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1.  Performance and Cost Efficiency of KRAS Mutation Testing for Metastatic Colorectal Cancer in Routine Diagnosis: The MOKAECM Study, a Nationwide Experience 
PLoS ONE  2013;8(7):e68945.
Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs.
96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists.
This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0.
The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment.
PMCID: PMC3723748  PMID: 23935912
2.  Frequent EGFR Positivity and Overexpression in High-Grade Areas of Human MPNSTs 
Sarcoma  2008;2008:849156.
Malignant peripheral nerve sheath tumours (MPNSTs) are highly malignant and resistant. Transformation might implicate up regulation of epidermal growth factor receptor (EGFR). Fifty-two MPNST samples were studied for EGFR, Ki-67, p53, and survivin expression by immunohistochemistry and for EGFR amplification by in situ hybridization. Results were correlated with clinical data. EGFR RNA was also quantified by RT-PCR in 20 other MPNSTs and 14 dermal neurofibromas. Half of the patients had a neurofibromatosis type 1 (NF1). EGFR expression, detected in 86% of MPNSTs, was more frequent in NF1 specimens and closely associated with high-grade and p53-positive areas. MPNSTs expressed more EGFR transcripts than neurofibromas. No amplification of EGFR locus was observed. NF1 status was the only prognostic factor in multivariate analysis, with median survivals of 18 and 43 months for patients with or without NF1. Finally, EGFR might become a new target for MPNSTs treatment, especially in NF1-associated MPNSTs.
PMCID: PMC2526168  PMID: 18769552
3.  ERCC1, XRCC1 and GSTP1 Single Nucleotide Polymorphisms and Survival of Patients with Colon Cancer Receiving Oxaliplatin-Based Adjuvant Chemotherapy 
Journal of Cancer  2014;5(6):425-432.
Background: While single nucleotide polymorphisms (SNP) in genes involved in DNA repair or drug metabolism have been shown to influence survival of metastatic colon cancer patients treated with FOLFOX, data on adjuvant setting are scarce.
Methods: This study evaluated the correlation between disease-free survival (DFS) of 210 unselected stage III colon cancer patients receiving FOLFOX chemotherapy, and ERCC1-118 (rs11615, c.354T>C), XRCC1-399 (rs25487, c.1196G>A) and GSTP1-105 (rs1695, c.313A>G) polymorphisms. SNP were determined on tumor DNA using a PCR-based RFLP technique.
Results: In univariate analysis, a trend towards longer DFS was observed for ERCC1 (C/T + T/T) versus (C/C) (HR=2.29; p=0.06), and XRCC1 (A/A) versus (G/G + G/A) (HR=1.61; p=0.16), but not for GSTP1 genotypes; a statistically significant p value was obtained when combining ERCC1 and XRCC1 favorable genotypes (0 versus ≥ 1 favorable genotypes, HR=2.42; p=0.02). After adjustment on tumor stage, lymph node ratio and differentiation grade, multivariate analysis showed that combining ERCC1 and XRCC1 genotypes gave a p value slightly above the threshold for statistical significance (HR=2.03; p=0.06), which was lower than for tumor stage, lymph node ratio or differentiation grade.
Conclusion: The association of ERCC1 and XRCC1 polymorphisms may influence the prognosis of stage III colon cancer patients treated with FOLFOX adjuvant chemotherapy. Yet, these findings need to be confirmed in independent prospective studies.
PMCID: PMC4026996  PMID: 24847383
colon cancer; adjuvant FOLFOX; polymorphism; ERCC1; XRCC1; GSTP1.
4.  Thymus and Mediastinal Node Involvement in Childhood Langerhans Cell Histiocytosis: Long-Term Follow-Up From the French National Cohort 
Pediatric Blood & Cancer  2013;60(11):1759-1765.
Mediastinal involvement (MI) in Langerhans cell histiocytosis (LCH) has been rarely reported. Here, we describe the clinical, radiological, and biological presentation, and the outcome of childhood LCH with MI.
From the French LCH register, which includes 1,423 patients aged less than 18 years, we retrieved the medical charts of patients with mediastinal enlargement detected on chest X-rays.
Thirty-seven patients were retrieved, including 18 males; median age of diagnosis was 0.7 years, and median follow-up time was 6.2 years. The prevalence of MI varied with the age at diagnosis, ranging from 7% below 1 year old to less than 1% at >5 years. Thirteen cases (35%) were diagnosed because of MI-related symptoms, including respiratory distress (N = 4), superior venous cava syndrome (N = 2), and/or cough and polypnea (N = 10). CT scans performed in 32 cases at diagnosis showed tracheal compression (N = 5), cava thrombosis (N = 2), and/or calcification (N = 16). All patients presented multi-system disease at LCH diagnosis, and 35/37 were initially treated with vinblastine and corticosteroids. Death occurred in five cases, due to MI (N = 1) or hematological refractory involvement (N = 4). The overall 5-year survival was 87.1%, and immunodeficiency was not detected as a sequel.
MI in LCH mainly occurs in young children, and diagnosis was based on CT showing thymus enlargement and calcifications. Pediatr Blood Cancer 2013;60:1759–1765. © 2013 Wiley Periodicals, Inc.
PMCID: PMC3824083  PMID: 23813854
Langerhans cell histiocytosis; mediastinal nodes; survey; thymus
5.  Diagnostic criteria, specific mutations, and genetic predisposition in gastrointestinal stromal tumors 
In 1998, gastrointestinal stromal tumor (GIST) emerged as a distinct oncogenetic entity and subsequently became a paradigm of targeted therapies in solid tumors. Diagnosis of GIST relies on both histology and immunohistochemistry. Ninety-five percent of GISTs express either KIT or DOG-1. Approximately 80%–90% of GISTs harbor gain-of-function mutations of either KIT or platelet-derived growth factor receptor alpha polypeptide (PDGFRA) receptor tyrosine kinase (RTK). More than 100 different mutations have been described, some of which are associated with specific clinical and/or histological characteristics. Detection of KIT or PDGFRA mutations is recommended in advanced GISTs because they are highly predictive of tumor response to RTK inhibitors, as well as in KIT-negative cases to confirm diagnosis. In most cases, GISTs are sporadic, but in rare cases, they are related with genetic predisposition, such as neurofibromatosis type 1, Carney triad, Carney–Stratakis syndrome, and inherited KIT or PDGFRA germline mutations.
PMCID: PMC3681166  PMID: 23776354
gastrointestinal stromal tumors; KIT; PDGFRA; genetic predispositions; imatinib
6.  A novel X‐linked recessive form of Mendelian susceptibility to mycobaterial disease 
Journal of Medical Genetics  2007;44(2):e65.
Mendelian susceptibility to mycobacterial disease (MSMD) is associated with infection caused by weakly virulent mycobacteria in otherwise healthy people. Causal germline mutations in five autosomal genes (IFNGR1, IFNGR2, STAT1, IL12RB1, IL12B) and one X‐linked (NEMO) gene have been described. The gene products are physiologically related, as they are involved in interleukin 12/23‐dependent, interferon γ‐mediated immunity. However, no genetic aetiology has yet been identified for about half the patients with MSMD.
A large kindred was studied, including four male maternal relatives with recurrent mycobacterial disease, suggesting X‐linked recessive inheritance. Three patients had recurrent disease caused by the bacille Calmette–Guérin vaccine, and the fourth had recurrent tuberculosis. The infections showed tropism for the peripheral lymph nodes.
Known autosomal and X‐linked genetic aetiologies of MSMD were excluded through genetic and immunological investigations. Genetic linkage analysis of the X‐chromosome identified two candidate regions, on Xp11.4–Xp21.2 and Xq25–Xq26.3, with a maximum LOD score of 2.
A new X‐linked recessive form of MSMD is reported, paving the way for the identification of a new MSMD‐causing gene.
PMCID: PMC2598058  PMID: 17293536
7.  Improvement of the quality of BRAF testing in melanomas with nationwide external quality assessment, for the BRAF EQA group 
BMC Cancer  2013;13:472.
Knowledge about tumour gene mutation status is essential for the treatment of increasing numbers of cancer patients, and testing quality has a major impact on treatment response and cost. In 2012, 4,629 tests for BRAF p.V600 were performed in France, in patients with melanomas.
Two batches of unstained melanoma sections were sent, in May and November 2012, to the 46 laboratories supported by the French National Institute of Cancer (INCa). An external quality assessment (EQA) evaluated mutation status, response times and compliance with INCa recommendations.
All the French laboratories involved in testing participated in the EQA. Fourteen different methods were used to detect BRAF mutations, most consisting of combinations of in-house techniques. False responses were noted in 25/520 cases (4.8%), 11 of which concerned confusion between p.V600E and p.V600K. Thus, 2.7% of responses would have led to inappropriate treatment. Within six months, mean response times decreased from 22 to 12 days (P<0.001), and the percentage of samples evaluated by a pathologist for tumour cell content increased, from 75.2% to 96.9% (P<0.001).
Despite the use of non-certified methods, the false response rate was low. Nationwide EQA can improve the quality of molecular pathology tests on tumours.
PMCID: PMC3852250  PMID: 24119386
8.  Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples 
PLoS ONE  2013;8(4):e61103.
Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations.
Materials and Methods
Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples.
Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway.
Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways.
PMCID: PMC3625162  PMID: 23593401
9.  Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor 
Diagnostic Pathology  2012;7:156.
Lymphoid infiltration is a prognostic marker in solid tumors, such as colorectal, breast and lung carcinomas. However, lymphoid infiltration is heterogeneous and the reproducibility of quantification based on single counts within a tumor is very low. We aimed to develop a reproducible method for evaluating lymphoid infiltration in tumors.
Virtual slides were obtained from tissue sections from the localized colorectal carcinomas of 117 patients, stained for CD3 and CD45R0. We assessed the variation of lymphoid cell density by automatic counts in 1 mm-wide, 5 μm-long segments of the invasive front, along an axis 4 mm in length running perpendicular to the invasive front of the tumor.
We plotted curves of the variation of lymphocyte density across the tumor front. Three distinct patterns emerged from this linear quantification of lymphocyte (LQLI). In pattern 1, there was a high density of lymphocytes within the tumor. In pattern 2, lymphocyte density peaked close to the invasive margin. In pattern 3, lymphocytes were diffusely distributed, at low density. It was possible to classify all the tumors studied, and interobserver reproducibility was excellent (kappa =0.9). By contrast, single counts of CD3+ cells on tissue microarrays were highly variable for a given LQLI pattern, confirming the heterogeneity of lymphoid infiltration within individual tumors. In univariate analysis, all pathologic features (stage, metastatic lymph node ratio (LNR), vascular embolism, perineural invasion), CD3+ cell density, LQLI patterns for CD3+ and CD45R0+ cells) were found to have a significant effect on disease-free survival (DFS). In multivariate analysis, only the LQLI pattern for CD3+ cells (HR: 6.02; 95% CI: 2.74-13.18) and metastatic lymph node ratio (HR: 6.14; 95% CI: 2.32-16.2) were associated with DFS.
LQLI is an automated, reproducible method for the assessment of lymphoid infiltration. However, validation of its prognostic value in larger series is required before its introduction into routine practice for prognostic evaluation in patients with colorectal carcinomas.
Virtual slides
The virtual slide(s) for this article can be found here:
PMCID: PMC3542251  PMID: 23148481
Tumor infiltration; Lymphocytes; Invasive margin; Linear quantification; Colorectal cancer; Image analysis; Automated count
10.  Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease 
Nature immunology  2011;12(3):213-221.
Germline mutations in CYBB, the human gene encoding the gp91phox subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This ‘experiment of nature’ indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.
PMCID: PMC3097900  PMID: 21278736
11.  Gene expression profiling identifies emerging oncogenic pathways operating in extranodal NK/T-cell lymphoma, nasal type 
Blood  2009;115(6):1226-1237.
Biopsies and cell lines of NK/T-cell lymphoma, nasal-type (NKTCL) were subject to combined gene expression profiling and array-based comparative genomic hybridization analyses. Compared to PTCL, NOS, NKTCL had higher transcript levels for NK-cell markers and cytotoxic molecules, especially granzyme H, a novel sensitive biomarker of NKTCL. Compared to normal NK cells, tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, EBV-induced genes and PDGFRA. Notably, PDGFRα and its phosphorylated form were confirmed at the protein level, and in vitro the MEC04 NKTCL-cell line was sensitive to imatinib. Deregulation of the AKT, JAK-STAT and NF-κB pathways suggested by bioinformatical analysis, was corroborated by nuclear expression of phosphorylated AKT, STAT3 and RelA in NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). Several features of NKTCL uncovered by this analysis (overexpression of VEGFA and its receptor KDR by the tumor cells, overexpression of MET-HGF) suggest perturbation of angiogenic pathways. Integrative analysis also evidenced deregulation of the tumor suppressor HACE1 in the frequently deleted 6q21 region. This study highlights emerging oncogenic pathways in NKTCL and identifies novel diagnostic and therapeutic targets.
PMCID: PMC2826234  PMID: 19965620
12.  Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence 
PLoS Pathogens  2008;4(11):e1000204.
Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.
Author Summary
Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for dramatic health problems globally. It is estimated that this pathogen infects one-third of the human population and causes three million deaths annually. Most individuals remain asymptomatic for several years before developing an active disease. In such individuals, the bacilli are not cleared but rather persist in a dormant state. Major goals of TB research are to (i) understand how the bacilli remain alive for years within infected individuals, and (ii) find how to prevent their reactivation and hence clinical disease. During dormancy, most of the bacilli are confined to granulomas that consist of well-defined aggregates of different host immune cells. Granulomas prevent spreading of bacilli. In this study, we analyzed the role of a particular cell population found within granulomas, the “foamy macrophages”. These cells are filled with droplets of lipids, a well-known nutrient for persistent bacilli. We found that within these cells, the bacilli do not replicate, but remain alive and seem to internalize host lipids. The foamy macrophages might thus constitute a reservoir for persisting bacilli within their human host, and could provide a relevant model for screening of new antimicrobials against non-replicating persistent mycobacteria.
PMCID: PMC2575403  PMID: 19002241
13.  Prosthetic Hip Infection Caused by Tropheryma whipplei▿  
Journal of Clinical Microbiology  2008;46(4):1556-1557.
We report a case of prosthetic hip infection due to Tropheryma whipplei in a 74-year-old man not previously known to have Whipple's disease. Diagnosis was based on systematic 16S rRNA gene amplification and sequencing of samples obtained during revision hip arthroplasty.
PMCID: PMC2292971  PMID: 18287323
14.  Importance of T Cells, Gamma Interferon, and Tumor Necrosis Factor in Immune Control of the Rapid Grower Mycobacterium abscessus in C57BL/6 Mice▿  
Infection and Immunity  2007;75(12):5898-5907.
Mycobacterium abscessus is an emerging rapidly growing mycobacterium that causes tuberculous-like lesions in humans. We studied the immune control of this organism in C57BL/6 mice challenged intravenously with 107 CFU. Bacteria were eliminated from both the spleen and the liver within 90 days, and liver histology showed organized granulomatous lesions. A T- and B-cell requirement was investigated by challenging Rag2−/−, Cd3ɛ−/−, and μMT−/− mice. Rag2−/− and Cd3ɛ−/− mice were significantly impaired in the ability to clear M. abscessus from the liver and spleen, and μMT−/− mice were significantly impaired in the ability to clear M. abscessus from the liver, suggesting that infection control was primarily T cell dependent in the spleen and both T and B cell dependent in the liver. The liver granulomatous response was similar to that of wild-type controls in μMT−/− mice but completely absent in Cd3ɛ−/− and Rag2−/− mice. We studied the involvement of gamma interferon (IFN-γ) and tumor necrosis factor (TNF) by challenging C57BL/6 mice deficient in the IFN-γ receptor (Ifngr1−/−) and in TNF (Tnf−/−). Ifngr1−/− mice were significantly impaired in M. abscessus control both in the spleen and in the liver, and granulomas were profoundly altered. The effect was even more substantial in Tnf−/− mice; they failed to control M. abscessus infection in the liver and died within 20 to 25 days after infection with many hepatic inflammatory foci and major lesions of ischemic necrosis in the liver and kidney. These features were not observed with the closely related species M. chelonae. T-cell immunity, IFN-γ, and TNF are central factors for the control of M. abscessus in C57BL/6 mice, as they are for the control of pathogenic slowly growing mycobacteria.
PMCID: PMC2168332  PMID: 17875636
15.  IFN-γ Mediates the Rejection of Haematopoietic Stem Cells in IFN-γR1-Deficient Hosts 
PLoS Medicine  2008;5(1):e26.
Interferon-γ receptor 1 (IFN-γR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-γR1 deficiency.
Methods and Findings
We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr1+/+ donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr1−/− recipients. However, Ifngr1−/− mice previously infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rejected HSCT. Like infected IFN-γR1-deficient humans, infected Ifngr1−/− mice displayed very high serum IFN-γ levels before HSCT. The administration of a recombinant IFN-γ-expressing AAV vector to Ifngr1−/− naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr1−/− × Ifng−/− double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-γ depletion in infected Ifngr1−/− mice in vivo allowed subsequent engraftment.
High serum IFN-γ concentration is both necessary and sufficient for graft rejection in IFN-γR1-deficient mice, inhibiting the development of heterologous, IFN-γR1-expressing, haematopoietic cell lineages. These results confirm that IFN-γ is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-γR1-deficient patients through IFN-γ depletion from the blood. They further raise the possibility that depleting IFN-γ may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor.
Claire Soudais and colleagues investigated the mechanism of rejection of hematopoietic stem cell transplants in patients with interferon-gamma receptor 1 (IFN-γR1) deficiency and show that IFN-γ is an anti-hematopoietic cytokine in vivo.
Editors' Summary
Normally, the body's immune system efficiently recognizes and kills bacteria and viruses, but in some rare inherited disorders (“primary immunodeficiencies”) part of the immune system works poorly or is missing. This leaves affected individuals susceptible to infections. People with one of these disorders—interferon-gamma receptor 1 (IFN- γR1) deficiency—are very susceptible to infections with mycobacteria. Except for Mycobacterium tuberculosis and M. leprae (which cause tuberculosis and leprosy, respectively), mycobacteria rarely cause human disease. However, most people with IFN-γR1 deficiency die during childhood from multiple, widespread mycobacterial infections, because IFN-γR1 deficiency disables a specific part of their immune system. When most bacteria enter the body, immune system cells called macrophages engulf and kill them, but mycobacteria actually multiply inside macrophages. This infection stimulates lymphocytes and other immune system cells to release IFN-γ, which binds to IFN-γR1 on uninfected macrophages, activates them, and recruits them to the infection site. Here, they form a “granuloma,” a mass of macrophages and activated lymphocytes that “walls off” the infection. Granuloma formation does not occur in patients with IFN-γR1 deficiency, so mycobacteria (including the usually benign tuberculosis vaccination strain M. bovis BCG) spread throughout the body with disastrous consequences.
Why Was This Study Done?
The only effective treatment for patients with IFN-γR1 deficiency is hematopoietic stem cell transplantation (HSCT). HSCs are the source of all the immune system cells, so transplantation of HSCs from a donor with a normal IFNGR1 gene can provide a patient who has IFN-γR1 deficiency with a new immune system that can combat mycobacterial infections. Unfortunately, in this particular immune deficiency, the new HSCs cannot engraft, even when the patient's own immune system is disabled before HSCT by intensive chemotherapy, and when the donor cells come from a close relative and are a good immunological match. In this study, the researchers have investigated why rejection is so common in IFN-γR1 deficiency using a mouse strain called C57BL/6 Ifngr1−/−—C57BL/6 denotes the genetic background of these mice and Ifngr1−/− indicates that, like human patients, these mice make no IFN-γR1.
What Did the Researchers Do and Find?
Ifngr1−/− mice, the researchers report, cannot control M. bovis BCG infections and do not form mature granulomas just like human patients with IFN-γR1 deficiency. Wild-type C57BL/6 (Ifngr1+/+) mice, however, rapidly control M. bovis BCG infections and form mature granulomas. Ifngr1+/+ HSC transplanted into mycobacteria-free Ifngr1−/− mice survived well and protected the mice against later mycobacterial challenge but Ifngr1−/− mice infected with M. bovis BCG before HSCT rejected the transplanted HSCs. Mycobacteria-infected Ifngr1−/− mice and human patients with IFN-γR1 deficiency have blood high levels of IFN-γ. Could this be responsible for HSCT rejection? To find out, the researchers expressed IFN-γ in uninfected Ifngr1−/− mice before HSCT. As in infected mice, these grafts failed. Conversely, transplanted HSCs survived when transplanted into Ifngr1−/− mice that had been genetically altered to express no IFN-γ, even when these mice were infected with M. bovis BCG before transplantation. Finally, when the researchers used antibodies (proteins made by the immune system that recognize specific molecules) to remove circulating IFN-γ from infected Ifngr1−/− mice, HSCT worked well in the animals with the lowest IFN-γ levels.
What Do These Findings Mean?
These findings indicate that in a mouse model of IFN-γR1 deficiency, high circulating IFN-γ concentrations drive the rejection of transplanted HSCs and prevent the development of antimycobacterial immunity, probably by directly killing the transplanted cells and/or stopping them multiplying. They also suggest how HSCT could be improved in patients with IFN-γR1 deficiency although, as with all animal studies, the situation in people might turn out to be very different. Importantly, antibodies that reduce circulating IFN-γ are already being used to treat other human immune diseases, so the clinical use of these antibodies in patients with IFN-γ deficiency before HSCT is feasible. Finally, the researchers speculate that the use of IFN-γ–depleting antibodies might be beneficial in other situations where HSCT often fails such as when a close relative is not available as a donor. However, this possibility will need to be thoroughly tested in mice before human clinical trials can be started.
Additional Information.
Please access these Web sites via the online version of this summary at
General information about primary immunodeficiencies is available from the US National Institute of Child Health and Human Development
Online Mendelian Inheritance in Man (OMIM) provides information about familial predisposition to mycobacterial disease
Wikipedia has pages on hematopoietic stem cell transplantation and on interferon-γ (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Human Genetics of Infectious Diseases Lab focuses on the genetic basis of predicposition or resistance to infectious diseases in humans
PMCID: PMC2214797  PMID: 18232731
16.  Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease 
PLoS Genetics  2006;2(8):e131.
The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding.
Mendelian susceptibility to mycobacterial disease is a rare syndrome. It is defined by the occurrence of severe disease caused by low virulence mycobacteria in otherwise healthy individuals, in whom antiviral immune response is not affected. Eleven known genetic defects, affecting five genes, have been involved in this type of deficient response to infection, involving immune-mediator molecules IL12 and interferon gamma: IL12B, IL12RB1, IFNGR1, IFNGR2, and STAT1. The signal transducer and activator of transcription-1 (STAT1) amino acid change L706S was previously shown to cause disease by impairing STAT1 phosphorylation. Here, we report two new STAT1 mutations that impair STAT1 DNA-binding activity. We show, by functional analysis of the three STAT1 mutant alleles, that they are intrinsically deleterious for both interferon gamma–induced antimycobacterial immunity, which is mediated through gamma-activated factor and for interferon alpha–induced antiviral immunity, which is mediated through interferon-stimulated genes factor 3. Interestingly, the three alleles are dominant for interferon gamma–induced gamma-activated factor–mediated antimycobacterial immunity, but recessive for interferon alpha–induced interferon-stimulated genes factor 3–mediated antiviral immunity at the cellular and clinical levels. These two new STAT1 alleles, which affect the binding of STAT1 to DNA, define distinct novel genetic causes of Mendelian susceptibility to mycobacterial disease and provide further insight into the molecular mechanism of disease.
PMCID: PMC1550284  PMID: 16934001
17.  Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell–dependent antitumor effects 
Journal of Clinical Investigation  2004;114(3):379-388.
Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell–dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-γ production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.
PMCID: PMC489961  PMID: 15286804
18.  In a novel form of IFN-γ receptor 1 deficiency, cell surface receptors fail to bind IFN-γ 
Journal of Clinical Investigation  2000;105(10):1429-1436.
Complete IFN-γ receptor ligand-binding chain (IFNγR1) deficiency is a life-threatening autosomal recessive immune disorder. Affected children invariably die of mycobacterial infection, unless bone marrow transplantation is undertaken. Pathogenic IFNGR1 mutations identified to date include nonsense and splice mutations and frameshift deletions and insertions. All result in a premature stop codon upstream from the segment encoding the transmembrane domain, precluding cell surface expression of the receptors. We report herein two sporadic and two familial cases of a novel form of complete IFNγR1 deficiency in which normal numbers of receptors are detected at the cell surface. Two in-frame deletions and two missense IFNGR1 mutations were identified in the segment encoding the extracellular ligand-binding domain of the receptor. Eight independent IFNγR1-specific mAb’s, including seven blocking antibodies, gave recognition patterns that differed between patients, suggesting that different epitopes were altered by the mutations. No specific binding of 125I–IFN-γ to cells was observed in any patient, however, and the cells failed to respond to IFN-γ. The mutations therefore cause complete IFNγR1 deficiency by disrupting the IFN-γ–binding site without affecting surface expression. The detection of surface IFNγR1 molecules by specific antibodies, including blocking antibodies, does not exclude a diagnosis of complete IFNγR1 deficiency.
PMCID: PMC315467  PMID: 10811850

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