Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

Abstract

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell-activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non-Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non-specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi-nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR-enzyme-linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl-2 oncogene coupled with a variable segment of the IgH to assess the Bcl-2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P<0.01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.

Fig. 2

Sensitivity of polymerase chain reaction (PCR) technique for V–D–J rearrangement determination; 1, 10, 10², 10³, 10⁴ and 10⁵ clonal B cells from lymphoblastic lymphoma (LM) cell lines were mixed with 10⁵ normal peripheral blood mononuclear cells (PBMC). Lanes 1–6 correspond to the following clonal B cell/PBMC ratios: 1:1, 1:10, 1:1000, 1:10 000, 1:10⁴ and 1:10⁵ Sensitivity determinations using FR1c/JH_1–6 primers (a); FR2/VLJH primers (b) and FR3/LJH primers (c).

Fig. 1

Semi-nested polymerase chain reaction (PCR) amplifications of DNA from labial salivary glands biopsies of Sjögren's syndrome (SS) patients and subject controls. (a) FR3-VLJH2 protocol: one band indicating monoclonal immunoglobulin heavy chain (IgH) rearrangement from pSS (lanes 1–4, 7 and 8) and a smear of bands corresponding to polyclonal IgH rearrangement from CS (lanes 5, 6); (b,c) FR2-VLJH protocol: one or two bands indicating monoclonal IgH rearrangement from SS (b, lanes 1–3; c, lanes 7, 8 and 12) and a smear of bands corresponding to polyclonal B cell expansion (b, lanes 4–6) or containing polyclonal and clonal IgH rearrangement from CS (c, lanes 9–11). Negative control in lane 13 and positive control, lane 14; (d) FR1c/JH_1–6 protocol: one or two bands indicating monoclonal IgH rearrangement from primary SS (pSS) (lanes 2–4) and from secondary SS (sSS) (lanes 7–9) and weak smear or bands corresponding to polyclonal B and clonal B cell expansion from chronic non-specific sialadenitis (CS) (lanes 5, 6 and 11). Positive control in lane 1 and negative control in lane 10. MW: molecular weight marker.