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Micro-environment seems to exert an important influence on human mesenchymal stem cell (MSC) differentiation and proliferative capacity in bone marrow as well as in culture ex vivo. Oct-4, Rex-1, and TERT genes are well-known for the maintenance of pluripotentiality differentiation and the proliferative capacity of embryonic stem cells. Some previous data report expression of these embryonic factors in selected clones from bone marrow adult stem cells. Our goal was to study expression of Oct-4, Rex-1, and TERT in primary cultured human MSC according to the serum concentration. In addition, we have studied the expression of Gata-4 since this factor plays a key role in organogenesis. We hypothesized that low serum concentration with appropriate growth factors may induce an undifferentiated status with a re-expression of embryonic factors and extend differentiation capacity. Thus, using a defined culture medium, we report on the increased expression of Oct-4, Rex-1, and Gata-4 in human MSC. We have correlated this expression to an increase in differentiation efficiency towards osteogenic and adipogenic phenotypes. Our data suggest that the culture medium used permits the emergence of adult stem cells with a high differentiation capacity and expression of embryonic factors. These cells may have important implications for cell therapy.

We have studied the occurrence and association of 11q deletions with other chromosomal imbalances in Stage 4 neuroblastomas. To this purpose we have performed comparative genomic hybridization (CGH) analysis on 50 Stage 4 neuroblastomas and these data were analyzed together with those from 33 previously published cases. We observed a high incidence of 11q deletion in Stage 4 neuroblastoma without MYCN amplification (59%) whereas 11q loss was only observed in 15% of neuroblastomas with MYCN-amplification (p = 0.0002) or 11% of cases with 1p deletion detected by CGH (p = 0.0001). In addition, 11q loss showed significant positive correlation with 3p loss (p = 0.0002). Event-free survival was poor and not significantly different for patients with or without 11q deletion. Our study provides further evidence that Stage 4 neuroblastomas with 11q deletions represent a distinct genetic subgroup that typically shows no MYCN-amplification nor 1p deletion. Moreover, it shows that neuroblastomas with 11q deletion also often present 3p deletion. This genetic subgroup shows a similar poor prognosis as MYCN amplified 4 neuroblastomas., (Copyright 2001 Wiley-Liss, Inc.)

A pilot study of neuroblastoma mass screening was initiated in January 1990 in the Rhône French district. The expected number of births per year is 26,000. The study is designed for a 5-year period with three major goals: 1) measurement of the compliance rate of a voluntary test at 4 months of age; 2) evaluation of the technical value of high-pressure liquid chromatography (HPLC) as a screening method; and 3) detailed biological characterization of all detected tumors. 61,551 children were screened between May 1, 1990 and December 31, 1993. Participation was 69% in 1990, 81.5% in 1991, and over 83% in 1992. HPLC was a satisfactory assay method. The number of clinical examinations required for positive tests as defined in the protocol is 1 per 3,621 tests. The false positive rate is 1 per 3,583 tests. Eight neuroblastomas were discovered by-screening (one stage I, three stage II, one stage III, three stage IVs). All are alive and well but were good prognosis cases according to the main prognostic factors. Five patients were discovered before screening (so called Halo effect): one stage I, one stage III, three stage IVs. One died of disease and four are alive in complete remission after treatment. Two patients were false negative (one stage III with N-myc amplification, one stage IV with bad prognosis features) and three cases of neuroblastoma were missed because of noncompliance with the screening program. This pilot study concludes on the feasibility of a mass screening program in France. The estimated cumulative incidence of neuroblastoma at 3 years is 1 per 4,375 living births and overdiagnosis is probable. All the detected cases were of good prognosis and the false negative ones were poor prognosis cases.

Somatic mutations of von Hippel Lindau (VHL) tumor-suppressor gene have been identified in kidney cancers from North America and Japan. We studied VHL gene mutation in 31 kidney tumors from France. Of these tumors, 45% (14/31) displayed mutations, 60% of which occurred at A:T base pairs. The frequency and the spectrum of mutations identified in kidney tumors from Europe were similar to those reported for tumors from other geographical locations.

Chromosome I abnormalities are indicators of prognosis in neuroblastoma (NB) but are not yet routinely exploited because conventional methods are technically demanding. We evaluated the pertinence of interphase cytogenetics fluorescence in situ hybridization (FISH) for the analysis of chromosome I in NB, compared with conventional methods. Deletion of Ip was detected in 8 of 9 cell lines analyzed by both FISH and restriction fragment length polymorphism (RFLP), but was evidenced in only 2 cases by conventional cytogenetics, painting analysis being required to reveal the other cases. The chromosome I number evaluated by FISH reflected the total chromosome modal number obtained by cytogenetics. Twenty-eight specimens obtained from ultrasound-guided punctures, surgical biopsies of the primary tumor and bone-marrow aspirates were studied by FISH on frozen cytocentrifuged smears; 12 had a chromosome I trisomy and 16 a disomy. Requirements for a reliable control analysis of Ip deletion by RFLP were met in only 23 cases. The retention of 2 alleles was observed in 15 cases and Ip deletion in 7, by both techniques. In one case, an interstitial deletion of Ip was evidenced only by RFLP, and one of 5 cases analyzed only by FISH had a Ip deletion. Although FISH might be improved by using additional probes, it presents major advantages for routine exploitation. Determining Ip deletion in individual cells makes it possible to analyze small and heterogeneous tumoral specimens; the technique requires only a few hours and can easily be standardized in non-specialized laboratories. The number of chromosome I homologues per cell might serve as a rapid screening for ploidy.

Immunohistological expression of integrins has been analyzed on 45 neuroblastoma specimens representative of the different clinical and histological forms of the tumor. None of the specimens expressed the alpha 5 chain of the integrins. The beta 1 chain was expressed on all specimens, the alpha 1 chain on 44 specimens and the alpha 3 chain on 42; the 4 specimens which lacked alpha 1 or alpha 3 were stage-4 neuroblastomas. The alpha 2 chain was expressed on 18 specimens, and the alpha 6 chain on 17; 15 reacted with both. Their reactivity was related to the maturation of the tumor rather than the stage of the disease: they were expressed on low-grade, well-differentiated specimens; stage 3-4 neuroblastoma specimens analyzed at diagnosis were negative, but usually expressed both chains when analyzed after in vivo differentiation by chemotherapy. alpha v reacted with 18 specimens and beta 3 with 12, without strict relation with the stage of the disease and/or its degree of differentiation; 9 well-differentiated specimens expressed the beta 4 chain; only 4 well-differentiated specimens expressed the alpha 4 chain. The 4 specimens which lacked alpha 1-beta 1 or alpha 3-beta 1 expression had n-myc amplification, whereas those which expressed either alpha 4, beta 4, beta 3 or alpha v had no amplification. Furthermore, the expression of the 3 heterodimers alpha 4-beta 1, alpha v-beta 3 and alpha 6-beta 4 was essentially observed on primary tumors which developed in the mediastinum. The expression of alpha 2-beta 1 and alpha 6-beta 1 was observed on both n-myc-positive and -negative specimens. beta 1 and alpha 3 were diffusely expressed on all counterparts of these tumors, from undifferentiated neuroblasts to ganglion and Schwann cells. The alpha 1 chain reacted with undifferentiated and intermediate neuroblasts as well as with Schwann cells, but ganglion cells were negative. alpha 2 and alpha 6 chains were negative on undifferentiated neuroblasts, variably expressed on intermediate neuroblasts, and restricted to Schwann cells in ganglioneuroma. The expression of alpha 4 and beta 4 was restricted to Schwann cells. alpha v and beta 3 occasionally reacted with undifferentiated and intermediate neuroblasts; alpha v was strongly positive on Schwann cells but negative on ganglion cells, whereas beta 3 was positive on both neuronal and non-neuronal populations.

LFA-3, ICAM-1, HLA.ABC and HLA.DR expression was analyzed on 66 neuroblastoma specimens. HLA.ABC was expressed on 26 specimens, HLA.DR on 2, LFA-3 on 20 and ICAM-1 on 10. HLA.ABC and LFA-3 were positive on ganglioneuroblastoma or ganglioneuroma, but they were negative on neuroblastoma, independently of the clinical staging; HLA.ABC and LFA-3 were induced in vivo by chemotherapy in parallel with tumoral cell differentiation, in both the primary and the metastases. The expression of ICAM-1 was restricted to 5 of the 10 low-grade stage-1 or stage-2 specimens, 1 stage-3 specimen, and the primary tumors of 2 patients with stage-4 disease, analyzed hence at diagnosis and after chemotherapy (4 specimens); metastatic cells obtained in 1 of these patients were negative. HLA.ABC and LFA-3 expressed on both mycN-negative and -positive specimens, whereas ICAM-1 was restricted to MYCN-negative specimens. LFA-3 diffusely stained partially differentiated neuroblasts, Schwann cells and ganglion cells. The expression of HLA.ABC on differentiated neuroblasts varied from one sample to another and within the same tumor; Schwann cells were strongly positive, but ganglion cells were negative. In positive samples, ICAM-1 was expressed on differentiated neuroblasts and Schwann cells, but negative on ganglion cells; however, most of the differentiated tumors were ICAM-1-negative, suggesting ICAM-1 induction by unknown local signal. The 4 markers were negative on undifferentiated neuroblasts. The distribution of these 4 markers on clinical specimens was in agreement with their reactivity on fetal tissues, as well as with results obtained on neuroblastoma cell lines before and after in vitro treatment with IFN-gamma.

The expression of receptors for proliferation and differentiation factors was analyzed by indirect immunofluorescence on 29 Burkitt lymphoma (BL) cell lines previously classified into 3 groups on the basis of their reactivity with 8 monoclonal antibodies (MAbs), including anti-CALLA, BL13 and TU1. BL13 and HB5 antibodies recognize different epitopes of the EBV/CR2 receptors. The determinant recognized by BL13 has been previously shown to be expressed only on cell lines of the first two groups, supposed to derive from the germinal center and to be negative on a third group of lines of putative BM origin and established from sporadic cases of BL. In contrast, and as expected from its reactivity on normal B cells in the BM or in the lymph nodes, HB5 antibody reacts with all BL lines except one. The receptor for transferrin is expressed on the 29 lines. Two new MAbs, Bac-1 and B1H5, could recognize respectively receptors for BCGF1 and BCGF2. Bac-1 reacts with 15 of 17 BL lines belonging to the first two groups and 7 of 12 BL lines of the third group; 14 of 15 EBV + lines express Bac-1. No BL line expresses B1H5. The IL2 receptor is weakly expressed on 5 EBV + cell lines and one EBV (-) line. All delta are BCGF1-positive. The almost constant expression of BCGF1 receptor on EBV + cell lines is the only strict relation between the expression of receptors for growth factors and their characteristics (i.e. EBV association, translocation, ethnic origin and clinical presentation). The maturation stage or the origin of BL cell lines in relation to the expression of growth factor receptors and the functional significance of these receptors will be discussed.