Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations

Abstract

We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.

Pooled analysis of p53 mutations in hematological malignancies

“…Myelodysplastic syndrome. The myelodysplastic syndrome (MDS) cohort of patients included subtypes: refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-T), and a single patient with chronic 1993; Cesarman et al, 1993;Lo Coco et al, 1993;O'Connor et al, 1993;Gaidano et al, 1993;Nakamura et al, 1993;Bhatia et al, 1993;Villuendas et al, 1993;Trumper et al, 1993;Ballerini et al, 1993;Baldini et al, 1994;Farrugia et al, 1994;Watanabe et al, 1994;Chang et al, 1994;De Re et al, 1994;Matolcsy et al, 1994;Matsushima et al, 1994;Edwards et al, 1994;Koga et al, 1994;Preudhomme et al, 1995;Kocialkowski et al, 1995;Knowles et al, 1995;Louie et al, 1995).…”

Apoptosis‐related genes and proteins in Hodgkin's disease

“…The p53 gene has been investigated in HD in several studies (28,45,56,57). Most of these have shown that H-RS cells frequently express p53 protein, but that point mutations are absent or rare (56 (17) studied p53 by singlecell PCR and demonstrated identical point mutations in exon 7 in five of seven H-RS cells isolated from one of seven HD cases. Chen et al ( 1996) (12) isolated areas rich in H-RS cells by micromanipulation and demonstrated that point mutations in exons 6 to 8 could be identified in 6 of 23 cases.…”

“…These results have been obtained by analysis of crude extracts of genomic DNA from tissue specimens in which the H-RS cells only constitute a small minority. More recently, different approaches have been taken by Trumper et al( 1993) ( 17) and Chen et al( 1996) ( 12) Trumper et al (1993).…”

The Clinical Significance of Mutations of the P52 Tumour Suppressor Gene in Haematological Malignancies

“…In Hodgkins disease, P53 mutations may be confined to Reed-Sternberg cells in many cases (Gupta et al, 1993a, b). Because those cells only constitute a small part of the cell infiltrate, P53 may not be recognized by conventional methods (SSCP, ICC, IHC) without prior enrichment of the Reed-Sternberg cell population Trumper et al, 1993).…”