of the pancreas at early onset of disease (1988). Malignancy of the pancreas was considered at that time and an operation was performed. The pathological diagnosis was pancreatitis with massive lymphocyte infiltration. About 1 year later the swellings of bilateral submandibular glands emerged (1989). Seven years later, he showed lymphoadenopathy and renal dysfunction (1996). He was diagnosed with so-called pseudolymphoma by histology and immunohistology study. Patient 2 (78 years old, female) showed bilateral enlargements of the lacrimal and parotid glands following the first onset of 1·5 years. Massive lymphocyte infiltration with MESA was proved, and Mikulicz's disease and SS were diagnosed at that time. Patient 3 (79 years old, female) was SS with MALT lymphoma development in the subsequent left parotid gland. Patients 4–6 were SS with nodal B cell lymphoma (Table 1). No steroids or other immunosuppressive mediations were taken at the time of sampling.

Table 1.

Clinical, laboratory, histological features of patients with SS-ssociated pathological lymphocyte infiltration.

No.	A/S*	Dye eye/Dye mouth	RF/ANA	SSA/SSB	Salivary gland involvement†/focus score‡	tissue biopsy	pathologic diagnosis
1	61/M	+/+	n.d/+	−/−	+/1.7	pancreas	pancreatitis
						L-submandibular gland	MESA
						R-submandibular gland	MESA
						lymph node (cervical)	LPD
						kidney	LPD
2	78/F	+/+	+/+	−/−	+/4.0	lacrimal gland	MESA
						MSG	MESA
						lacrimal gland	MESA
						MSG	Mikulicz' disease
3	79/F	+/+	+/+	+/−	+/1.0	R-parotid gland	MESA
						R-parotid gland	MESA
						L-parotid gland	MALT lymphoma
4	64/F	+/+	+/+	−/−	+/3.4	MSG	MESA
						LND	marginal zone B lymphoma
5	67/M	+/+	+/−	−/−	+/1.0	MSG	MESA
						LND	mantle cell lymphoma
6	85/M	+/+	−/−	−/−	+/1.0	MSG	MESA
						LND	mantle cell lymphoma

^*A/S means patient age/sex;

^†+ means at least one objective evidence of salivary gland involvement according to AECC criterion;

^‡+ means focus score > 1.

MSG: minor salivary gland, MESA: myoepithelial sialadenitis, LND: lymph node, LPD: lymphoproliferative disorder.

DNA extraction

Available specimens were studied from six patients, including the initial and subsequent biopsies of the tissues with sequential observation (Table 1). All patients provided informed consent and all the specimens were obtained as part of the diagnostic histological procedure. DNA extraction was performed by DEXPAT (DNA extraction from paraffin − embedded tissue) (Takara Biochemicals, Tokyo, Japan) and polymerase chain reaction (PCR) was performed on the soluble DNA. The integrity of the DNA following extraction from paraffin-embedded tissue sections was confirmed using control DNA primers for gene coding for the cell adhesion molecule E-cadherin (uvomorulin, chromosome 16q22), as described previously [12].

PCR for Ig monoclonanity

Determination of monoclonality through an evaluation of immunoglobulin heavy chain gene rearrangement is a commonly performed and useful diagnostic assay [13]. A modified seminested PCR technique was used to amplify the CDR3 region comprising the V–D–J region of the Ig heavy chain gene (IgVH–CDR3) [12]. For the first round of amplification, the framework 3 consensus primer (Fr3 5′-ACA CGG C [C/T] [G/C] TGT ATT ACT G-3′) and a downstream consensus primer directed at the joining region (LJH: 5′-TGA GGA GAC GGT GAC C-3′) were used. For the second round of PCR, the Fr3 primer was used in conjunction with an inner downstream primer (VLJH: 5′-GTG ACC AGG TNC CTT GGC CCC CAG-3′). In each round the PCR mixture (Takara Biochemicals) contained 50 µl total reaction mixture. The first PCR contained 100 ng of DNA and the second 2 µl of the first round reaction product. Reactions were carried out in a thermocycler (DNA Engine PT-200, Bio-Rad, Hercules, CA, USA), beginning with an initial denaturation of 98°C for 2 min preceding the addition of the DNA polymerase and terminated by an extension step of 72°C for 10 min. Thirty-one first-round and 31 second-round cycles consisting of 96°C for 1 min, 60°C for 1 min and 72°C for 1 min were performed. Use of the primers would be expected to generate a fragment of between 80 and 120 base pairs (bp) in length. The reaction products were analysed on a 10% non-denaturing polyacrylamide gel electrophoresis, stained with ethidium bromide and viewed under ultraviolet light. The dilution experiment by the mixture RAJI cells in normal peripheral blood mononuclear cells (PBMC) revealed that our method could detect one malignant RAJI cell in 10 000 PBMC by applying 100 ng genomic DNA (data not shown).

Cloning and sequencing of PCR products and intraclonal variation analysis

Bands corresponding to VH genes were isolated from 1·5% low-melting agarose gel, and the DNA was purified using a QIAquick PCR Purification Kit (Qiagen, Tokyo, Japan). Approximately 20 ng of purified DNA was cloned using the T-A Cloning Kit (Invitrogen, Tokyo, Japan). Up to 10 clones were picked up at random. Sequencing reactions were performed with M13 prime on the ABI 310 Genetic Analyser [7].

Results

Molecular analysis of B cell clonality

B cell clonality was studied by PCR in various involved tissues in six patients. In patient 1, oligoclonal or monoclonal B cell bands were found initially by PCR in tissues of pancreas (1988); 1 year later (1989) in the left and right submandibular glands, and subsequently 7 years later (1996) in the lymph node and kidney (Fig. 1a). After cloning and sequencing, five distinct B cell clones (including one non-functional rearrangement clone with stop code, clone 4 in Fig. 1b) were identified in the left submandibular glands, two distinct clones were observed in the right submandibular glands and one clone in the pancreas. Seven years later (1996), one of five clones in the left submandibular glands was also found in lymph node and kidney, and two clones with only one amino acid difference was identified in lymph node (Fig. 1b). Meanwhile, marked lymphocytic infiltration was identified by biopsy of the lymph node and kidney, although immunohistochemical staining did not show monoclonality. This case presented oligoclonal or monoclonal B cell expansion with the presence of one simimlar clone in glandular and extraglandular tissues during the course of SS.

Fig. 1.

B cell clonality of patient 1. (a) Polymerase chain reaction (PCR) results regarding B cell monoclonality; #the presence of the same subclone. (b) Sequences of PCR products: the same clone is shown in italics and bold type; 1 amino acid difference is underlined. *Clone 4 was a non-functional clone with a stop codon (Sub.m.gl: submandibular gland; LND: lymph node).

In patient 2, swellings of bilateral lacrimal glands emerged twice. The first biopsies of lacrimal gland and minor salivary gland (MSG) revealed the polyclonal pattern of B cell expansion with mild lymphocyte infiltration identified by histology. After 1·5 years, the same size of B cell monoclonal bands were found by PCR in lacrimal gland and MSG (Fig. 2a). Sequence of CDR3 revealed that monoclonal B cells in the second biopsies of lacrimal glands and MSG were derived possibly from the same progenitor, because each had almost identical CDR3 with only one nucleotide difference, resulting in one amino acid difference (Fig. 2b). Histology of the second MSG showed massive lymphocyte infiltration with lymphoepithelial lesion. Mikulicz's disease type of SS was diagnosed at this time. This case may represent clonal progression from polyclonal to monoclonal B cell expansion in glandular tissues and development of the same clone in different tissues at the same onset.

Fig. 2.

B cell clonality of patient 2. (a) Polymerase chain reaction (PCR) results regarding B cell monoclonality; #the presence of the same subclone. (b) Sequences of PCR products: the same clone is shown in italics and bold type; 1 amino acid difference is underlined (MSG: minor salivary gland; gl. gland).

In patient 3, one of the biopsies of the right parotid glands presented polyclonal B cell expansion, while another part of the same-side parotid glands was oligoclonal. About 1 month later, monoclonal B cells expansion was identified in the left parotid glands (Fig. 3a). DNA sequence of CDR3 identified that the sequential clone in left parotid glands is the same, with one of two clones in the initial right parotid gland (Fig. 3b). The sequential clone in the left parotid gland was proved to be MALT lymphoma by pathological diagnosis. Although it was difficult to draw a conclusion of clonal progression, we detected expansion of the same clone in a different site of the parotid glands within a 1-month interval.

Fig. 3.

B cell clonality of Patient 3. (a) Polymerase chain reaction (PCR) results of B cell monoclonality; #the presence of the same subclone. (b) Sequences of PCR products: the same clone is shown in italics and bold type.

Among three SS patients with nodal B cell lymphoma (patients 4–6), patient 4 (marginal zone B cell lymphoma) showed different monoclonal B cell bands between MSG and lymph node, while patients 5 and 6 (Mantle cell lymphoma) presented the same size of bands in MSG and lymph node (data not shown). Cloning and DNA sequencing data indicated different monoclonal lymphocytes expansion between MSG and lymph node, because each had a distinct IgH–CDR3 sequence (Fig. 4a).

Fig. 4.

(a) DNA sequence of the IgVH–CDR3 region between MSG and lymph node in three SS patients with nodal lymphoma. (b) Comparison of clone 7 in patient 1 and clone 1 in patient 3. The conserved sequences are framed.

IgH–CDR3 amino acid sequences

We compared the IgVH–CDR3 amino acid sequence of our cases to those presented on GenBank using the NCBI protein-blast program. By applying homology criteria by Bende et al., i.e. CDR3 region at least seven amino acids, at least 75% amino acid sequence homology and a length difference between the CDR3 sequences not exceeding three amino acids, we found the conserved sequence motifs in CDR3 region between clone 7 in patient 1 and clone 1 in patient 3 (Fig. 4b) [14]. In addition, clones 6 and 8 from patient 1 showed homology to those reported on GenBank for patients with rheumatoid arthritis [15], and clone 5 to those patients with systemic lupus erythematosus [16].

Discussion

The risk for development of malignant lymphoma in SS patients was reported to be 16 times higher than in a comparable population [3]. It has been reported that salivary lymphoepithelial lesions from SS patients showed high frequent evidence of clonal immunoglobulin gene rearrangement and revealed morphological and/or immunophenotypic evidence of low-grade lymphoma [17,18]. Although many studies had tried to predict the progression to a malignant lymphoproliferative disorder by B cell clonality analysis in MESA-associated infiltrates, prediction and diagnosing of lymphoma still remain to be defined.

In this study, we analysed B cell clonality by focusing on the CDR3 region of IgVH. Because this region is unique to each B cell clone and specific to antigen recognition, the sequence of CDR3 offers the useful clonal signature of an individual B cell [13]. We observed clonal progression of B cells during lymphoma development in SS patients by sequence analysis of the variable region of IgH CDR3 gene. Patient 1 presented monoclonal B cell expansion in pancreas and oligoclonal B cell expansion in submandibular glands. In this patient, one identified subclone still existed in lymph node and kidney after 7 years (Fig. 1). Patient 2 showed progressive expansion of B cells from polyclonal to monoclonal in lacrimal glands and MSG with no evidence so far of malignancy. Sequence analysis indicates a strong possibility that clones in MSG (2002) and lacrimal gland (2002) might be derived from the same progenitor, because only one amino acid difference of CDR3 region was identified between MSG (2002) and lacrimal gland (2002) clone in this patient (Fig. 2). Although we had attempted to target PCR amplification of the longer VH–FR1 to clarify this possibility, we could not sequence the VH–FR1 sequence because of the poor amount of DNA. In patient 3 the sequential malignant clone in the left parotid glands was the same, with one of two clones in the initial right parotid gland (Fig. 3b), providing evidence of expansion of the same clone in different sites of parotid glands. Clonality analysis of these patients may suggest that monoclonal B cell population could spread from one glandular site to another site during the course of SS. Another possibility is that the microenvironment in SS may trigger monoclonal B cell proliferation, because the same clone was identified in different tissues during the course of disease. It has been reported that MESA and neoplastic B cell clones showed a marked VH gene restriction along with the similar CDR3 sequences, indicating that, even from different patients, MESA-associated clones may bind the same or similar antigens and are selected for clonal expansion [7]. In this regard, we found the conserved amino acids sequence motifs in CDR3 in different clones from patients 1 and 3 (Fig. 4b).

Recent studies have proposed that disturbed B cell biology, defects in apoptosis, T cell modulation, persistent antigenic stimulation and the effects of various molecules contribute to the development of lymphoma in SS. In particular, the B cell activating factor of the tumour necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) have been receiving increasing attention because dysregulated BAFF expression in SS has been described to lead to disease progression and perpetuation of humoral autoimmunity [19]. BAFF is a member of the tumour necrosis factor (TNF) superfamily, also called B lymphocyte stimulator (BLys), which specifically regulates B lymphocyte proliferation and survival, and has emerged as a potential survival factor for B cell lymphomas. Continuous B cell activation resulting from increased BAFF may lead to the development of B cell lymphoma in SS [20]. Furthermore, it has been considered that gradual progression from polyclonal to monoclonal in lacrimal or minor salivary glands may lead to lymphoma development in SS patients. From a variety of studies on clonality analysis in SS patients, patients with the same and persistent monoclonal B cell expansion in follow-up biopsy specimens are at higher risk of developing lymphoma. Malignant clones may be brought from clones in initial lymphoproliferative glandular tissues (Table 2) [7,8,10,21–27]. In contrast to this classic story, patients 1 and 2 had been followed without evidence of lymphoma for more than 10 and 4 years, respectively. Although it remains to be determined whether they will develop into lymphoma over a longer follow-up period, the simple detection of B cell clonality cannot be used as a criterion for diagnosis of B cell lymphoma.

Table 2.

The literatures of monoclonality and lymphoma development.

Author	Year of Publication	Journal	Number of Cases	SS Lymphoma	Same Clone†	Diferent Clone‡
Diss PC	1993	New Engl J Med	1	1SS→MALT lymphoma	1
Pablos JL	1994	Arthritis Rheum	14	1SS→B cell neoplasm		1
Jordan R	1995	Oral Surg Oral Med Oral Pathol Oral Radiol Endod	11	4SS→lymphoma	4§
Lasota J	1997	Mod Pathol	1	1SS→MALT lymphoma		1
De Vita S	1997	Arthritis Rheum	6	1SS→Lympoma (DLB)	1
Bathler DW	1998	Blood	7	5SS→lymphoma	4	1
Aiello A	1999	Blood	1	1SS→MALT lymphoma→FL	1
Gellrich S	1999	Arthritis Rheum	2	0 0	0	0
Gasparotto D	2003	Arthritis Rheum	1	1SS→Lymphoma (MZB)	1
Hansen A	2006	Arthritis Rheum	1	1SS→Lymphoma (MZB)	1
			45	16	13	3

^†Same clone: malignant clone is from the initial lymphoproliferative glandular tissues by PCR and/or sequence data to identify same monoclonal or mutated monoclonal B cell expansion with conserved amino acids sequence motifs in their CDR3.

^‡Different clone: different size of monoclonal bands by PCR or same size bands with distinct lgVH-CDR3 between initial lymphoproliferation and lymphoma.

^§Without sequence data, the result is from PCR. DLB, diffuse large B cell lymphoma; FL, follicular lymphoma; MZB, marginal zone B cell lymphoma.

Our three patients with nodal lymphoma, who presented different B cell expansions between MSG and lymphoma tissue (lymph node), contradict this classic story. Additional steps such as genetic events and the effects of various molecules, possibly the BAFF/APRIL system, may be necessary for lymphoma development [19,28,29]. Our data sustain previous hypotheses [10] that microenvironments in SS may serve the conditions to sustain lymphoproliferation and transformation not only in salivary and lacrimal glands but also in extraglandular tissue, and malignant clones can arise from visceral organs and lymph nodes beyond its original target organ, the glandular tissues [10,28].