We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

We report duplication of the APP locus on chromosome 21 in five families with autosomal dominant early-onset Alzheimer disease (ADEOAD) and cerebral amyloid angiopathy (CAA). Among these families, the duplicated segments had a minimal size ranging from 0.58 to 6.37 Mb. Brains from individuals with APP duplication showed abundant parenchymal and vascular deposits of amyloid-beta peptides. Duplication of the APP locus, resulting in accumulation of amyloid-beta peptides, causes ADEOAD with CAA.

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.

The clinical gradient of the germline TP53 mutations, which should be validated by other studies, suggests that it might be appropriate to stratify the clinical management of LFS according to the class of the mutation.

Clinical classification of sequence variants identified in hereditary disease genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch Syndrome genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist variant classification, and recognized by microattribution. The scheme was refined by multidisciplinary expert committee review of clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants not obviously protein-truncating from nomenclature. This large-scale endeavor will facilitate consistent management of suspected Lynch Syndrome families, and demonstrates the value of multidisciplinary collaboration for curation and classification of variants in public locus-specific databases.

Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors. We have developed a simple functional assay for p53 mutation in which human p53 expressed in Saccharomyces cerevisiae activates transcription of the ADE2 gene. Consequently, yeast colonies containing wild-type p53 are white and colonies containing mutant p53 are red. Since this assay tests the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations. By combining this approach with gap repair techniques in which unpurified p53 reverse transcription-PCR products are cloned by homologous recombination in vivo it is possible to screen large numbers of samples and multiple clones per sample for biologically important mutations. This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue. In addition, the assay detects temperature-sensitive mutants, which give pink colonies. We show here that this form of p53 functional assay can be used rapidly to detect germline mutations in blood samples, somatic mutations in tumors, and mutations in cell lines.

To determine the prevalence of early-onset Alzheimer disease (EOAD) and of autosomal dominant forms of EOAD (ADEOAD), we performed a population-based study in the city of Rouen (426,710 residents). EOAD was defined as onset of disease at age <61 years, and ADEOAD was defined as the occurrence of at least three EOAD cases in three generations. Using these stringent criteria, we calculated that the EOAD and ADEOAD prevalences per 100,000 persons at risk were 41.2 and 5.3, respectively. We then performed a mutational analysis of the genes for amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) in 34 families with ADEOAD ascertained in France. In 19 (56%) of these families, we identified 16 distinct PSEN1 missense mutations, including 4 (Thr147Ile, Trp165Cys, Leu173Trp, and Ser390Ile) not reported elsewhere. APP mutations, including a novel mutation located at codon 715, were identified in 5 (15%) of the families. In the 10 remaining ADEOAD families and in 9 additional autosomal dominant Alzheimer disease families that did not fulfill the strict criteria for ADEOAD, no PSEN1, PSEN2, or APP mutation was identified. These results show that (1) PSEN1 and APP mutations account for 71% of ADEOAD families and (2) nonpenetrance at age <61 years is probably infrequent for PSEN1 or APP mutations.