Summary We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia. Human leukemic bone marrow was transplanted intrafemorally into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20 and CD34 were isolated from patient samples and engrafted mice before serial transplantation into primary or subsequent (up to quaternary) recipients. Surprisingly, blasts representative of all the different maturational stages were able to reconstitute and re-establish the complete leukemic phenotype in vivo. Sorted blast populations mirrored normal B precursor cells with transcription of a number of stage-appropriate genes. These observations have informed a model for leukemia-propagating stem cells in childhood ALL.

Keywords: Catalysis / Ene reaction / Lewis acids / Natural products / Nitrogen heterocycles 1,3-Disubstituted tetrahydro-oxazolo-isoquinolinones 19a,b were obtained from phenylalanine in seven steps and 42% overall yield by Katritzky's benzotriazole method. The tricyclic oxazolidinone 19a was further converted into amino alcohol 10 by employing a chemoselective reduction of the ester group with LiBH 4 /MeOH. Compound 10 and the corresponding 1-unsubstituted tetrahydroisoquinoline alcohol 11 were converted into aldehydes 27 and 33, which cyclized

Alkaloids U 0600Towards a Total Synthesis of Quinocarcin: Diastereoselective Synthesis of Functionalized Azepino[1,2-b]isoquinolines. -Key structural skeletons (II) and (III) of alkaloid quinocarcin (IV) are prepared by Lewis acid catalyzed hetero-ene reaction of aldehydes (I). The stereoselectivity in the reaction of aldehyde (Ia) is highly dependent on the type of Lewis acid, whereas aldehyde (Ib) affords only diastereomer (IIb) regardless of the Lewis acid. -(KOEPLER, O.; LASCHAT*, S.; BARO, A.; FISCHER, P.; MIEHLICH, B.; HOTFILDER, M.; LE VISEUR, C.; Eur. J. Org.

Sarcomas are rare tumours in patients under the age of 20. Because of their characteristic chromosomal translocations Ewing's sarcomas have been a paradigm for understanding the biology of sarcomas. Since the survival rates in Ewing's sarcoma patients with metastasis and relapse are still unsatisfactory, new therapeutic options have to be developed. Model systems based on fibroblast cell lines have helped in understanding the biology of the transforming protein EWS/FLI1. Cell lines established from primary Ewing's sarcoma are now in use to test new therapeutic options. Since these models only poorly reflect the conditions within the patients, xenograft mouse models have recently been developed to study the biology of Ewing's sarcoma in a more relevant in vivo environment. These new model systems will help define novel therapeutic targets and support the preclinical evaluation of new treatments.

Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×103 − 1×105 CD34+CD19− cells, 2×104 − 6×106 CD34+CD19+ lymphoid progenitors and 3×104 − 2×106 more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 103 CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.