Inequalities in survival and care across social determinants of health in a cohort of advanced lung cancer patients in Quebec (Canada): A high-resolution population-level analysis.

BACKGROUND: Advanced lung cancer patients exposed to breakthrough therapies like EGFR tyrosine kinase inhibitors (EGFR-TKI) may experience social inequalities in survival, partly from differences in care. This study examined survival by neighborhood-level socioeconomic and sociodemographic status, and geographical location of advanced lung cancer patients who received gefitinib, an EGFR-TKI, as first-line palliative treatment. Differences in the use and delay of EGFR-TKI treatment were also examined. METHODS: Lung cancer patients receiving gefitinib from 2001 to 2019 were identified from Quebec's health administrative databases. Accounting for age and sex, estimates were obtained for the median survival time from treatment to death, the probability of receiving osimertinib as a second EGFR-TKI, and the median time from biopsy to receiving first-line gefitinib. RESULTS: Among 457 patients who received first-line treatment with gefitinib, those living in the most materially deprived areas had the shortest median survival time (ratio, high vs. low deprivation: 0.69; 95% CI: 0.47-1.04). The probability of receiving osimertinib as a second EGFR-TKI was highest for patients from immigrant-dense areas (ratio, high vs. lowdensity: 1.95; 95% CI: 1.26-3.36) or from Montreal (ratio, other urban areas vs. Montreal: 0.39; 95% CI: 0.16-0.71). The median wait time for gefitinib was 1.27 times longer in regions with health centers peripheral to large centers in Quebec or Montreal in comparison to regions with university-affiliated centers (95% CI: 1.09-1.54; n = 353). CONCLUSION: This study shows that real-world variations in survival and treatment exist among advanced lung cancer patients in the era of breakthrough therapies and that future research on inequalities should also focus on this population.

Advanced Lung Cancer Patients' Use of EGFR Tyrosine Kinase Inhibitors and Overall Survival: Real-World Evidence from Quebec, Canada.

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are breakthrough palliative treatments for advanced lung cancer patients with tumors harboring mutations in the EGFR gene. Using healthcare administrative data, three cohorts were created to describe the use of three EGFR-TKIs that are publicly funded in Quebec for specific indications (i.e., 1st-line gefitinib, 1st-line afatinib, and post-EGFR-TKI osimertinib). The main objective was to compare overall survival (OS) among patients receiving these treatments to those in previous experimental and real-world studies. The patients who received EGFR-TKIs for indications of interest between 1 April 2001, and 31 March 2019 (or 31 March 2020, for post-EGFR-TKI osimertinib) were included to estimate the Kaplan-Meier-based median OS for each cohort. An extensive literature search was conducted to include comparable studies. For the gefitinib 1st-line (n = 457), the afatinib 1st-line (n = 80), and the post-EGFR-TKI osimertinib (n = 119) cohorts, we found a median OS (in months) of 18.9 (95%CI: 16.3-21.9), 26.6 (95%CI: 13.7-NE) and 19.9 (95%CI: 17.4-NE), respectively. Out of the 20 studies that we retained from the literature review and where comparisons were feasible, 17 (85%) had similar OS results, which further confirms the value of these breakthrough therapies in real-world clinical practice.

Stereotactic Ablative Radiation Therapy for the Treatment of Early-stage Non-Small-Cell Lung Cancer: CEPO Review and Recommendations.

BACKGROUND: Lung cancer is the second most diagnosed cancer and the leading cause of cancer-related mortality in Canada. Surgical resection is the treatment of choice for patients with stage I non-small-cell lung cancer (NSCLC). However, 20% to 30% of them are deemed medically inoperable and may be offered radiation therapy. Standard external-beam radiation therapy (EBRT) is associated with high rates of local recurrence and poor long-term survival. Stereotactic ablative radiation therapy (SABR) is increasingly being proposed for inoperable patients, and the use of this treatment modality for operable patients is also being contemplated. The objective of this guideline is to review the efficacy and safety of SABR in these two clinical situations and to develop evidence-based recommendations. METHOD: A review of the scientific literature published up to December 2013 was performed. A total of 44 publications were included. RECOMMENDATIONS: Considering the evidence available to date, the Comité de l'évolution des pratiques en oncologie recommends the following: (1) for medically operable patients with stage T1-2N0M0 NSCLC, surgery remains the standard treatment because comparative data regarding the efficacy of SABR and surgery are currently insufficient for SABR to be considered an equivalent alternative to surgery for these patients; (2) for medically inoperable patients with stage T1-2N0M0 NSCLC or medically operable patients who refuse surgery, SABR should be preferred to standard EBRT (grade B recommendation); (3) the biological equivalent dose (BED(10)) used for SABR treatment should be at least 100 Gy (grade B recommendation); (4) for patients with a central tumor, a large-volume tumor (large planning target volume) or severe pulmonary comorbidity, a risk-adaptive schedule should be used (dose reduction or increase in the number of fractions; grade B recommendation); (5) the choice of using SABR to treat NSCLC should be discussed within tumor boards; treatment with SABR (or with standard EBRT) should not be considered for patients whose life expectancy is very limited because of comorbidities (grade D recommendation).

Transarterial embolization therapies for the treatment of hepatocellular carcinoma: CEPO review and clinical recommendations.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world and its incidence rate has consistently increased over the past 15 years in Canada. Although transarterial embolization therapies are palliative options commonly used for the treatment of HCC, their efficacy is still controversial. The objective of this guideline is to review the efficacy and safety of transarterial embolization therapies for the treatment of HCC and to develop evidence-based recommendations. METHOD: A review of the scientific literature published up to October 2013 was performed. A total of 38 studies were included. RECOMMENDATIONS: Considering the evidence available to date, the CEPO recommends the following: (i) transarterial chemoembolization therapy (TACE) be considered a standard of practice for the palliative treatment of HCC in eligible patients; (ii) drug-eluting beads (DEB)-TACE be considered an alternative and equivalent treatment to conventional TACE in terms of oncological efficacy (overall survival) and incidence of severe toxicities; (iii) the decision to treat with TACE or DEB-TACE be discussed in tumour boards; (iv) bland embolization (TAE) not be considered for the treatment of HCC; (v) radioembolization (TARE) not be considered outside of a clinical trial setting; and (vi) sorafenib combined with TACE not be considered outside of a clinical trial setting.

Utilisation de la radiothérapie stéréotaxique d'ablation pour le traitement du cancer du poumon non à petites cellules / Use of stereotactic ablation radiotherapy for the treatment of non-small cell lung cancer

CONTEXTE ET OBJECTIFS: La Société canadienne du cancer estime que 8 200 nouveaux cas de cancer du poumon seront diagnostiqués au Québec en 2014 (26 100 cas au Canada) et que 6 400 décès seront enregistrés (20 500 décès au Canada). Chez les hommes comme chez les femmes, le cancer du poumon occupe le deuxième rang des néoplasies les plus fréquemment diagnostiquées et demeure la première cause de décès par cancer. Environ 85 % des cancers du poumon sont de type non à petites cellules (CPNPC). Parmi les patients chez qui on a nouvellement diagnostiqué un CPNPC, seulement 15 % à 20 % présentent une maladie de stade précoce (stade I). Le traitement standard pour ces patients est la résection chirurgicale. Toutefois, 20 % à 30 % d'entre eux sont considérés comme inopérables en raison de comorbidités et ils se voient offrir un traitement de radiothérapie. Lorsque ce traitement est administré par radiothérapie externe usuelle, les taux de récidive locale à 3 ans sont élevés, atteignant 29 % à 57 %, et les taux de survie globale à 3 ans sont de l'ordre de 20 % à 50 %. Les patients inopérables décèdent souvent de leurs comorbidités plutôt que de leur cancer du poumon. La radiothérapie stéréotaxique d'ablation (stereotactic ablative radiation therapy, SABR), aussi connue sous l'appellation stereotactic body radiation therapy, SBRT), est une modalité de radiothérapie externe hypofractionnée, guidée par imagerie et hautement conformationnelle, qui consiste à irradier une cible extracrânienne en une seule séance ou en un petit nombre de fractions. L'intérêt de cette méthode est l'utilisation de doses très élevées (dites ablatives) par fraction, administrées avec une très grande précision. L'utilisation de très hautes doses pour cibler des tumeurs mobiles pose toutefois des défis d'innocuité importants. Afin d'assurer une irradiation sécuritaire et reproductible, un ensemble de stratégies ont été mises au point en ce qui a trait à la planification du traitement et au ciblage de la tumeur (p. ex. immobilisation du patient, systèmes d'asservissement respiratoire, ciblage de la tumeur en temps réel). Jusqu'à tout récemment, la recherche s'est surtout intéressée à l'évaluation de l'efficacité et de l'innocuité de la SABR pour le traitement du CPNPC chez les patients inopérables. Les bons résultats de contrôle local obtenus chez ces patients ont amené les milieux à s'interroger sur la possibilité que la SABR puisse constituer une option à la chirurgie pour les patients opérables, et sur le fait que la SABR pourrait limiter la toxicité de la radiothérapie dans un contexte de réirradiation thoracique. L'objectif de ce guide de pratique est d'évaluer l'efficacité et l'innocuité de la SABR pour le traitement du CPNPC dans ces trois situations cliniques.RÉSULTATS: La revue de la littérature a permis de répertorier 48 articles rapportant les résultats de 20 études prospectives, 21 études rétrospectives et 4 méta-analyses. Aucune étude randomisée n'a été répertoriée. Évaluation de la SABR pour le traitement du CPNPC chez des patients inopérables: Pour le traitement des patients inopérables, à 3 ans, la SABR a permis d'obtenir des taux de contrôle local oscillant autour de 88 % et des taux de survie spécifique au cancer d'environ 82 %. Les toxicités de grade 3 ou plus ont été de 5 % ou moins dans la majorité des études. Une toxicité accrue a été observée chez les patients présentant une tumeur centrale. Toutefois, lorsqu'un ajustement du protocole de radiothérapie en fonction du risque était prévu, les taux de toxicité sévère étaient faibles. Les études rétrospectives comparant la SABR à la radiothérapie externe usuelle pour le traitement de patients inopérables ont toutes montré un avantage de survie globale en faveur de la SABR. De même, une méta-analyse d'études observationnelles a démontré un avantage de survie globale et de survie spécifique au cancer en faveur de la SABR. Une comparaison indirecte de visu des résultats d'études observationnelles évaluant la SABR et la radiothérapie externe usuelle suggère également une supériorité de la SABR au niveau du contrôle local, de la survie globale et de la survie spécifique au cancer. Évaluation de la SABR pour le traitement du CPNPC chez des patients opérables: Les études ayant évalué l'efficacité de la SABR pour le traitement de patients opérables ont présenté des données de contrôle local et de survie d'une ampleur similaire à celles observées pour les patients inopérables. La SABR a été comparée à la chirurgie à l'aide d'analyses de cohortes de patients appariés, principalement selon des scores de propension, et donc sujettes à certaines limites méthodologiques. Les résultats oncologiques n'ont généralement pas été significativement différents entre les groupes, mais le profil d'innocuité a été favorable à la SABR. Évaluation de la réirradiation thoracique avec la SABR? En ce qui a trait à l'évaluation de la SABR dans un contexte de réirradiation, les études comptaient peu de patients, étaient de faible niveau de preuve et les populations et traitements étudiés étaient hétérogènes. Dans les 16 premiers mois, les taux de récidive locale ont varié entre 1,4 % et 29 %. Toutefois, des taux de toxicité sévère allant jusqu'à 33 % ont été observés. MÉTHODE: Une revue de la littérature scientifique publiée jusqu'en décembre 2013 a été réalisée dans l'outil de recherche Pubmed. Les études évaluant la SABR pour le traitement du CPNPC chez les patients inopérables, les patients opérables et les patients ayant déjà reçu une radiothérapie thoracique ont été retenues. Les études comparant la SABR à la radiothérapie externe usuelle ou la chirurgie ont aussi été considérées. Les études évaluant la SABR pour l'administration d'une surimpression, pour le traitement de patients présentant une atteinte ganglionnaire (N+) ou pour le traitement de métastases pulmonaires, ont été exclues. Les études à caractère économique ont aussi été rejetées. Les abrégés de communications présentées à l'occasion des principaux congrès internationaux de même que les sites Web d'organismes publiant des revues systématiques, des lignes directrices et des consensus d'experts ont également été consultés. CONCLUSION: En conclusion, la SABR permet d'obtenir un bon contrôle local qui semble se traduire en un bénéfice de survie. Les données actuellement disponibles suggèrent que la SABR serait plus efficace que la radiothérapie externe usuelle pour le traitement des patients inopérables ou pour ceux refusant la chirurgie. Toutefois, en ce qui a trait au traitement des patients opérables, le CEPO juge la preuve insuffisante pour que la SABR puisse être considérée comme une option équivalente à la chirurgie. Finalement, les données sur l'utilisation de la SABR dans un contexte de réirradiation sont trop fragmentaires pour conclure à propos de son efficacité. La toxicité sévère observée appelle à la prudence. RECOMMANDATIONS: Considérant les données probantes disponibles à ce jour, le Comité de l'évolution des pratiques en oncologie (CEPO) recommande: 1. Pour les patients médicalement opérables atteints d'un CPNPC de stade T1-2N0M0, que la chirurgie demeure le traitement standard; les données comparatives sur l'efficacité de la SABR et de la chirurgie sont présentement insuffisantes pour que la SABR soit considérée comme une option équivalente à la chirurgie pour ces patients; 2. Pour les patients atteints d'un CPNPC de stade T1-2N0M0 médicalement inopérables ou pour ceux médicalement opérables mais refusant la chirurgie, que la SABR soit préférée à la radiothérapie externe usuelle (recommandation de grade B); 3. Que la dose biologique équivalente (BED10) totale d'un traitement de SABR soit d'au moins 100 Gy (recommandation de grade B); 4. Pour les patients présentant une tumeur de localisation centrale, une tumeur de grand volume (grand PTV) ou une comorbidité pulmonaire sévère, qu'un ajustement de dose (réduction) ou de fractionnement (augmentation du nombre de fractions) soit apporté (recommandation de grade B); 5. Que l'utilisation de la SABR pour le traitement du CPNPC soit discutée en comité des thérapies du cancer; un traitement de SABR (de même que de radiothérapie externe usuelle) ne devrait pas être envisagé pour les patients qui ont une espérance de vie très limitée au regard de leurs comorbidités (recommandation de grade D). La littérature actuellement disponible ne permet pas de formuler une recommandation à propos de la place de la SABR dans un contexte de réirradiation thoracique.(AU)

BACKGROUND AND OBJECTIVES: The Canadian Cancer Society estimates for 2014 that 8,200 new cases of lung cancer will be diagnosed in Québec (26,100 cases in Canada) and that 6,400 lung cancer deaths will be recorded (20,500 deaths in Canada). In both men and women, lung cancer is the second most diagnosed neoplasia and the leading cause of cancer-related mortality. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Only 15 to 20% of patients newly diagnosed with NSCLC present with early-stage disease (stage I). Surgical resection is the standard treatment for these patients. However, 20 to 30% of them are deemed inoperable because of comorbidities and are offered radiation therapy. When standard external-beam radiation therapy is administered, the 3-year local recurrence rates are high, being 29 to 57%, and the overall survival rates are in the order of 20 to 50%. Inoperable patients often die from comorbidities instead of lung cancer. Stereotactic ablative radiation therapy (SABR, also known as stereotactic body radiation therapy, SBRT) is a highly conformal, image-guided, hypofractionated form of external-beam radiotherapy in which an extracranial body target is irradiated in a single or few fraction(s). The advantage of this method is the use of very high (ablative) per-fraction doses delivered with very high precision. However, the use of very high doses to target a mobile tumour is challenging because of major safety concerns. To ensure safe and reproducible irradiation, various treatment-planning and tumor-targeting strategies have been developed (e.g., immobilization of the patient, respiratory gating systems, and real-time tumour tracking methods). Until quite recently, research has focused mainly on evaluating the efficacy and safety of SABR for the treatment of NSCLC in inoperable patients. The high local control rates obtained in these patients led the field to consider the possibility that SABR could be an alternative to surgery for operable patients and that it could reduce radiation-related toxicity in the context of thoracic reirradiation. The objective of this clinical guideline is to evaluate the efficacy and safety of SABR for the treatment of NSCLC in these three clinical situations. METHODS: A review of the scientific literature published up to December 2013 was carried out using the PubMed search tool. The search was limited to studies evaluating SABR for the treatment of NSCLC in inoperable patients, operable patients and patients previously treated with thoracic radiotherapy. Studies comparing SABR with standard external-beam radiotherapy or with surgery were also considered for inclusion. Studies evaluating the use of SABR for the delivery of a boost dose, for the treatment of patients with N+ stage disease or for the treatment of lung metastases were excluded. Economic studies were also excluded. Abstracts from papers presented at major international conferences and the websites of organizations that publish systematic reviews, clinical guidelines and expert consensuses were also consulted. RESULTS: The literature review identified 48 articles reporting the results of 20 prospective studies, 21 retrospective studies and 4 meta-analyses. No randomized controlled studies were found. Evaluation of SABR for the treatment of NSCLC in inoperable patients: For the treatment of inoperable patients, SABR was associated with 3-year local control rates of about 88% and cancer-specific survival rates of approximately 82%. Grade 3 or higher toxicity rates were 5% or less in most of the studies. Increased toxicity was observed in patients with a central tumour. However, severe-toxicity rates were low when a risk-adapted treatment protocol was used. All the retrospective studies comparing SABR with standard external-beam radiotherapy for the treatment of inoperable patients showed an overall survival benefit in favour of SABR. Moreover, a meta-analysis of observational studies showed an overall and cancer-specific survival benefit in favour of SABR. An indirect comparison of results from observational studies evaluating SABR and standard external-beam radiotherapy also suggests that SABR has superior efficacy in terms of local control, overall survival and cancer-specific survival. Evaluation of SABR for the treatment of NSCLC in operable patients: The studies that evaluate the efficacy of SABR for the treatment of operable patients provided survival and local control data of similar magnitude to those observed for inoperable patients. SABR was compared with surgery by propensity score-matching approaches in most of the studies. These analyses therefore have certain methodological limitations. In general, the oncological outcomes were not significantly different between the groups, but the safety profile was favorable to SABR. Evaluation of thoracic reirradiation with SABR: The studies that evaluated SABR in a reirradiation setting had small samples, were of low evidence grade and were highly heterogeneous in terms of patient characteristics and treatments. Local recurrence during the first 16 months occurred at rates varying from 1.4 to 29%. However, severe-toxicity rates of up to 33% were observed. CONCLUSION: In conclusion, SABR offers good local control, which seems to be associated with a survival benefit. The available data suggest that SABR shows better efficacy than standard external-beam radiotherapy in inoperable patients and in operable patients who refuse surgery. However, with regard to the treatment of operable patients, the CEPO considers that the current body of evidence is not sufficient to recognize SABR as an equivalent alternative to surgery. Lastly, the data on the use of SABR in reirradiation settings is too fragmentary to draw any conclusions regarding its efficacy. The observed severe toxicity data urge caution. RECOMMENDATIONS: Considering the currently available evidence, the Comité de l'évolution des pratiques en oncologie (CEPO) recommends: 1. For medically operable patients with stage T1-2N0M0 NSCLC, that surgery remains the standard treatment, since comparative data on the efficacy of SABR and surgery are currently insufficient for SABR to be considered an equivalent alternative to surgery for these patients; 2. For medically inoperable patients with stage T1-2N0M0 NSCLC or medically operable patients who refuse surgery, that SABR be preferred to standard external-beam radiotherapy (grade B recommendation); 3. That the total biological equivalent dose (BED10) used for SABR treatment be at least 100 Gy (grade B recommendation); 4. For patients with a central tumour, a large-volume tumour (large PTV) or severe pulmonary comorbidity, that a risk-adapted protocol be used (dose reduction or increase in the number of fractions) (grade B recommendation); 5. That the option of using SABR to treat NSCLC be discussed in tumor boards; treatment with SABR (or with standard external-beam radiotherapy) should not be considered for patients whose life expectancy is very limited by comorbidities (grade D recommendation). The currently available literature does not permit any recommendations to be made regarding the role of SABR in a thoracic reirradiation setting.(AU)

Adjuvant treatment for endometrial cancer: literature review and recommendations by the Comité de l'évolution des pratiques en oncologie (CEPO).

OBJECTIVE: Despite the very good prognosis of endometrial cancer, a number of patients with localized disease relapse following surgery. Therefore, various adjuvant therapeutic approaches have been studied. The objective of this review is to evaluate the efficacy and safety of neoadjuvant and adjuvant therapies in patients with resectable endometrial cancer and to develop evidence-based recommendations. METHODS: A review of the scientific literature published between January 1990 and June 2012 was performed. The search was limited to published phase III clinical trials and meta-analyses evaluating the efficacy of neoadjuvant or adjuvant therapies in patients with endometrial carcinoma or carcinosarcoma. A total of 23 studies and five meta-analyses were identified. RESULTS: The selected literature showed that in patients with a low risk of recurrence, post-surgical observation is safe and recommended in most cases. There are several therapeutic modalities available for treatment of endometrial cancers with higher risk of recurrence, including vaginal brachytherapy, external beam radiotherapy, chemotherapy, or a combination of these. CONCLUSIONS: Considering the evidence available to date, the CEPO recommends the following: (1)post-surgical observation for most patients with a low recurrence risk; (2)adjuvant vaginal brachytherapy for patients with an intermediate recurrence risk; (3)adjuvant pelvic radiotherapy with or without vaginal brachytherapy for patients with a high recurrence risk; addition of adjuvant chemotherapy may be considered as an option for selected patients (excellent functional status, no significant co-morbidities, poor prognostic factors); (4)adjuvant chemotherapy and pelvic radiotherapy with or without brachytherapy and para-aortic irradiation for patients with advanced disease;

Identification of a SIRT1 mutation in a family with type 1 diabetes.

Type 1 diabetes is caused by autoimmune-mediated ß cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to ß cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo.

AMPK is a metabolic sensor that helps maintain cellular energy homeostasis. Despite evidence linking AMPK with tumor suppressor functions, the role of AMPK in tumorigenesis and tumor metabolism is unknown. Here we show that AMPK negatively regulates aerobic glycolysis (the Warburg effect) in cancer cells and suppresses tumor growth in vivo. Genetic ablation of the α1 catalytic subunit of AMPK accelerates Myc-induced lymphomagenesis. Inactivation of AMPKα in both transformed and nontransformed cells promotes a metabolic shift to aerobic glycolysis, increased allocation of glucose carbon into lipids, and biomass accumulation. These metabolic effects require normoxic stabilization of the hypoxia-inducible factor-1α (HIF-1α), as silencing HIF-1α reverses the shift to aerobic glycolysis and the biosynthetic and proliferative advantages conferred by reduced AMPKα signaling. Together our findings suggest that AMPK activity opposes tumor development and that its loss fosters tumor progression in part by regulating cellular metabolic pathways that support cell growth and proliferation.

Disruption of Igfbp1 fails to rescue the phenotype of Sirt1-/- mice.

Sirtuin 1 (SIRT1) is an NAD-dependent histone deacetylase (HDAC) whose activity is thought to forestall the onset of a variety of age-related diseases. Mice carrying null mutations of the Sirt1 gene suffer high rates of neonatal lethality and those that survive are sterile, growth retarded, lean and their livers express high levels of insulin-like growth factor binding protein-1 (IGFBP1). IGFBP1 binds and regulates the bioavailability of Igfs. Interestingly, Igfbp1 transgenic mice largely phenocopy Sirt1-/- mice, suggesting the possibility that the over-expression of IGFBP1 in Sirt1-/- mice might be responsible for many of their phenotypes. We interbred Sirt1 heterozygote mice to Igfbp1-deficient mice to test the hypothesis that the disruption of one or both alleles of Igfbp1 would rescue the phenotype of Sirt1-/- mice. We report that mono- or bi-allelic disruption of the Igfbp1 gene had no effect on the embryonic and neonatal lethality of Sirt1-/- mice. However, we show that mice lacking at least one allele of both Sirt1 and Igfbp1 genes have a much higher incidence of malocclusion.

sirt1-null mice develop an autoimmune-like condition.

The sirt1 gene encodes a protein deacetylase with a broad spectrum of reported substrates. Mice carrying null alleles for sirt1 are viable on outbred genetic backgrounds so we have examined them in detail to identify the biological processes that are dependent on SIRT1. Sera from adult sirt1-null mice contain antibodies that react with nuclear antigens and immune complexes become deposited in the livers and kidneys of these animals. Some of the sirt1-null animals develop a disease resembling diabetes insipidus when they approach 2 years of age although the relationship to the autoimmunity remains unclear. We interpret these observations as consistent with a role for SIRT1 in sustaining normal immune function and in this way delaying the onset of autoimmune disease.

SirT1 regulates energy metabolism and response to caloric restriction in mice.

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.

Connections between ETV6-modulated genes: identification of shared features.

Accumulating genetic and functional evidence point to ETV6 as being the tumour suppressor gene targeted by the deletions at chromosome 12p12-13 found in various cancers, particularly childhood leukemia. ETV6 is a ubiquitously expressed transcription factor (TF) of the ETS family with very few known targeted genes. We recently compiled a list of 87 ETV6-modulated genes that can be classified into a number of subgroups based on their coordinated expression patterns. In the present report, we hypothesized that genes presenting a similar profile of modulation could also share biological features, promoter sequence similarities and/or, common transcription factor binding sites (TFBSs). Using an exploratory approach based on hierarchical clustering of expression data, Gene Ontology (GO) terms, sequence similarity and evolutionary conserved putative TFBSs, we found that many genes presenting a similar expression profile also share biological features and/or conserved predicted TFBSs but rarely show detectable promoter sequence similarities. We also calculated the proportion of ETV6-modulated genes that have any conserved TFBSs of the Jaspar database in their regulatory sequence and compared these proportions to those calculated for two other gene lists, ETV6 non-modulated and ETS-regulated. We found that the NF-kB, c-REL and p65 TFBSs, which all bind TFs of the REL class, were under-represented among the ETV6-modulated genes compared to the ETV6-non-modulated genes, while the Broad-complex 1 TFBS appeared to be over-represented. NF-Y and Chop/cEBP TFBSs were over-represented in the promoters of ETV6-modulated genes compared to ETS-regulated genes. These analyses will help direct further studies intending to understand the role of ETV6 as a transcriptional regulator and aid in constructing the ETV6-regulatory gene network.

In vivo footprinting analysis of the Glypican 3 (GPC3) promoter region in neuroblastoma cells.

Glypican 3 (GPC3) is an X-linked gene that has its peak expression during development and is down-regulated in all studied tissues after birth. We have shown that GPC3 was expressed in neuroblastoma and Wilms' tumor. To understand the mechanisms regulating the transcription of this gene in neuroblastoma cells, we have focused our study on the identification of putative transcription factors binding the promoter. In this report we performed in vivo dimethylsulfate, UV type C irradiation and DNaseI footprinting analyses coupled with ligation-mediated PCR on nearly 1000 bp of promoter in two neuroblastoma cell lines, SJNB-7 (expressing GPC3) and SK-N-FI (not expressing GPC3). Nucleosome signature footprints were observed in the most distal part of the studied region in both cell lines. We detected eight large differentially protected regions, suggesting the presence of binding proteins in both cell lines but more DNA-protein interactions in GPC3-expressing cells. Sp1 was previously shown to be able to bind some of these regions. Here by combining electromobility shift assays and chromatin immunoprecipitations we showed that the transcription factor NFY was part of the DNA-protein complex found in footprinted regions upstream of the described minimal promoter. These studies performed on chromatin in situ suggest that NFY and yet unknown cell type-specific factors may play an important role in the regulation of GPC3.

Identification of transcripts modulated by ETV6 expression.

Deletions at chromosome 12p12-13 are observed in 26-47% of childhood pre-B acute lymphoblastic leukaemia (ALL) cases, suggesting the presence of a tumour suppressor gene (TSG). Accumulating genetic and functional evidence points to ETV6 as being the most probable TSG targeted by the deletions. ETV6 is a ubiquitously expressed transcription factor of the ETS family with very few known targets. To understand its function and to elucidate the impact of its absence in leukaemia, we conducted a study to identify targeted genes. Following the induction of ETV6 expression, global expression was evaluated at different time points. We identified 87 modulated genes, of which 10 (AKR1C1, AKR1C3, IL18, LUM, PHLDA1, PTGER4, PTGS2, SPHK1, TP53 and VEGF) were validated by real-time quantitative reverse transcription-polymerase chain reaction. To assess the significance of the validated candidate genes in leukaemia, their expression patterns were determined, as well as that of ETV6, in pre-B ALL patients. The expression of IL18, LUM, PTGER4, SPHK1 and TP53 was significantly correlated with that of ETV6, further suggesting that ETV6 could regulate the expression of these genes in leukaemia. This work constitutes another step towards the understanding of the functions of ETV6 and the impact of its inactivation in childhood leukaemia.

A detailed transcriptional map of the chromosome 12p12 tumour suppressor locus.

Loss of heterozygosity of the short arm of chromosome 12 is a frequent event in a wide range of haematological malignancies and solid tumours. In previous studies, the shortest commonly deleted region was delimited to a 750-kb interval, defined by the markers D12S89 and D12S358, in pre-B acute lymphoblastic leukaemia patients, suggesting the presence of a tumour suppressor locus. Here we report the construction of a transcriptional map that integrates the data obtained by genomic sequence analysis, EST database search, comparative analysis and exon amplification. We identified seven putative transcriptional units as well as six pseudogenes. Four of these candidate genes were already known: ETV6, encoding an ets-like transcription factor, LRP6, a member of the LDL receptor gene family, BCL-G, a recently identified pro-apoptotic gene and MKP-7, encoding a new member of the dual-specificity phosphatase family. The products encoded by the three new genes identified in this study, LOH1CR12, LOH2CR12 and LOH3CR12, have no clear homology to known proteins. The gene predictions were all confirmed by expression analysis using RT-PCR and Northern blot. This transcriptional map is a crucial step toward the identification of the tumour suppressor gene at 12p12.