Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B‐cell lymphoma and leukaemia: a novel strategy and its implications

Abstract

Summary. In the search of B-cell lymphoma/leukaemia dissemination to cerebrospinal fluid (CSF), we used the highly sensitive semi-nested PCR (snPCR) for the analysis of IgH gene rearrangements. This method detects a rearranged IgH gene from a single B lymphocyte which may or may not represent the neoplastic B-cell population. We therefore performed multiple snPCR (three to five) experiments on the same CSF sample, postulating that the detection of a band of the same size and sequence in different PCR runs was highly indicative of a clonal population. 17 consecutive cases with a differential diagnosis of primary (PCNSL) (n¼10) or secondary (SCNSL) (n¼7) CNS lymphoma or leukaemia were investigated by the new strategy. The clonal nature of the B-cell population was confirmed in 3/10 of suspected PCNSL, and in six other cases the PCR study was indicative of reactive lymphocytosis. One case revealed a clonal B-cell population in the clinical context of an autoimmune disorder. Evidence of clonal B-cell population was found in 4/7 of suspected SCNSL. In one of these cases the detected band and its sequence proved identical to that of the primary nodal lymphoma. We believe that the evaluation of B-cell clonality in CSF requires multiple snPCR amplification on the same sample to compare the size of the products and, if necessary, the DNA sequences to ascertain the diagnosis of malignancy in equivocal cytologic and clinical findings.

PCR Analysis of Immunoglobulin Heavy Chain (IgH) and TcR-γ Chain Gene Rearrangements in the Diagnosis of Lymphoproliferative Disorders: Results of a Study of 525 Cases

“…Indeed, the sensitivity of PCR in detecting clonal IgH rearrangement is strongly dependent on the relative number of polyclonal lymphoid cells, which are coamplified with the clonal population if present in the sample (24,33,34). As demonstrated in this study, these polyclonal lymphoid cells can be responsible for a pseudoclonal PCR profile (see Figs.…”

“…As reported elsewhere, the sensitivity of the IgH and TcR-␥ PCR for the detection of a clonally rearranged cell population is tissue dependent (24,33,34). Indeed, this sensitivity is dependent on the number of polyclonal cells present in the sample, which is coamplified with the clonal cell population if present.…”

“…2 and 3). Therefore, when IgH gene rearrangement study was performed using tissue specimens containing a relatively small number of polyclonal B cells (i.e., skin and gastrointestinal biopsies), we used a multiple-PCR strategy that involves three to five PCR reactions from the same sample (24). Using this strategy, the demonstration of the same rearranged band in every experiment is highly indicative of the presence of clonal B-cell population in the analyzed sample.…”

“…In our previous reports, we demonstrated that using the sn-PCR assay for the detection of IgH gene rearrangement, a clonalrearranged B-cell population of approximately Ն1% of total cells is detectable in a background of lymph node-based polyclonal B-cells (22). In the absence of polyclonal cell population, this technique was sufficiently sensitive to produce a detectable band from a single IgH gene-rearranged B-cell (24). The applications of the same conditions of PCR amplifications for TcR-␥ genes yielded comparable results (22 and data not shown).…”

“…1), and 70 to 110 bp for FR1c/J H (5), FR2a/J H , and FR3a/J H (2, 3), respectively. For the study of IgH gene rearrangements in tissue specimens with a relatively small polyclonal B-cell population, such as skin and gastrointestinal samples, we used the multiple sn-PCR experiment (24). Three to five FR1c/J H , FR2a/J H , and FR3a/J H PCR amplifications were performed from the same sample, and the demonstration of a band of the same size in all the different PCR runs was considered as indicative of a clonal cell population (Figs.…”