Molecular Analyses for BCell Clonality

The evaluation of clonal B-cell expansion is an important preliminary step to better investigate the patho-biologic events implicated in the different stages of progression and in different microenvironments. The assessment of clonality may be dependent on the technique used to identify monoclonal B-cell proliferation in tissue lymphoid infiltrates, and includes immunophenotypic and immunogenotypic analysis [83],

7.2.1. Immunophenotypic analysis

Clonal expansions are characterized by identical rearrangements of the genes codifying for the Igs or T-cell receptors (TCR), thus resulting in homogeneous phenotypic expression of these gene products [181]. Immunophenotypic analysis of peripheral blood cells or resuspended cells from various tissues (lymph nodes, spleen, bone marrow, etc.) is widely employed in the clinical laboratory for the diagnosis and the characterization of lymphoproliferative disorders. Phenotypic characterization of clonality on B lineage cells involves the use of mono- or polyclonal antibodies against the heavy and light chains of the Igs. Immunophenotyping employs peroxidase-conjugated antiserum specific for heavy (IgM, IgA, IgG) or light (X or k) chains applied directly on tissue sections to identify monoclonal populations. The appearance and expression of the Igs during ontogeny of normal B lymphocytes is well established, and permits a clear classification of the developmental stages of their B-cell neoplastic counterparts [182, 183]. Thus, all of the B-cell expansions capable of synthesizing and expressing Igs are well characterized by their cytoplasmic or membrane Ig expression, with the characteristic light-chain restriction (e.g., the expression of either k or X light chain, but not both). Also, the intensity of membrane Ig expression can be correlated with the level of B-cell differentiation and, obviously, with different types of B-cell neoplastic expansion. In SS, some authors had used in past years immunologic phenotyping in order to determine the nature of the lymphoproliferative disorder. Discrepancies between histopathology and immunophenotypic analysis may be resolved by immunogenotypic analysis.

7.2.2. Immunogenotypic analysis B-cell

Immunogenotyping is a more sensitive technique in which clonal rearrangements of DNA can be detected in biopsy tissue extracts by molecular hybridization, using DNA probes specific for immunoglobulin heavy and light chains. The DNA is first digested in vitro by bacterial restriction enzymes, and the restriction fragments of DNA are separated by size using agarose gel electrophoresis, then transferred on to a membrane and exposed to Ig-specific DNA probes. The probe binds only to bands on the membrane that contain homologous DNA sequences. The membrane is then dried, and autoradiography is performed to identify rearrangement bands representing clonal populations. This technique can detect 1 % or more of cells in the biopsy specimen containing the same Ig rearrangement compared with 10% for the immunoperoxidase technique. The immunogenotypic analysis included Southern blot analysis, PCR and in situ hibridization (ISH).

Past studies have used Southern DNA restriction fragment length polymorphism methods to detect clonal expansion of B cells [49, 90] in biopsy specimens lacking clinical lymphoma (i.e., MESA). The molecular evaluation of B-cell clonality by Southern blot hybridization analysis, an approach developed 10-12 years ago, represented a major advance in the study of B-cell lymphomagenesis. Using this technique, the presence of clonal populations in instances other than overt lymphoma could be demonstrated [90, 125, 184, 185], This type of study requires a large amount of fresh tissue, which limits the investigation to patients. Fishleder et al. [90] showed the presence of immunoglobulin-gene rearrengements in the salivary gland lesions that were considered to be benign lymphoepithelial lesions without lymphoma-tous involvement, according to their histologic criteria. These authors further indicated that detectable immunoglobulin-gene rearrengements does not represent overt lymphoid neoplasia. Finally, Bodeutsch et al. [107] described that progression into systemic monoclonal gammopathy or malignant lymphoma exclusively occurred in the subgroup of patients with monotypic plasma cell populations, defined by a k : X ratio of 1:3.

More recently, the PCR has been successfully used for the rapid assessment of B-cell clonality in B-cell tumors by the amplification of immunoglobulin heavy (IgH) and light chain variable-diversity-joining (VDJ) region gene rearrangements [131,195, 196], PCR offers a more sensitive technique for the detection of monoclonal gene rearrangements than Southern blotting and has shown to detect monoclonality in up to 85% of MALT lymphomas [187, 188], De Vita et al. [125] investigated the usefulness of PCR in the detection of B-cell clonality in 7 patients with parotid swelling and suspected malignant lymphoma in the course of SS. The use of PCR to demonstrate clonal immunoglobulin-gene rearrangement has allowed the detection of small monoclonal B-cell pop ulations in paraffin-embedded tissue and this has provided a marker with which to follow the progress of a neoplastic B-cell clone in serial archival biopsy specimens.

Finally, Speight et al. [189] have used ISH for k and X chain mRNA in labial salivary glands, and suggested that the determination of k : X ratios in labial minor salivary glands may provide important prognostic information. Jordan et al. [35] found a high frequency of light-chain restriction detected by ISH in 13 out of 70 cases of SS (19%) and, of the 13 SS cases showing restriction, 4 (31 %) have subsequently developed extrasalivary gland lymphoma. The positive predictive value of this test to identify patients at risk of lymphoma was 31 % with a sensitivity of 67% and a specificity of 86%. Results of immunogenotypic analysis always should be interpreted in the context of the clinical situation and in conjunction with the results of morphological examination. On the whole, it appears that small B-cell clonalities (as detected by highly sensitive molecular approaches) in fully benign lesions may be of little clinical importance and, in our opinion, molecular analyses should not be primarily targeted to such biopsy samples. T-cell repertoire

Few studies have focused on the T-cell repertoire of patients with SS in the course of B-cell lymphopro-liferation [190-192], T-cell expansion may be in turn sustained by chronic antigenic triggering in the local microenvironment, as demonstrated in the model of H. py/ori-associated B-cell lymphoproliferation in the stomach [78, 193]. Thus, the study of T-cell clonal expansion is of great importance in investigating the stages of B-cell lymphoproliferation (fully benign, pseudolymphoma or definitely malignant) which may be still T-cell- and antigen-dependent. Two different, although complementary, approaches are currently employed for the assessment of TCR expression, i.e., reverse transcriptase (RT)-PCR-based molecular analyses and cytofluorometry and/or immunohisto-chemistry studies using monoclonal antibodies [194, 195]. Concerning T-cell expansion in autoimmune diseases predisposing to B-cell malignancies, conflicting results have been obtained in RA [196-198] and few studies have focused on SS [199-201], Methodological differences, as well as the presence of a vast majority of bystander T cells in the tissue lesion (besides the putative pathogenetic initiating T cells), the stage of the disease, the possible role of multiple environmental antigens or autoantigens, and the genetic background of the individual patient, make it understandable why no consensus has been reached regarding restricted or preferential TCR V gene usage [105, 197,201,202],

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment