Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development.

We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

Miscreant myeloproliferative disorder stem cells.

Myeloproliferative disorders (MPDs), typified by robust marrow and extramedullary hematopoiesis, have a propensity to progress to acute leukemia. Although the hematopoietic stem cell (HSC) origin of MPDs was suggested over 30 years ago, only recently the HSC-specific effects of MPD molecular mutations have been investigated. The pivotal role of BCR-ABL in chronic myeloid leukemia (CML) development provided the rationale for targeted therapy, which greatly reduced mortality rates. However, BCR-ABL inhibitor-resistant CML HSCs persist that may be a reservoir for relapse. This has provided the impetus for investigating molecular mechanisms governing the production of recalcitrant HSC. Comparatively little was known about the molecular events driving BCR-ABL-negative MPDs until seminal studies revealed that a large proportion of MPD patients harbor a JAK2-activating point mutation, JAK2V617F. Although JAK2 activation appears to be central to BCR-ABL-negative MPD pathogenesis, its effects may be cell type and context specific. Recent evidence suggests that acquired mutations misdirect differentiation and survival of the MPD-initiating stem cell resulting in the production of aberrant self-renewing progenitors that subvert the microenvironment leading to leukemia stem cell generation and leukemic transformation. Thus, combined therapies targeting aberrant molecular pathways may be required to redirect miscreant MPD stem cells.

Inhibition of murine hematopoiesis by CAMAL, an inhibitor of human hematopoiesis.

CAMAL (common antigen of myelogenous acute leukemia) is an antigenic preparation isolated in this laboratory from the bone marrow or peripheral blood leucocytes of persons with myeloid leukemias and shown using an immunoperoxidase slide test to be diagnostic of these leukemias. Material further purified from CAMAL preparations, which migrates in the range of 30-35 kilodaltons (kDa) by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE), which is referred to as P30-35 CAMAL, and was previously shown to be inhibitory to colony formation by progenitor cells from normal healthy human donors in vitro. This inhibitory activity was directed toward neutrophilic granulocyte colonies (CFU-G) in particular. We now report that P30-35 CAMAL is inhibitory to colony formation by murine progenitor cells in vitro. Colonies from P30-35 CAMAL-treated cultures of murine bone marrow cells were reduced in number and in size, an effect similar to that seen in cultures of human cells. As in assays using human cells, murine CFU-G appeared to be preferentially targeted by the inhibitory activity of P30-35 CAMAL. In addition, day 10 spleen colony formation was inhibited by P30-35 CAMAL in an ex vivo assay. Hence, the effects of P30-35 CAMAL on murine progenitor cells appear to parallel the effects observed using human cells. These observations support the possibility that CAMAL might be a regulatory protein in hematopoiesis which is conserved between species.

Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells.

Benzoporphyrin derivatives (BPDs) are photosensitizers, which fluoresce strongly at 690 nm, and may be candidates for various applications of photodynamic therapy (PDT). Fluorescence-activated cell sorting (FACS) analysis, subsequent to ultraviolet light excitation, revealed pronounced differences in red fluorescence between leukemic cell lines (HL60, K562 and L1210), leukemic clinical isolates, and normal human or murine bone marrow cells incubated with BPD. These observed differences in BPD-mediated fluorescence provide the rationale for sorting leukemic from normal cells via FACS or may constitute a novel method for extracorporeal purging of remission marrow by photodynamic therapy in autologous bone marrow transplantation.