[Clinical characteristics and follow-up of 60 patients with recent diagnosis of giant cell arteritis, NEWTON study]. / Caractéristiques et profils évolutifs des patients avec un diagnostic récent d'artérite à cellules géantes, étude NEWTON.

INTRODUCTION: The management of giant cell arteritis (GCA) has evolved with the arrival of tocilizumab (TCZ) and the use of PET/CT. Our objective is to describe the characteristics and followup of patients with recent diagnosis of GCA in current care. PATIENTS AND METHODS: The NEWTON cohort is a monocentric retrospective cohort based on data collected from 60 GCA patients diagnosed between 2017 and 2022 according to the ACR/EULAR 2022 criteria. RESULTS: The median age at diagnosis was 73 [68.75; 81] years old. At diagnosis, the main manifestations were unusual temporal headaches in 48 (80 %) and an inflammatory syndrome in 50 (83 %) patients. Temporal artery biopsy confirmed the diagnosis in 49/58 (84 %) patients. Doppler of the temporal arteries found a halo in 12/23 (52 %) patients. The PET/CT found hypermetabolism in 19/43 (44 %) patients. Prednisone was stopped in 17.5 [12.75; 24.25] months. During follow-up, 22 (37 %) patients received TCZ. At least one complication of corticosteroid therapy was observed in 22 (37 %) patients. After a median follow-up of 24 [12; 42] months, 25 (42 %) patients relapsed. At the end of the follow-up, 29 (48.3 %) patients were weaned from corticosteroid therapy and 15 (25 %) were on TCZ. CONCLUSION: Despite the increasing use of TCZ in the therapeutic arsenal and of the PET/CT in the imaging tools of GCA patients, relapses and complications of corticosteroid therapy remain frequent, observed in more than a third of patients.

Vaccination to improve the persistence of CD19CAR gene-modified T cells in relapsed pediatric acute lymphoblastic leukemia.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome.

BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio-facio-cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild-type BRAF but is less strongly activating than the cancer-associated p.V600E mutation.

Impact of the European clinical trials directive on prospective academic clinical trials associated with BMT.

The European Clinical Trials Directive (EU 2001; 2001/20/EC) was introduced to improve the efficiency of commercial and academic clinical trials. Concerns have been raised by interested organizations and institutions regarding the potential for negative impact of the Directive on non-commercial European clinical research. Interested researchers within the European Group for Blood and Marrow Transplantation (EBMT) were surveyed to determine whether researcher experiences confirmed this view. Following a pilot study, an internet-based questionnaire was distributed to individuals in key research positions in the European haemopoietic SCT community. Seventy-one usable questionnaires were returned from participants in different EU member states. The results indicate that the perceived impact of the European Clinical Trials Directive has been negative, at least in the research areas of interest to the EBMT.

High dose chemotherapy with autologous hematopoietic stem cell support for solid tumors other than breast cancer in adults.

Since the early 1980s high dose chemotherapy with autologous hematopoietic stem cell support was adopted by many oncologists as a potentially curative option for solid tumors, supported by a strong rationale from laboratory studies and apparently convincing results of early phase II studies. As a result, the number and size of randomized trials comparing this approach with conventional chemotherapy initiated (and often abandoned before completion) to prove or disprove its value was largely insufficient. In fact, with the possible exception of breast carcinoma, the benefit of a greater escalation of dose of chemotherapy with stem cell support in solid tumors is still unsettled and many oncologists believe that this approach should cease. In this article, we critically review and comment on the data from studies of high dose chemotherapy so far reported in adult patients with small cell lung cancer, ovarian cancer, germ cell tumors and sarcomas.

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes.

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.

Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG).

Deletion of the long arm of chromosome 20 represents the most common chromosomal abnormality associated with the myeloproliferative disorders (MPDs) and is also found in other myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Previous studies have identified a common deleted region (CDR) spanning approximately 8 Mb. We have now used G-banding, FISH or microsatellite PCR to analyse 113 patients with a 20q deletion associated with a myeloid malignancy. Our results define a new MPD CDR of 2.7 Mb, an MDS/AML CDR of 2.6 Mb and a combined 'myeloid' CDR of 1.7 Mb. We have also constructed the most detailed physical map of this region to date--a bacterial clone map spanning 5 Mb of the chromosome which contains 456 bacterial clones and 202 DNA markers. Fifty-one expressed sequences were localized within this contig of which 37 lie within the MPD CDR and 20 within the MDS/AML CDR. Of the 16 expressed sequences (six genes and 10 unique ESTs) within the 'myeloid' CDR, five were expressed in both normal bone marrow and purified CD34 positive cells. These data identify a set of genes which are both positional and expression candidates for the target gene(s) on 20q.

Large deletions at the t(9;22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia.

The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation. Patients with CML show considerable heterogeneity both in their presenting clinical features and in the time taken for evolution to blast crisis. In this study, metaphase fluorescence in situ hybridization showed that a substantial minority of patients with CML had large deletions adjacent to the translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations, or on both. The deletions spanned up to several megabases, had variable breakpoints, and could be detected by microsatellite polymerase chain reaction in unfractionated bone marrow and purified peripheral blood granulocytes. The deletions were likely to occur early and possibly at the time of the Philadelphia (Ph) chromosome translocation: deletions were detected at diagnosis in 11 patients, were found in all Ph-positive metaphases, and were more prevalent in patients with variant Ph chromosomes. Kaplan-Meier analysis showed a median survival time of 36 months in patients with a deletion; patients without a detectable deletion survived > 90 months. The survival-time difference was significant on log-rank analysis (P =. 006). Multivariate analysis demonstrated that the prognostic importance of deletion status was independent of age, sex, percentage of peripheral blood blasts, and platelet count. Our data therefore suggest that an apparently simple, balanced translocation may result not only in the generation of a dominantly acting fusion oncogene but also in the loss of one or more genes that influence disease progression. (Blood. 2000;95:738-743)

A two-dimensional protein map of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

A detailed physical and transcriptional map of the region of chromosome 20 that is deleted in myeloproliferative disorders and refinement of the common deleted region.

Acquired deletions of the long arm of chromosome 20 are the most common chromosomal abnormality seen in polycythemia vera and are also associated with other myeloid malignancies. Such deletions are believed to mark the site of one or more tumor suppressor genes, loss of which perturbs normal hematopoiesis. A common deleted region (CDR) has previously been identified on 20q. We have now constructed the most detailed physical map of this region to date--a YAC contig that encompasses the entire CDR and spans 23 cM (11 Mb). This contig contains 140 DNA markers and 65 unique expressed sequences. Our data represent a first step toward a complete transcriptional map of the CDR. The high marker density within the physical map permitted two complementary approaches to reducing the size of the CDR. Microsatellite PCR refined the centromeric boundary of the CDR to D20S465 and was used to search for homozygous deletions in 28 patients using 32 markers. No such deletions were detected. Genetic changes on the remaining chromosome 20 may therefore be too small to be detected or may occur in a subpopulation of cells.

Molecular genetics and cytogenetics of myeloproliferative disorders.

The myeloproliferative disorders are believed to represent clonal malignancies resulting from transformation of a pluripotent stem cell. X-inactivation patterns of peripheral blood cells have been proposed as a useful diagnostic tool but this method is limited by the finding of a clonal X-inactivation pattern in a significant proportion of normal elderly women. There is no pathognomonic chromosomal abnormality associated with the myeloproliferative disorders. However, consistent acquired cytogenetic changes include del(20q), del(13q), trisomy 8 and 9 and duplication of segments of 1q, all of which have been observed at diagnosis or before cytoreductive therapy and therefore represent early lesions which contribute to the pathogenesis of these disorders. Although, the acquired molecular defects underlying most myeloproliferative disorders have not yet been elucidated, translocations associated with the rare 8p11 syndrome have permitted identification of a novel fusion protein. The role of a number of candidate genes in the other myeloproliferative disorders has also been studied, but no mutations have been identified so far. It is likely that a number of genes will be involved, given the varied phenotypes of the diseases. Identification of causal genes will be of considerable interest to both clinicians, who currently lack a specific and sensitive diagnostic test, and scientists interested in fundamental issues of stem cell behaviour.

Clonal haemopoiesis in normal elderly women: implications for the myeloproliferative disorders and myelodysplastic syndromes.

Studies of X chromosome inactivation patterns are central to many aspects of our understanding of the pathogenesis of haematological malignancies. In patients with myeloproliferative disorders and myelodysplastic syndromes the demonstration of skewed X inactivation patterns in multiple haemopoietic lineages has been taken to indicate a stem cell origin for these groups of diseases. However, stem cell depletion or selection pressures can also produce skewed X inactivation patterns and might increase with age. We have therefore used the HUMARA assay to study X inactivation patterns of elderly patients with myeloproliferative disorders together with an age-matched control group of normal elderly women. A clonal pattern (clonal granulocytes and polyclonal T cells) was observed in 23.1% of normal women and 63.4% of patients with myeloproliferative disorders. This is the first report of X inactivation patterns in purified subpopulations of blood cells in normal elderly women. These results have three significant implications. Firstly, the finding of clonal granulocytes and polyclonal T cells in normal elderly women is likely to reflect age-related stem cell depletion or selection pressures. Secondly, the demonstration of clonal granulocytes and polyclonal T cells is not a useful diagnostic marker for myeloproliferative disorders or myelodysplastic syndromes in elderly women. Thirdly, our data raise the possibility that clonal blood cell patterns may precede rather than follow mutations which subsequently give rise to myelodysplastic or myeloproliferative phenotypes.