B cell–derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that >50% of CLLs display hypermutated immunoglobulin variable region (IgV) sequences and a more favorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of IgV hypermutation. To further investigate the phenotype of CLLs, their cellular derivation and their relationship to normal B cells, we have analyzed their gene expression profiles using oligonucleotide-based DNA chip microarrays representative of ∼12,000 genes. The results show that CLLs display a common and characteristic gene expression profile that is largely independent of their IgV genotype. Nevertheless, a restricted number of genes (<30) have been identified whose differential expression can distinguish IgV mutated versus unmutated cases and identify them in independent panels of cases. Comparison of CLL profiles with those of purified normal B cell subpopulations indicates that the common CLL profile is more related to memory B cells than to those derived from naive B cells, CD5⁺ B cells, and GC centroblasts and centrocytes. Finally, this analysis has identified a subset of genes specifically expressed by CLL cells of potential pathogenetic and clinical relevance.

We have investigated the frequency of p53 mutations in B- and T-cell human lymphoid malignancies, including acute lymphoblastic leukemia, the major subtypes of non-Hodgkin lymphoma, and chronic lymphocytic leukemia. p53 exons 5-9 were studied by using genomic DNA from 197 primary tumors and 27 cell lines by single-strand conformation polymorphism analysis and by direct sequencing of PCR-amplified fragments. Mutations were found associated with (i) Burkitt lymphoma (9/27 biopsies; 17/27 cell lines) and its leukemic counterpart L3-type B-cell acute lymphoblastic leukemia (5/9), both of which also carry activated c-myc oncogenes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, in particular, its stage of progression known as Richter's transformation (3/7). Mutations were not found at any significant frequency in other types of non-Hodgkin lymphoma or acute lymphoblastic leukemia. In many cases, only the mutated allele was detectable, implying loss of the normal allele. These results suggest that (i) significant differences in the frequency of p53 mutations are present among subtypes of neoplasms derived from the same tissue; (ii) p53 may play a role in tumor progression in B-cell chronic lymphocytic leukemia; (iii) the presence of both p53 loss/inactivation and c-myc oncogene activation may be important in the pathogenesis of Burkitt lymphoma and its leukemic form L3-type B-cell acute lymphoblastic leukemia.

The molecular mechanism involved in the process of antigen-driven somatic hypermutation of Ig genes is unknown, but it is commonly believed that this mechanism is restricted to the Ig loci. B cell lymphomas commonly display multiple somatic mutations clustering in the 5′-regulatory region of BCL-6, a proto-oncogene encoding for a POZ/Zinc finger transcriptional repressor expressed in germinal center (GC) B cells and required for GC formation. To determine whether BCL-6 mutations represent a tumor-associated phenomenon or reflect a physiologic mechanism, we screened single human tonsillar GC B cells for mutations occurring in the BCL-6 5′-noncoding region and in the Ig variable heavy chain sequences. Thirty percent of GC B cells, but not naive B cells, displayed mutations in the 742 bp region analyzed within the first intron of BCL-6 (overall frequency: 5 × 10⁻⁴/bp). Accordingly, an expanded survey in lymphoid malignancies showed that BCL-6 mutations are restricted to B cell tumors displaying GC or post-GC phenotype and carrying mutated Ig variable heavy chain sequences. These results indicate that the somatic hypermutation mechanism active in GC B cells physiologically targets non-Ig sequences.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL. Publisher9s Note: There is a Blood Commentary on this article in this issue. Key Points ctDNA is as an easily accessible source of tumor DNA for cHL genotyping. ctDNA is a radiation-free tool to track residual disease in cHL. Visual Abstract