In this paper the functional relevance of a TNFA promoter polymorphism, a G/A polymorphic sequence at position -238, was tested by analysing its influence on TNF alpha production upon in vitro stimulation of monocytes from 78 healthy, unrelated individuals by lipopolysaccharide (LPS) or after allogenic stimulation in a panel of 32 healthy individuals. All TNFA-A positive individuals were either DR3 or DR7 positive, confirming the previously reported strong linkage disequilibrium of the TNFA-A allele with the two extended haplotypes (B18, F1C30, DR3) and (B57, SC61, DR7). No individuals homozygous for the TNFA-A allele were present in the panel. The mean level of TNF alpha production was not significantly different in TNFA-G/G homozygous and in TNFA-A/G heterozygous individuals after LPS stimulation of monocytes (P = 0.35) or after allogenic stimulation (P = 0.7). After LPS and allogenic stimulation DR3 positive individuals had a higher mean TNF production. This could not be further differentiated by typing for TNF -283.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the ®broblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/ MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the e ects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identi®ed so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identi®ed in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at di erent levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14). Oncogene (2001) 20, 3553 ± 3562.

Intensive HD-CT seems to enhance the response rate and survival when compared with conventional treatment in poor-prognosis pPNET. The poor results of this treatment in RMS and DSRCT do not support the inclusion of such an approach in these patient subsets. No definitive conclusions can currently be drawn concerning the clinical implications of the detection of fusion transcripts during treatment or follow-up.

A total of 10 desmoplastic small round-cell tumour patients were treated by high-dose chemotherapy with stem cell support. After high-dose chemotherapy, no complete response conversion was obtained and EWS-WT1 fusion transcript detection was positive in the peripheral blood during follow-up in all patients. High-dose chemotherapy did not seem to change the results in desmoplastic small round-cell tumour. Desmoplastic small round-cell tumour (DSRCT) is a rare and aggressive disease affecting mainly young male patients. Large masses in the abdomen with compressive symptoms are frequently observed. Morphological, immunohistochemical, and cytological characteristics have been previously described (Gerald et al, 1998). A recurrent chromosomal translocation t(11;22)(p13;q12) has been found in tumour cells with detection of a chimeric transcript EWS-WT1 using PCR.Treatment of DSRCT is a challenge because radical surgery is infrequently performed, and conventional chemotherapy induces short-lived responses (Farhat et al, 1996).Recently, the Memorial Sloan-Kettering Cancer Center group reported interesting results using an intensive alkylator-based protocol associated to surgery and radiotherapy (Kushner et al, 1996).Previously, we reported the results of high-dose chemotherapy in patients with small round-cell tumour (Bertuzzi et al, 2002). Seven out of 28 of them were affected by DSRCT.Here, we report the clinical and molecular results obtained in 10 adult DSRCT prospectively treated by high-dose chemotherapy and autologous peripheral stem cell transplantation. PATIENTS AND METHODS Eligibility criteriaPatients with DSRCT aged 15 -60 years were considered eligible for the study. Disease staging was determined using chest X-rays, technetium 99m bone scan, computed tomography or magnetic resonance imaging, and the evaluation of bone marrow specimens.The diagnosis was established on the basis of histochemical findings as previously described (Gerald et al, 1998).Informed consent for the treatment was obtained in accordance with our Institutional Review-Board guidelines. Treatment protocolThe treatment programme combined conventional chemotherapy, local treatment (surgery and/or radiotherapy), and myeloablative therapy with autologous stem-cell rescue. The chemotherapeutic programme consisted of a four-course induction phase with epirubicin 30 mg m À2 , days 1 -3; ifosfamide 3 g m À2 , days 1 -3; and vincristine 2 mg, day 1 (IVE), every 3 weeks, followed by a mobilisation phase with high-dose etoposide (2.4 g m À2 ) or cyclophosphamide (7 g m À2 ), with G-CSF support. Peripheralblood stem cells were collected using a Cobe Spectra CS 3000. Finally, patients in complete (CR) or partial response (PR) received high-dose chemotherapy with melphalan 160 mg m À2 plus mitoxantrone 60 mg m À2 or thio-tepa 600 mg m À2 , followed by the reinfusion of the peripheral-blood stem cells. G-CSF (300 mg day À1 ) starting on day þ 5 was administered when the number of reinfused CD34 þ cells was less than 5 Â 10 6 kg À1 .Local surgery or radio...

SUMMARYThe aim of the present study was to analyse the in vitro proliferation and cytokine production by alloantigen-stimulated peripheral blood mononuclear cells (PBMC) obtained from patients affected by systemic sclerosis (SSc) and patients with Raynaud's phenomenon (RP). In SSc patients the proliferation of PBMC stimulated in vitro with alloantigens was significantly increased compared with healthy subjects, while no differences were observed for RP patients. Lymphocytes from SSc patients also produced larger amounts of IFN-g compared with healthy controls. However, patients with clinically active disease had lower IFN-g levels than those found in clinically stable patients. Patients affected by RP showed significantly higher levels of IFN-g than healthy subjects. Analysis at the clonal level of the lymphocyte subsets involved in alloantigen stimulation in one patient affected by active SSc, and one subject with RP confirmed the results obtained using PBMC. In particular, in the RP patient but not in the SSc patient, we observed a population of CD4 þ T cells which proliferated to alloantigens in vitro and produced high levels of IFN-g. We suggest that T lymphocytes producing high levels of IFN-g might play a protective role in RP patients and in established scleroderma.