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MSCNET Sintesi della relazione

Project ID: 37602
Finanziato nell'ambito di: FP6-LIFESCIHEALTH
Paese: Denmark

Final Report Summary - MSCNET (Myeloma Stem Cell Network - A translational programme identifying and targeting the early myeloma cell hierarchy)

Multiple myeloma (MM) is a neoplasia defined by the accumulation of malignant plasma cells in the bone marrow. Many recent therapeutic advances have extended survival, but are not curative and new approaches are being sought that can underpin disease resolution. Understanding pathogenesis in MM will benefit these searches. Although malignant cells, which represent terminally differentiated B-cells, are readily identifiable by morphological criteria, it is as yet not known with certainty whether a less differentiated cell may actually be feeding tumour growth. This uncertainty has arisen due to a number of observations suggestive of a less mature clonal precursor. More recently, the question of whether such a 'feeder' cell may in fact represent one or more clonal 'stem' cell had also emerged based on 'the stepwise oncogenesis' and as in other cancers, could represent a paradigm shift in understanding MM origins and progression. The recent focus on potential stem cells in cancer has generated much interest and rapid translational investigations including novel biotechnologies.

Following a range of scientific meetings and technical workshops, we now share the relevant competences and experiences related to Multiparametric flow cytometry (MFC), real-time Polymerase chain reaction (PCR) assays, cDNA-chip and PepChip technology and storage of biological samples with specific clinical information. Furthermore, we share models for in vitro differentiation of B cells and an animal model for in vivo transplantation studies. By these platforms, we have generated a common database with analytic information about potential B lineage subsets (naïve, large centroblast, small centroblast, centrocytes, memory B-cell and plasmablast / cells) in peripheral blood and bone marrow of impact for pathogenesis, including a well characterised panel of myeloma cancer cell lines. Potential stem cell genes have been identified in the current literature and available databases and PCR assays established for single cell (N = 1-100) analysis for stem cell genes, transcription factors and differentiation markers.

First, in a model of Light-chain MM (LC MM), where CD138+ tumour cells express only the light chain, we sought molecular evidence for less mature cells bearing both Ig heavy and light chains, in different B-cell subsets (naïve, IgM+, IgM- B-cells, plasmablasts, plasma cells). In 5/6 cases, no evidence was found for such a cell, and in the remaining cases, IgH and L chain expression was restricted to the CD138+ fraction, suggesting that the progenitor cell in LCMM is a plasmablast / plasma cell.

Second, in a model of Ras mutated MM patients studied by single cell cDNA libraries, we identified that memory B-cells do lacks 'late' oncogene like (K-RAS) mutations but express the 'early' oncogene presumably representing a clonotypic remnant that is only partially transformed. This demonstrated that CD19+ / 38- cells do not but CD19- / 38+ cells harbour the Ras mutated clone. This observation and the indications from the study of LC MM indicate a stepwise evolution of MSC within the myeloma hierarchy in parallel with the step wise oncogenesis.

Third, in the 5T2 and 5T33 mice model we have succeeded in identification of tumour V(D)J transcripts ready for transplantation experiments. In the mouse transplantation model, CD138+ cells rapidly engrafted disease (3-4 weeks), whereas CD138- cell mediated engraftment was delayed (4-12 weeks), which was supported by in vitro clonogenic assay. This demonstrated that the marker CD138 is not sufficient to identify a myeloma stem cell.

Fourth, in a study of 18 myeloma cell lines, we determined the frequency of culture initiating cells concluding that CD138+ cells had a 10-100 fold higher frequency than CD138- cells. This demonstrated that the marker CD138 is not sufficient to identify myeloma cell line culture initiating cells.

MSCNET sought to initiate detailed studies to define both the B-MSC and PMSC, in primary tumour samples as well as in disease models. With regards to the B-MSC, we were unable to identify clonally-derived progeny in distinct B-cell subsets representative of several maturation stages leading to plasma cells, and this study has been accepted as a speaker presentation at ASH 2010, the foremost platform for cutting-edge research in haematology.

We identified a clonal hierarchy in 5T murine myeloma, and showed that CD138+ malignant plasma cells are sufficient to propagate tumour growth. In this model, however, the 'stem cell' component appears more complex, as CD138- cells can also engraft, although at a slower rate and this question will be addressed in future collaborations among MSCNET membres, directly facilitated by the network's activities during tenure of this program. In relation to the oncogenic hits driving 'stem cells', MSCNET has provided pertinent state-of-art observations. Our study on LC-MM raises the conceptual issue of a sIg+ B-MSC as being an unlikely feeder cell in this form of disease, as the genetic lesions that such a cell would need to acquire to feed the light chain phenotype on a cyclic basis (as proposed by the CSC paradigm) appears highly unlikely.

The analysis of KRAS mutations in memory B-cells that are aberrant and therefore, tumour-derived has provided pivotal insights in agreement with our LC-MM predictions. This data showed that KRAS mutations are only found in malignant plasma cells, not memory Bcells (the B-MSC counterpart). This cements the proposal that a MSC which is a B-MSC is highly unlikely to acquire the repertoire of mutations to establish features associated with presentation disease every time it cycles to feed the tumour bulk. As argued in the KRAS study, this B-MSC appear to be a residue of the cell of origin giving rise to MM, but appear to have no malignant potential.

In conclusion, MSCNET favoured the P-MSC model as driving malignant growth and has delineated multiple genetic pathways by which malignant PCs can support this potential, which currently is clinical validated.

Informazioni correlate


Hans Erik JOHNSEN, (Professor)
Tel.: +45-99326875
Fax: +45-99326801