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We conducted a dose escalation Phase 1 study of autologous PBMCs loaded by microfluidic squeezing (Cell Squeeze® technology) with HPV16 E6 and E7 antigens (SQZ-PBMC-HPV), in HLA-A*02+ patients with advanced/metastatic HPV16+ cancers. Preclinical studies in murine models had shown such cells resulted in stimulation and proliferation of antigen specific CD8+ cells, and demonstrated antitumor activity. Administration of SQZ-PBMC-HPV was every 3 weeks. Enrollment followed a modified 3+3 design with primary objectives to define safety, tolerability, and the recommended Phase 2 dose. Secondary and exploratory objectives were antitumor activity, manufacturing feasibility, and pharmacodynamic evaluation of immune responses. Eighteen patients were enrolled at doses ranging from 0.5 × 106 to 5.0 × 106 live cells/kg. Manufacture proved feasible and required < 24 h within the overall vein-to-vein time of 1 - 2 weeks; at the highest dose, a median of 4 doses were administered. No DLTs were observed. Most related TEAEs were Grade 1 - 2, and one Grade 2 cytokine release syndrome SAE was reported. Tumor biopsies in three patients showed 2 to 8-fold increases in CD8+ tissue infiltrating lymphocytes, including a case that exhibited increased MHC-I+ and PD-L1+ cell densities and reduced numbers of HPV+ cells. Clinical benefit was documented for the latter case. SQZ-PBMC-HPV was well tolerated; 5.0 × 106 live cells/kg with double priming was chosen as the recommended Phase 2 dose. Multiple participants exhibited pharmacodynamic changes consistent with immune responses supporting the proposed mechanism of action for SQZ-PBMC-HPV, including patients previously refractory to checkpoint inhibitors.
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Neoplasias , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Antígenos HLA-A , Papillomavirus Humano 16 , Leucocitos Mononucleares , Neoplasias/complicaciones , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/complicacionesRESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a life-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulation due to pathogenic variants in genes that encode complement components and regulators. Among these genes, the factor H (FH) gene, CFH, presents with the highest frequency (15% to 20%) of variants and is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remains challenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. To address this knowledge gap, we expressed and functionally characterized 105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign and, for each, we fully documented the nature of the pathogenicity. Twenty-six previously characterized FH variants were used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%) alter FH expression or function in vitro and, therefore, are proposed to be pathogenic. We show that rarity in control databases is not informative for variant classification, and we identify important limitations in applying prediction algorithms to FH variants. Based on structural and functional data, we suggest ways to circumvent these difficulties and, thereby, improve variant classification. Our work highlights the need for functional assays to interpret FH variants accurately if clinical care of patients with aHUS is to be individualized and optimized.
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Síndrome Hemolítico Urémico Atípico/genética , Factor H de Complemento/genética , Síndrome Hemolítico Urémico Atípico/metabolismo , Síndrome Hemolítico Urémico Atípico/patología , Factor H de Complemento/química , Factor H de Complemento/metabolismo , Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Modelos Moleculares , Mutación Puntual , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BNT162b2 is a highly efficacious mRNA vaccine approved to prevent COVID-19. This brief report describes the immunogenicity and anti-viral protective effect of BNT162b2 in hACE2 transgenic mice. Prime-boost immunization with BNT162b2 elicited high titers in neutralizing antibodies against SARS-CoV-2, which correlated with viral clearance and alleviated lung lesions in these mice after viral challenge.
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An effective vaccine is needed to end the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we assess the preliminary safety, tolerability and immunogenicity data from an ongoing single-center (in Jiangsu province, China), parallel-group, double-blind phase 1 trial of the vaccine candidate BNT162b1 in 144 healthy SARS-CoV-2-naive Chinese participants. These participants are randomized 1:1:1 to receive prime and boost vaccinations of 10 µg or 30 µg BNT162b1 or placebo, given 21 d apart, with equal allocation of younger (aged 18-55 years) and older adults (aged 65-85 years) to each treatment group (ChiCTR2000034825). BNT162b1 encodes the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) and is one of several messenger RNA-based vaccine candidates under clinical investigation. Local reactions and systemic events were generally dose dependent, transient and mild to moderate. Fever was the only grade 3 adverse event. BNT162b1 induced robust interferon-γ T cell responses to a peptide pool including the RBD in both younger and older Chinese adults, and geometric mean neutralizing titers reached 2.1-fold (for younger participants) and 1.3-fold (for the older participants) that of a panel of COVID-19 convalescent human sera obtained at least 14 d after positive SARS-CoV-2 polymerase chain reaction test. In summary, BNT162b1 has an acceptable safety profile and produces high levels of humoral and T cell responses in an Asian population.
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Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Vacuna BNT162 , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/efectos adversos , China/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , ARN Mensajero/genética , ARN Mensajero/inmunología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Adulto Joven , Vacunas de ARNmRESUMEN
OBJECTIVE: To determine at the phenotypic, functional, and transcriptional levels whether abatacept, a CTLA-4Ig molecule that binds with high affinity to CD80/86 on antigen-presenting cells (APCs) and is used to treat rheumatoid arthritis, induces a state of immunologic tolerance in T cells and dendritic cells in mice. METHODS: We investigated the capacity of abatacept to regulate the development of antigen-specific immunologic tolerance in vivo using murine models of priming and tolerance to generate highly purified antigen-specific T cell populations and CD11c+ APCs. These were combined with detailed immunologic and full genome transcriptional analyses. RESULTS: We found that abatacept inhibited T cell activation, but did not render T cells anergic or lead to the generation of Treg cells. However, it induced a sustained inhibition of T cell activation due to the inability of these cells to progress through the cell cycle following T cell receptor stimulation. We also observed that this state was accompanied by an inhibition of dendritic cell activation due to their reduced licensing by T cells. CONCLUSION: This study provides detailed insight into the mode of action of abatacept, demonstrating that its effectiveness is not due to the induction of T cell tolerance, but rather to a sustained inhibition of T cell activation that results in reduced functionality of APCs, with significant implications for its clinical application.
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Abatacept/farmacología , Células Presentadoras de Antígenos/efectos de los fármacos , Antirreumáticos/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Traslado Adoptivo , Animales , Antígenos/inmunología , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Modelos Inmunológicos , ARN/aislamiento & purificación , Linfocitos T/inmunología , Análisis de Matrices Tisulares , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Most high-throughput methods which are used in molecular biology generate gene lists. Interpreting large gene lists can reveal mechanistic insights and generate useful testable hypotheses. The process can be cumbersome and challenging. Multiple commercial and open solution currently exist that can aid researchers in the functional annotation of gene lists. The process of gene set annotation includes dataset preparation, which is method specific, gene list annotation and analysis and interpretation of the significant associations that were found. In this chapter, we demonstrate how WebGestalt can be applied to gene lists generated from transcriptional profiling data.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Anotación de Secuencia Molecular/métodos , Programas Informáticos , Sitios de Unión , Biopsia , Ontología de Genes , Redes Reguladoras de Genes , Antígenos HLA-D/fisiología , Humanos , Ipilimumab , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Factores de Transcripción/fisiologíaRESUMEN
BACKGROUND: Treatment with ipilimumab, a fully human anti-CTLA-4 antibody approved for the treatment of advanced melanoma, is associated with some immune-related adverse events (irAEs) such as colitis (gastrointestinal irAE, or GI irAE) and skin rash, which are managed by treatment guidelines. Nevertheless, predictive biomarkers that can help identify patients more likely to develop these irAEs could enhance the management of these toxicities. METHODS: To identify candidate predictive biomarkers associated with GI irAEs, gene expression profiling was performed on whole blood samples from 162 advanced melanoma patients at baseline, 3 and 11 weeks after the start of ipilimumab treatment in two phase II clinical trials (CA184004 and CA184007). Overall, 49 patients developed Grade 2 or higher (grade 2+) GI irAEs during the course of treatment. A repeated measures analysis of variance (ANOVA) was used to evaluate the differences in mean expression levels between the GI irAE and No-GI irAE groups of patients at the three time points. RESULTS: In baseline samples, 27 probe sets showed differential mean expression (≥ 1.5 fold, P ≤ 0.05) between the GI irAE and No-GI irAE groups. Most of these probe sets belonged to three functional categories: immune system, cell cycle, and intracellular trafficking. Changes in gene expression over time were also characterized. In the GI irAE group, 58 and 247 probe sets had a ≥ 1.5 fold change in expression from baseline to 3 and 11 weeks after first ipilimumab dose, respectively. In particular, on-treatment expression increases of CD177 and CEACAM1, two neutrophil-activation markers, were closely associated with GI irAEs, suggesting a possible role of neutrophils in ipilimumab-associated GI irAEs. In addition, the expression of several immunoglobulin genes increased over time, with greater increases in patients with grade 2+ GI irAEs. CONCLUSIONS: Gene expression profiling of peripheral blood, sampled before or early in the course of treatment with ipilimumab, resulted in the identification of a set of potential biomarkers that were associated with occurrence of GI irAEs. However, because of the low sensitivity of these biomarkers, they cannot be used alone to predict which patients will develop GI irAEs. Further investigation of these biomarkers in a larger patient cohort is warranted.
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Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Biomarcadores de Tumor/sangre , Tracto Gastrointestinal/patología , Perfilación de la Expresión Génica , Sistema Inmunológico/metabolismo , Melanoma/genética , Proteínas Ligadas a GPI/metabolismo , Tracto Gastrointestinal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema Inmunológico/efectos de los fármacos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Ipilimumab , Isoantígenos/metabolismo , Recuento de Leucocitos , Melanoma/sangre , Melanoma/tratamiento farmacológico , Neutrófilos/metabolismo , Curva ROC , Receptores de Superficie Celular/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
PURPOSE: Ipilimumab, a fully human monoclonal antibody specific to CTLA-4, has been shown to improve overall survival in metastatic melanoma patients. As a consequence of CTLA-4 blockade, ipilimumab treatment is associated with proliferation and activation of peripheral T cells. To better understand various tumor-associated components that may influence the clinical outcome of ipilimumab treatment, gene expression profiles of tumors from patients treated with ipilimumab were characterized. EXPERIMENTAL DESIGN: Gene expression profiling was performed on tumor biopsies collected from 45 melanoma patients before and 3 weeks after the start of treatment in a phase II clinical trial. RESULTS: Analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab. Furthermore, ipilimumab appeared to induce two major changes in tumors from patients who exhibited clinical activity: genes involved in immune response showed increased expression, whereas expression of genes for melanoma-specific antigens and genes involved in cell proliferation decreased. These changes were associated with the total lymphocyte infiltrate in tumors, and there was a suggestion of association with prolonged overall survival in these patients. Many IFN-γ-inducible genes and Th1-associated markers showed increased expression after ipilimumab treatment, suggesting an accumulation of this particular type of T cell at the tumor sites, which might play an important role in mediating the antitumor activity of ipilimumab. CONCLUSIONS: These results support the proposed mechanism of action of ipilimumab, suggesting that cell-mediated immune responses play an important role in the antitumor activity of ipilimumab.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Biopsia , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Ipilimumab , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Microambiente Tumoral/inmunologíaRESUMEN
Therapeutic development of a targeted agent involves a series of decisions over additional activities that may be ignored, eliminated or pursued. This paper details the concurrent application of two methods that provide a spectrum of information about the biological activity of a compound: biochemical profiling on a large panel of kinase assays and transcriptional profiling of mRNA responses. Our mRNA profiling studies used a full dose range, identifying subsets of transcriptional responses with differing EC(50)s which may reflect distinct targets. Profiling data allowed prioritization for validation in xenograft models, generated testable hypotheses for active compounds, and informed decisions on the general utility of the series.
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Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Quinasa 9 Dependiente de la Ciclina/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor IGF Tipo 1/genética , TriajeRESUMEN
Gene set enrichment analysis for analyzing large profiling and screening experiments can reveal unifying biological schemes based on previously accumulated knowledge represented as "gene sets". Most of the existing implementations use a fixed fold-change or P value cutoff to generate regulated gene lists. However, the threshold selection in most cases is arbitrary, and has a significant effect on the test outcome and interpretation of the experiment. We developed a new gene set enrichment analysis method, ie, FDR-FET, which dynamically optimizes the threshold choice and improves the sensitivity and selectivity of gene set enrichment analysis. The procedure translates experimental results into a series of regulated gene lists at multiple false discovery rate (FDR) cutoffs, and computes the P value of the overrepresentation of a gene set using a Fisher's exact test (FET) in each of these gene lists. The lowest P value is retained to represent the significance of the gene set. We also implemented improved methods to define a more relevant global reference set for the FET. We demonstrate the validity of the method using a published microarray study of three protease inhibitors of the human immunodeficiency virus and compare the results with those from other popular gene set enrichment analysis algorithms. Our results show that combining FDR with multiple cutoffs allows us to control the error while retaining genes that increase information content. We conclude that FDR-FET can selectively identify significant affected biological processes. Our method can be used for any user-generated gene list in the area of transcriptome, proteome, and other biological and scientific applications.
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SUMMARY: Dose-response information is critical to understanding drug effects, yet analytical methods for dose-response assays cannot cope with the dimensionality of large-scale screening data such as the microarray profiling data. To overcome this limitation, we developed and implemented the Sigmoidal Dose Response Search (SDRS) algorithm, a grid search-based method designed to handle large-scale dose-response data. This method not only calculates the pharmacological parameters for every assay, but also provides built-in statistic that enables downstream systematic analyses, such as characterizing dose response at the transcriptome level. AVAILABILITY: Bio::SDRS is freely available from CPAN (www.cpan.org). CONTACTS: ruiruji@gmail.com; bruc@acm.org SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online.
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Algoritmos , Perfilación de la Expresión Génica/métodos , Transcriptoma/efectos de los fármacos , Relación Dosis-Respuesta a DrogaRESUMEN
The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901) across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.
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Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Expresión Génica/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Inhibidores de Proteínas Quinasas/farmacología , Algoritmos , Benzamidas/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular , Análisis por Conglomerados , Dasatinib , Difenilamina/análogos & derivados , Difenilamina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Mesilato de Imatinib , Piperazinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacologíaRESUMEN
Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary.
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Cardiomiopatías/genética , Segregación Cromosómica/fisiología , Citocinas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , ARN no Traducido/genética , Animales , Animales Modificados Genéticamente , Estudios de Casos y Controles , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Análisis por Conglomerados , Citocinas/fisiología , Embrión no Mamífero , Marcadores Genéticos/fisiología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Inflamación/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , ARN Largo no Codificante , ARN no Traducido/fisiología , Función Ventricular Izquierda/genética , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Neuroinflammatory and neuroimmune mechanisms, as exemplified by infiltrating immune cells and activation of resident endothelial/glial cells, respectively, are known to be involved in the establishment and maintenance of chronic pain. An immune system pathway that may be involved in the activation of both immune and glial cells is complement. The complement pathway is made up of a large number of distinct plasma proteins which react with one another to opsonize pathogens and induce a series of inflammatory responses to help fight infection. Cleaved products and complexes produced by complement activation are responsible for a range of effects including mediation of immune infiltration, activation of phagocytes, opsonization/lysis of pathogens and injured cells, and production of vasoactive amines such as histamine and serotonin. Gene-expression microarray-analysis performed on the rat spinal nerve ligation (SNL) model of neuropathic pain revealed that multiple complement components including the C1 inhibitor, C1q alpha, beta, and gamma, C1r, C1s, C2, C3, C4, C7, and factors B, D, H, and P, were up-regulated while DAF was down-regulated. Regulation of C3 and DAF was confirmed by real-time RT-PCR and in situ hybridization. To test the hypothesis that complement plays a role in neuropathic pain, SNL rats were treated with cobra venom factor (CVF) to deplete plasma of complement component C3. Pain behavior was significantly attenuated in SNL rats treated with CVF as was complement activity at the ipsilateral dorsal root ganglia. Our results suggest the complement pathway might be a novel target for the development of neuropathic pain therapeutics.
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Activación de Complemento/fisiología , Modelos Animales de Enfermedad , Neuralgia/inmunología , Neuralgia/fisiopatología , Nervios Espinales/fisiología , Animales , Activación de Complemento/genética , Ligadura , Neuralgia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sistema Nervioso Periférico/inmunología , Sistema Nervioso Periférico/patología , Sistema Nervioso Periférico/fisiología , Ratas , Nervios Espinales/inmunología , Nervios Espinales/patologíaRESUMEN
MOTIVATION: Gene expression profiling is an important tool for gaining insight into biology. Novel strategies are required to analyze the growing archives of microarray data and extract useful information from them. One area of interest is in the construction of gene association networks from collections of profiling data. Various approaches have been proposed to construct gene networks using profiling data, and these networks have been used in functional inference as well as in data visualization. Here, we investigated a non-parametric approach to translate profiling data into a gene network. We explored the characteristics and utility of the resulting network and investigated the use of network information in analysis of variance models and hypothesis testing. RESULTS: Our work is composed of two parts: gene network construction and partitioning and hypothesis testing using sub-networks as groups. In the first part, multiple independently collected microarray datasets from the Gene Expression Omnibus data repository were analyzed to identify probe pairs that are positively co-regulated across the samples. A co-expression network was constructed based on a reciprocal ranking criteria and a false discovery rate analysis. We named this network Reference Gene Association (RGA) network. Then, the network was partitioned into densely connected sub-networks of probes using a multilevel graph partitioning algorithm. In the second part, we proposed a new, MANOVA-based approach that can take individual probe expression values as input and perform hypothesis testing at the sub-network level. We applied this MANOVA methodology to two published studies and our analysis indicated that the methodology is both effective and sensitive for identifying transcriptional sub-networks or pathways that are perturbed across treatments.