Primary cutaneous follicle center cell lymphomas and large B cell lymphomas of the leg descend from germinal center cells. A single cell polymerase chain reaction analysis.

Primary cutaneous B cell lymphomas are defined as non-Hodgkin lymphomas that occur in the skin without extracutaneous involvement for 6 mo after diagnosis. They are characterized by a less aggressive course and better prognosis than their nodal counterparts. According to the European Organization for Research and Treatment of Cancer classification, the major subentities of primary cutaneous B cell lymphoma are follicle center cell lymphomas, immunocytomas, and large B cell lymphomas of the leg, which differ considerably regarding their clinical behavior, the former two being indolent, the latter being of intermediate malignancy. In this study, we applied a single cell polymerase chain reaction approach to analyze immunoglobulin V(H)/V(L) genes in 532 individual B lymphocytes from histologic sections of four follicle center cell lymphomas localized on the head and trunk, and four large B cell lymphomas on the leg. We found: (i) in six of eight patients a clonal heavy chain, and in seven of eight patients a clonal light chain rearrangement, all being potentially productive; (ii) no bias in VH gene usage, in four of seven light chain rearrangements the V kappa germline gene IGVK3-20*1 was used; (iii) no biallelic rearrangements; (iv) all V(H)/V(L) genes are extensively mutated (mutation rate 5.4-16.3%); (v) intraclonal diversity in six of eight cases (three of each group); and (vi) low replacement vs silent mutation ratios in framework regions indicating preservation of antigen-receptor structure, as in normal B cells selected for antibody expression. Our data indicate a germinal center cell origin of primary cutaneous follicle center cell lymphomas and large B cell lymphomas independent of those belonging to one of these subentities.

Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.

B cell neoplasias descending from germinal center cells harbor the hallmark of intraclonal diversity resulting from ongoing mutation in the variable parts of their immunoglobulin-encoding genes. To characterize a primary cutaneous follicle center B cell lymphoma in more detail, we analyzed the respective VH and VL genes in single cells mobilized from four sequential biopsies, three taken from the skin and one obtained after internal dissemination from a retrobulbar infiltrate. The lymphoma cells were found to contain V5-51/D6-12/JH5b (heavy chain) and A27/Jkappa2 (light chain) gene rearrangements detected on both the genomic and the transcriptional level. To provide an accurate mutation analysis, the specific VH gene counterpart (V5-51UK) was cloned from the patient's germline. Analyzing 226 single cells, we found: (i) complete nucleotide identity when VH and VL genes of lymphoma cells from one particular biopsy were compared among each other; (ii) intraclonal diversity due to ongoing mutation comparing the sequences obtained from sequential biopsies; (iii) both VH and VL genes to be highly mutated. Deducing from the sequence data, we propose a scenario of the clonal evolution of the B cell tumor in this patient. From the molecular-biological point of view, this primary cutaneous follicle center B cell lymphoma shows the features of a germinal center cell lymphoma. To draw this conclusion from single cell PCR data, however, a sample of sequential biopsies had to be analyzed.

Mainly unmutated V(H) genes rearranged in B cells forming germinal centers in a cutaneous pleomorphic T-cell lymphoma.

B cells in skin lesions of a pleomorphic cutaneous T-cell lymphoma with reactive germinal center hyperplasia were analyzed for their immunoglobulin V(H)DJ(H) gene rearrangements by micromanipulation and single cell polymerase chain reaction (PCR) analysis. In B lymphocytes located in germinal center-like structures, we found in 11/16 different V(H)DJ(H) rearrangements completely unmutated VH genes, suggesting that those cells did not undergo antigen-driven selection. Two V(H) genes showed more than 98% germ-line identity. In only three cells V(H) segments were somatically mutated to a higher extent, but two of these rearrangements were non-productive. These results differ markedly from what we have previously detected in B cells present in mycosis fungoides, another entity of cutaneous T-cell lymphomas where the Ig gene repertoire resembles the situation in peripheral blood with a significantly higher proportion of mutated V(H) genes. When investigating the large atypical B cells strongly expressing CD30 which were detected within the T-cell zone outside the germinal centers, we found again, in most cases, that the rearranged VH genes were completely unmutated. The B cells were of polyclonal origin. Due to this comparable Ig gene repertoire and mutational pattern, we suggest that these cells descend from the germinal center centroblasts which migrated into the T-cell zone and obviously became stimulated to express the CD30 marker. The micromanipulation technique and molecular analysis on the single cell level may provide an important input into our understanding of the mechanisms of immune regulation in cutaneous lymphomas.

Analysis of V(H)-D-J(H) gene transcripts in B cells infiltrating the salivary glands and lymph node tissues of patients with Sjögren's syndrome.

OBJECTIVE: In patients with Sjögren's syndrome (SS), B lymphocytes have been found to infiltrate salivary glands, resulting in sialadenitis and keratoconjunctivitis. The disease is frequently associated with benign and neoplastic lymphoproliferation. The present study was undertaken to investigate whether clonal B cell expansion takes place in lymphocytic infiltrations of salivary glands under (auto- [?]) antigen stimulation, by analyzing in more detail the variable part (V(H)-D-J(H)) of the immunoglobulin heavy chain genes expressed in these B cells. METHODS: Biopsies of the labial salivary glands and lymph nodes were performed on 2 female patients with SS. The Ig gene rearrangements in these tissues were amplified by reverse transcriptase-polymerase chain reaction using specific primers. RESULTS: A total of 94 V(H)-D-J(H) transcripts were cloned and sequenced. Our data suggest a polyclonal origin of the B cell infiltrates. In 92 of the transcripts, V(H) genes were modified by somatic mutation. Further analysis showed counterselection for replacement mutations within the framework regions, suggesting that those B cells were stimulated and selected for functional expression of a surface Ig. In labial salivary glands from both patients, clonally related B cells became evident. Members of 1 particular clone were found in both the lip and lymph node material. CONCLUSION: These data provide evidence, on the nucleotide sequence level, that an antigen-triggered clonal B cell expansion takes place in the salivary glands of patients with SS who do not have histologic evidence of developing lymphoma. It may be speculated that those B cell clones expand during disease progression, resulting in lymphomagenesis.

Analysis of V(H) genes rearranged by individual B cells in dermal infiltrates of patients with mycosis fungoides.

In patients with cutaneous T cell lymphomas such as mycosis fungoides B cells can frequently be detected in the lymphocytic dermal infiltrate. To analyse their immunoglobulin heavy chain gene repertoire, single B cells were obtained from tissue sections of two typical patients with mycosis fungoides using hydraulic micromanipulation followed by specific amplification of the respective gene segments by single-cell polymerase chain reaction (PCR) technique. A total of 21 V(H)DJ(H) genes was sequenced. From each individual B cell a single productive V(H)DJ(H) rearrangement was obtained. There was no clonal relationship detected between any of these rearrangements suggesting polyclonality of the infiltrating B cells. The representation of V(H) families was in accordance with the germ-line complexity. A remarkably high number of V(H) genes (5/13 in patient 1; 3/8 in patient 2) was completely or nearly germ-line-identical. Five of seven V(H)4 family genes were nearly unmutated. On the other hand, most of the V(H)3 gene family members were somatically mutated in an antigen-driven manner. The proportion of germ-line-identical V(H) genes, the usage of individual V(H), D, J(H) gene elements, and the pattern of somatic mutations found in the B cells infiltrating skin lesions of patients with mycosis fungoides resembles the peripheral blood repertoire, suggesting a bystander role of these cells.

Molecular analysis of the immunoglobulin VH gene rearrangement in a primary cutaneous immunoblastic B-cell lymphoma by micromanipulation and single-cell PCR.

The immunoglobulin VH gene rearrangement in a primary cutaneous, large-cell (centroblastic and immunoblastic) B-cell lymphoma was analyzed using a micromanipulation/single-cell polymerase chain reaction technique. In all single B cells obtained from CD20-stained skin sections that gave a polymerase chain reaction product (eight of 27 in biopsy I), the same VHDJH rearrangement, consisting of DP-54-DIR1-JH3a genes, was detected, with no intraclonal nucleotide diversity. Comparison with the most closely related germline counterpart showed significantly altered complementarity determining gene regions as a result of somatic mutations, suggesting an antigen-driven selection and expansion ofthis particular B-cell clone. Interestingly, in a biopsy obtained from the patient 9 mo later, during disease progression (deep muscle infiltration), the lymphoma cells again contained the same VHDJH gene rearrangement (six of 18 in biopsy II) without any further somatic mutations. Therefore, it is suggested that the cutaneous lymphoma characterized throughout this study descended from postgerminal center B-cells.

VH genes expressed in peripheral blood IgE-producing B cells from patients with atopic dermatitis.

In this study we analysed the VH genes expressed in IgE-producing peripheral B cells in two patients with severe atopic dermatitis. We found a diverse antibody repertoire containing V(H) genes of five different VH families, no VH2 and 7 genes, with no biased usage of one particular VH gene family. Some VH germ-line segments were found to be used several times within the epsilon transcripts (V3-23, V3-30). Comparison of the IgE-encoding VH genes with the respective germ-line elements revealed the expression of nearly germ-line identical as well as somatically mutated VH genes. In these latter VH genes base substitutions were distributed over both complementarity determining and framework regions. Because of the similarities between the IgE committed B cell repertoire in atopic patients and the peripheral B cell repertoire in non-atopic individuals we conclude that the atopic VH gene repertoire probably reflects the result of an increased class switch of polyclonal B cells, including virgin and mature B cells, in response to a Th2 cytokine profile.