RESUMO
The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.
Assuntos
Acidúria Argininossuccínica , Hepatopatias , Adulto , Humanos , Animais , Camundongos , Acidúria Argininossuccínica/genética , Acidúria Argininossuccínica/terapia , Cisteína , Glutationa , MetabolômicaRESUMO
Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.
Assuntos
Doença de Fabry/genética , Doença de Fabry/terapia , RNA Mensageiro/uso terapêutico , alfa-Galactosidase/genética , Animais , Modelos Animais de Doenças , Endocitose , Terapia de Reposição de Enzimas , Terapia Genética , Humanos , Lipídeos/química , Lisossomos/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/farmacocinética , Distribuição Tecidual , Triexosilceramidas/metabolismoRESUMO
The advent of therapeutic mRNAs significantly increases the possibilities of protein-based biologics beyond those that can be synthesized by recombinant technologies (eg, monoclonal antibodies, extracellular enzymes, and cytokines). In addition to their application in the areas of vaccine development, immune-oncology, and protein replacement therapies, one exciting possibility is to use therapeutic mRNAs to program undesired, diseased cells to synthesize a toxic intracellular protein, causing cells to self-destruct. For this approach to work, however, methods are needed to limit toxic protein expression to the intended cell type. Here, we show that inclusion of microRNA target sites in therapeutic mRNAs encoding apoptotic proteins, Caspase or PUMA, can prevent their expression in healthy hepatocytes while triggering apoptosis in hepatocellular carcinoma cells.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Mensageiro/genética , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Caspases/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , MicroRNAs/uso terapêutico , Cultura Primária de Células , Proteínas Proto-Oncogênicas/genética , Células RAW 264.7 , RNA Mensageiro/uso terapêuticoRESUMO
Individualized targeted therapies for cancer patients require accurate and reproducible assessment of biomarkers to be able to plan treatment accordingly. Recent studies have shown highly variable effects of preanalytical variables on gene expression profiling and protein levels of different tissue types. Several publications have described protein degradation of tissue samples as a direct result of delay of formalin fixation of the tissue. Phosphorylated proteins are more labile and epitope degradation can happen within 30 min of cold ischemic time. To address this issue, we evaluated the change in antigenicity of a series of phosphoproteins in paraffin-embedded samples from breast tumors as a function of time to formalin fixation. A tissue microarray consisting of 93 breast cancer specimens with documented time-to-fixation was used to evaluate changes in antigenicity of 12 phosphoepitopes frequently used in research settings as a function of cold ischemic time. Analysis was performed in a quantitative manner using the AQUA technology for quantitative immunofluorescence. For each marker, least squares univariate linear regression was performed and confidence intervals were computed using bootstrapping. The majority of the epitopes tested revealed changes in expression levels with increasing time to formalin fixation. Some phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in post-translational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) are quite labile and loss of antigenicity can be reported within 1-2 h of cold ischemic time. Therefore specimen collection should be closely monitored and subjected to quality control measures to ensure accurate measurement of these epitopes. However, a few phosphoepitopes (like phospho-JAK2 and phospho-ER) are sufficiently robust for routine usage in companion diagnostic testing.
Assuntos
Neoplasias da Mama/metabolismo , Isquemia Fria , Epitopos/metabolismo , Fosfoproteínas/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Feminino , Imunofluorescência/métodos , Formaldeído , Humanos , Inclusão em Parafina , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Fatores de Tempo , Análise Serial de Tecidos , Fixação de TecidosRESUMO
BACKGROUND: We have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features. RESULTS: Here we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue. CONCLUSIONS: In summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.
RESUMO
While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.
Assuntos
Mama/patologia , Patologia/normas , Manejo de Espécimes/normas , Mama/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Formaldeído , Humanos , Inclusão em Parafina , Estudos Prospectivos , Receptores de Estrogênio/metabolismo , Fatores de Tempo , Fixação de TecidosRESUMO
BACKGROUND: Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. METHODS: A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. RESULTS: We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. CONCLUSIONS: Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Isquemia Fria , Antígeno Ki-67/análise , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Proteínas de Ancoragem à Quinase A/análise , Biópsia com Agulha de Grande Calibre , Neoplasias da Mama/cirurgia , Fatores de Confusão Epidemiológicos , Reações Falso-Negativas , Feminino , Fixadores , Imunofluorescência , Formaldeído , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Mastectomia Segmentar , Análise por Pareamento , Antígenos de Histocompatibilidade Menor , Estudos Prospectivos , Proteínas Proto-Oncogênicas/análise , Projetos de Pesquisa , Fatores de TempoRESUMO
PURPOSE: Measurement of autophagy in cancer and correlation with histopathologic grading or clinical outcomes has been limited. Accordingly, we investigated LC3B as an autophagosome marker by analyzing nearly 1,400 tumors from 20 types of cancer, focusing on correlations with clinical outcomes in melanoma and breast cancer. EXPERIMENTAL DESIGN: Staining protocols were developed for automated quantitative analysis (AQUA) using antibodies versus LC3 isoform B (LC3B) and Ki-67. Clinically annotated breast and melanoma tissue microarrays (TMA) and a multitumor array were used. An AQUA program was developed to quantitate LC3B distribution in punctate and diffuse compartments of the cell. RESULTS: LC3B staining was moderate to high in the large majority of tumors. The percentage of area occupied by punctate LC3B was elevated by 3- to 5-fold at high LC3B intensities. In breast cancer and melanoma TMAs, LC3B and Ki-67 showed strong correlations (P < 0.0001), and in multitumor TMAs, mitotic figures were most often seen in tumors with the highest LC3B expression (P < 0.002). In breast cancer, LC3B expression was elevated in node-positive versus node-negative primaries and associated with increased nuclear grade and shortened survival. In a melanoma TMA with no survival data, LC3B levels were highest in nodal, visceral, and cutaneous metastases. CONCLUSIONS: The results reveal a common expression of LC3B in malignancy and support emerging evidence that autophagy plays a significant role in cancer progression. High LC3B was associated proliferation, invasion and metastasis, high nuclear grade, and worse outcome. Thus, autophagy presents a key target of therapeutic vulnerability in solid tumors.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/secundário , Melanoma/secundário , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Proliferação de Células , Feminino , Humanos , Estimativa de Kaplan-Meier , Melanoma/metabolismo , Invasividade Neoplásica , Transporte Proteico , Neoplasias Cutâneas/patologia , Análise Serial de TecidosRESUMO
BACKGROUND: PI3K and mTOR are key components of signal transduction pathways critical for cell survival. Numerous PI3K inhibitors have entered clinical trials, while mTOR is the target of approved drugs for metastatic renal cell carcinoma (RCC). We characterized expression of p85 and p110α PI3K subunits and mTOR in RCC specimens and assessed pharmacologic co-targeting of these molecules in vitro. METHODS: We employed tissue microarrays containing 330 nephrectomy cases using a novel immunofluorescence-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. In RCC cell lines we assessed synergism between PI3K and mTOR inhibitors and activity of NVP-BEZ235, which co-targets PI3K and mTOR. RESULTS: p85 expression was associated with high stage and grade (P < 0.0001 for both). High p85 and high mTOR expression were strongly associated with decreased survival, and high p85 was independently prognostic on multi-variable analysis. Strong co-expression of both PI3K subunits and mTOR was found in the human specimens. The PI3K inhibitor LY294002 and rapamycin were highly synergistic in all six RCC cell lines studied. Similar synergism was seen with all rapamycin concentrations used. NVP-BEZ235 inhibited RCC cell growth in vitro with IC(50)s in the low ηM range and resultant PARP cleavage. CONCLUSIONS: High PI3K and mTOR expression in RCC defines populations with decreased survival, suggesting that they are good drug targets in RCC. These targets tend to be co-expressed, and co-targeting these molecules is synergistic. NVP-BEZ235 is active in RCC cells in vitro; suggesting that concurrent PI3K and mTOR targeting in RCC warrants further investigation.
Assuntos
Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Terapia de Alvo Molecular , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Automação , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Sinergismo Farmacológico , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Neoplasias Renais/patologia , Morfolinas/farmacologia , Análise Multivariada , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, with that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA® method. Epitopes phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, estrogen receptor (ER), Ki67, cytokeratin and GAPDH were assessed. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared with biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, ER and cytokeratin showed significant loss of antigenicity. This data suggest that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Isquemia Fria , Epitopos/análise , Imuno-Histoquímica/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Biópsia por Agulha , Feminino , Humanos , Pessoa de Meia-Idade , Manejo de EspécimesRESUMO
Immunohistochemistry is the most common method for companion diagnostic testing in breast cancer. The readings for estrogen receptor, progesterone receptor, and Her2 directly affect prescription of critical therapies. However, immunohistochemistry is highly sensitive to innumerable pre-analytic variables that result in loss of signal in these assays. Perhaps the most significant pre-analytic variable is cold ischemic time. The work of Pinhel and colleagues in the previous issue of Breast Cancer Research examines the effects of cold ischemic time and finds a chilling result. The authors show that while the classic markers may be only mildly affected, phospho-specific markers are highly sensitive to this artifact. As a result, it is likely that future companion diagnostic tests that include phospho-specific epitopes will be reliably done only in core needle biopsies that minimize ischemic time.
Assuntos
Neoplasias da Mama/diagnóstico , Isquemia Fria , Técnicas de Preparação Histocitológica , Imuno-Histoquímica/métodos , Anticorpos , Anticorpos Fosfo-Específicos , Biomarcadores Tumorais , Feminino , Humanos , Receptor ErbB-2/imunologia , Receptores de Estrogênio/imunologia , Receptores de Progesterona/imunologiaRESUMO
BACKGROUND: Despite the widespread use of immunohistochemistry (IHC), there are no standardization guidelines that control for antibody probe variability. Here we describe the effect of variable antibody reagents in the assessment of cancer-related biomarkers by IHC. METHODS: Estrogen receptor (ER), epidermal growth factor receptor (EGFR) 1, and human epidermal growth factor receptor 3 (HER3) were evaluated by quantitative immunofluorescence. Correlations between ER clones 1D5, SP1, F10, and ER60c, and EGFR monoclonal 31G7, 2-18C9, H11, and 15F8, and polyclonal 2232 antibodies were assessed in 642 breast cancer patients. HER3 was measured by RTJ1, RTJ2, SGP1, M7297, RB-9211, and C-17 antibodies in 42 lung cancer patients. Survival analysis was done with the use of multiple cutoff points to reveal any prognostic classification. RESULTS: All ER antibodies were tightly correlated (Pearson's r(2) = 0.94-0.96; P < 0.0001) and western blotting confirmed their specificity in MCF-7 and BT474 cells. All EGFR antibodies but 2232 yielded specific results in western blotting; however, only 31G7 and 2-18C9 were strongly associated (Pearson's r(2) = 0.61; P < 0.0001). HER3 staining was nonspecific and nonreproducible. High EGFR-expressing patients had a worse prognosis when EGFR was measured with H11 or 31G7 (log rank P = 0.015 and P = 0.06). There was no statistically significant correlation between survival and EGFR detected by 2-18C9, 15F8, or polyclonal 2232 antibodies. CONCLUSIONS: Antibody validation is a critical analytic factor that regulates IHC readings in biomarker studies. Evaluation of IHC proficiency and quality control are key components toward IHC standardization. IMPACT: This work highlights the importance of IHC standardization and could result in the improvement of clinically relevant IHC protocols.
Assuntos
Anticorpos , Biomarcadores Tumorais/análise , Imuno-Histoquímica/normas , Especificidade de Anticorpos , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Receptores ErbB/análise , Receptores ErbB/biossíntese , Feminino , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Estimativa de Kaplan-Meier , Receptor ErbB-3/análise , Receptor ErbB-3/biossíntese , Receptores de Estrogênio/análise , Receptores de Estrogênio/biossíntese , Análise Serial de TecidosRESUMO
Recent development of antiangiogenic therapy for renal cell carcinoma (RCC) has significantly improved the treatment of these often refractory tumors. However, not all patients respond to therapy and assays for predicting outcome are needed. As a first step, we analyzed a retrospective cohort of tumors and assessed the ability of VEGF and VEGF receptors (VEGF-R1, -R2 and -R3) to classify tumors. We analyzed tissue microarrays containing 330 RCCs using a novel method of automated quantitative analysis of VEGF and VEGF-R expression by fluorescent immunohistochemistry. Expression of markers was separately quantified within three tissue components: tumor cells, endothelial cells and adjacent normal epithelium. VEGF and VEGF receptors were tightly coexpressed both within tumors and within adjacent normal cells (all P-values <0.001). Tumor cell expression of VEGF-R1 and -R2 was strongly and inversely correlated with vessel area (P<0.0001). Unsupervised hierarchical clustering classified tumors by coordinated expression of VEGF and VEGF-Rs. The distribution of clear cell and papillary tumors was not significantly different between clusters. Clusters with high expression of VEGF and VEGF-Rs in the tumor cells exhibited poor survival when compared with the other clusters on uni- and multivariable analysis. VEGF and VEGF receptors exhibit a complex pattern of coordinated expression in RCC. Clustering tumors by VEGF and VEGF-R in tissue components demonstrates distinct tumor phenotypes with different outcomes, and may provide a means for determining which tumors will respond to what antiangiogenic therapies.
Assuntos
Carcinoma de Células Renais/classificação , Endotélio Vascular/metabolismo , Neoplasias Renais/classificação , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Renais/metabolismo , Pessoa de Meia-Idade , Análise Serial de TecidosRESUMO
Beta1,6-n-acetylglucosaminyltransferase-V (GnT-V) catalyzes the addition of complex oligosaccharide side chains to glycoproteins, regulating the expression and function of several proteins involved in tumor metastasis. We analyzed the expression of five cell-surface glycoprotein substrates of GnT-V, matriptase, beta1-integrin, epidermal growth factor receptor, lamp-1, and N-cadherin, on a tissue microarray cohort of 670 breast carcinomas with 30-year follow-up. Phaseolus vulgaris leukocytic phytohemagglutinin (LPHA), a lectin specific for beta1,6-branched oligosaccharides, was used to assay GnT-V activity. Our results show a high degree of correlation of the LPHA staining with matriptase, lamp-1, and N-cadherin expressions, but not with epidermal growth factor receptor or beta1-integrin expressions. In addition, many of the GnT-V substrate proteins exhibited strong coassociations. Elevated levels of GnT-V substrates were correlated with various markers of tumor progression, including positive node status, large tumor size, estrogen receptor negativity, HER2/neu overexpression, and high nuclear grade. Furthermore, LPHA and matriptase showed significant association with disease-related survival. Unsupervised hierarchical clustering of the GnT-V substrate protein expression and LPHA revealed two distinct clusters: one with higher expression of all markers and poor patient outcome and one with lower expression and good outcome. These clusters showed independent prognostic value for disease-related survival when compared with traditional markers of tumor progression. Our results indicate that GnT-V substrate proteins represent a unique subset of coexpressed tumor markers associated with aggressive disease.