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1.
Hepatology ; 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34618932

RESUMO

BACKGROUND: Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage, highlighting the need for more accurate surveillance tests. Although biomarkers for HCC early detection have promising data in phase II case-control studies, evaluation in cohort studies is critical prior to adoption in practice. METHODS: We leveraged a prospective cohort of patients with Child Pugh A or B cirrhosis who were followed until incident HCC, liver transplantation, death, or lost to follow-up. We used a prospective specimen-collection, retrospective-blinded-evaluation (PRoBE) design for biomarker evaluation of GALAD, longitudinal GALAD and the HES algorithm -compared to alpha fetoprotein (AFP) - using patient-level sensitivity and screening-level specificity. RESULTS: Of 397 patients with cirrhosis, 42 patients developed HCC (57.1% early-stage) over a median of 2.0 years. Longitudinal GALAD had the highest c-statistic for HCC detection (0.85, 95%CI 0.77 - 0.92), compared to single-timepoint GALAD (0.79, 95%CI 0.71 - 0.87), AFP (0.77, 95%CI 0.69 - 0.85), and HES (0.76, 95%CI 0.67 - 0.83). When specificity was fixed at 90%, the sensitivity for HCC of single-timepoint and longitudinal GALAD was 54.8% and 66.7%, respectively, compared to 40.5% for AFP. Sensitivity for HCC detection was higher when restricted to patients with biomarker assessment within 6 months prior to HCC diagnosis, with the highest sensitivities observed for single-timepoint (72.0%) and longitudinal GALAD (64.0%), respectively. Sensitivity of single-timepoint and longitudinal GALAD for early-stage HCC was 53.8% and 69.2%, respectively. CONCLUSION: GALAD demonstrated high sensitivity for HCC detection in a cohort of patients with cirrhosis. Validation of these results are warranted in large phase III datasets.

2.
Mol Cancer Res ; 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380744

RESUMO

Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths globally, and the incidence rate in the United States is increasing. Studies have identified inter- and intratumor heterogeneity as histologic and/or molecular subtypes/variants associated with response to certain molecular targeted therapies. Spatial HCC tissue profiling of N-linked glycosylation by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) may serve as a new method to evaluate the tumor heterogeneity. Previous work has identified significant changes in the N-linked glycosylation of HCC tumors but has not accounted for the heterogeneous genetic and molecular nature of HCC. To determine the correlation between HCC-specific N-glycosylation changes and genetic/molecular tumor features, we profiled HCC tissue samples with MALDI-IMS and correlated the spatial N-glycosylation with a widely used HCC molecular classification (Hoshida subtypes). MALDI-IMS data displayed trends that could approximately distinguish between subtypes, with subtype 1 demonstrating significantly dysregulated N-glycosylation versus adjacent nontumor tissue. Although there were no individual N-glycan structures that could identify specific subtypes, trends emerged regarding the correlation of branched glycan expression to HCC as a whole and fucosylated glycan expression to subtype 1 tumors specifically. IMPLICATIONS: Correlating N-glycosylation to specific subtypes offers the specific detection of subtypes of HCC, which could both enhance early HCC sensitivity and guide targeted clinical therapies.

3.
Mass Spectrom Rev ; 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34392557

RESUMO

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.

5.
J Clin Invest ; 131(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34255748

RESUMO

PFKP (phosphofructokinase, platelet), the major isoform of PFK1 expressed in T cell acute lymphoblastic leukemia (T-ALL), is predominantly expressed in the cytoplasm to carry out its glycolytic function. Our study showed that PFKP is a nucleocytoplasmic shuttling protein with functional nuclear export and nuclear localization sequences (NLSs). Cyclin D3/CDK6 facilitated PFKP nuclear translocation by dimerization and by exposing the NLS of PFKP to induce the interaction between PFKP and importin 9. Nuclear PFKP stimulated the expression of C-X-C chemokine receptor type 4 (CXCR4), a chemokine receptor regulating leukemia homing/infiltration, to promote T-ALL cell invasion, which depended on the activity of c-Myc. In vivo experiments showed that nuclear PFKP promoted leukemia homing/infiltration into the bone marrow, spleen, and liver, which could be blocked with CXCR4 antagonists. Immunohistochemical staining of tissues from a clinically well-annotated cohort of T cell lymphoma/leukemia patients showed nuclear PFKP localization in invasive cancers, but not in nonmalignant T lymph node or reactive hyperplasia. The presence of nuclear PFKP in these specimens correlated with poor survival in patients with T cell malignancy, suggesting the potential utility of nuclear PFKP as a diagnostic marker.

6.
Methods Mol Biol ; 2350: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331294

RESUMO

We describe a multiplexed imaging mass spectrometry approach especially suitable for fibrosis research. Fibrosis is a process characterized by excessive extracellular matrix (ECM) secretion. Buildup of ECM impairs tissue and organ function to promote further progression of disease. It is an ongoing analytical challenge to access ECM for diagnosis and therapeutic treatment of fibrosis. Recently, we reported the use of the enzyme collagenase type III to target the ECM proteome in thin histological tissue sections of fibrotic diseases including hepatocellular carcinoma, breast cancer, prostate cancer, lung cancer and aortic valve stenosis. Detection of collagenase type III peptides by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows localization of ECM peptide sequences to specific regions of fibrosis. We have further identified that the ECM proteome accessed by collagenase type III has on average 3.7 sites per protein that may be differentially N-glycosylated. N-glycosylation is a major posttranslational modification of the ECM proteome, influencing protein folding, secretion, and organization. Understanding both N-glycosylation signaling and regulation of ECM expression significantly informs on ECM signaling in fibrosis.


Assuntos
Biomarcadores , Matriz Extracelular/metabolismo , Histocitoquímica/métodos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Glicosilação , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Pesquisa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fluxo de Trabalho
7.
Sci Rep ; 11(1): 9751, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963260

RESUMO

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.

9.
Clin Lab Med ; 41(2): 247-266, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34020762

RESUMO

N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.

10.
Artigo em Inglês | MEDLINE | ID: mdl-33864933

RESUMO

Hepatocellular carcinoma (HCC) surveillance is associated with early tumor detection and improved survival in patients with cirrhosis.1 Surveillance is performed using semiannual abdominal ultrasound with or without α-fetoprotein (AFP); however, this strategy misses more than one-third of HCC at an early stage.2 These data highlight a need for novel surveillance strategies with higher accuracy for early HCC detection. GALAD and Doylestown Plus are novel biomarker panels that combine multiple biomarkers with patient demographic and clinical characteristics; both demonstrated promising accuracy in phase II case-control studies;3,4 however, case-control studies can overestimate biomarker performance, highlighting a need for phase III cohort and nested case-control studies.5 Our study aimed to compare multiple biomarkers (including AFP, GALAD, and Doylestown Plus) in a nested case-control study of patients with cirrhosis.

11.
Methods Mol Biol ; 2271: 303-316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908016

RESUMO

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.


Assuntos
Fixadores/química , Formaldeído/química , Glicoproteínas/análise , Glicosídeo Hidrolases/metabolismo , Inclusão em Parafina , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fixação de Tecidos , Animais , Configuração de Carboidratos , Glicosilação , Humanos , Isomerismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Projetos de Pesquisa , Especificidade por Substrato , Fluxo de Trabalho
12.
Methods Mol Biol ; 2271: 331-342, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908018

RESUMO

N-glycan imaging mass spectrometry (N-glycan IMS) enables the detection and characterization of N-glycans in thin histological tissue sections. N-glycan IMS is used to study N-glycan regulation and localization in tissue-specific regions, such as tumor and normal adjacent to tumor, or by cell type within a tissue. Once a specific tissue-localized N-glycan signature is found to be associated with by a disease state, it has been challenging to study modulation of the same N-glycan signature by conventional molecular biology techniques. Here we describe a protocol that adapts tissue N-glycan IMS analysis workflows to cells grown on glass slides in an array format. Cells are grown under normal conditions in a cell culture chamber, fixed to maintain normal morphology, and sprayed with a thin coating of PNGase F to release N-glycans for imaging mass spectrometry profiling.


Assuntos
Glicômica , Glicoproteínas/análise , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Células Cultivadas , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Projetos de Pesquisa , Fluxo de Trabalho
13.
J Mol Cell Cardiol ; 154: 6-20, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33516683

RESUMO

Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0-17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease.

14.
Catheter Cardiovasc Interv ; 98(3): E436-E443, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33512085

RESUMO

BACKGROUND: Monitored anesthesia care (MAC) has become more widely used during transcatheter aortic valve replacement (TAVR) to avoid the complications of general anesthesia (GA). METHODS: We included consecutive patients who underwent transfemoral-TAVR at our institution between January 2012 and April 2017. We compared outcomes with GA versus MAC. RESULTS: Of 998 patients, MAC was used in 43.9%. MAC was associated with shorter procedural time (96.9 ± 30.9 vs. 135 ± 64.6 mins; p < .001), fluoroscopy time (20.4 ± 8.9 vs. 29 ± 18.7 mins; p < .001), lower contrast volume (45.5 ± 27 vs. 60.4 ± 43 cc; p < .001), and decreased radiation exposure (12,869 ± 8,099 vs. 20,630 ± 16,276 cGy-cm2 ; p < .001). Patients who underwent MAC had a briefer median (IQR) intensive care unit stay [23.3 (21-24) vs. 23.4 (20.8-26) hrs; p < .001], and hospital stay [2 (2, 3) vs. 3 (2-6) days; p < .001], and were more frequently discharged to home (93.4% vs. 82.9%; p < .001). MAC was associated with lower mortality at 30 days (0.5% vs. 2.9%; log-rank p = .012; adjHR 0.22, 95% CI 0.06-0.82; p = .024), but not at 1 year (11.7% vs. 14.6%; log-rank p = .157) or 3 years (36.8% vs. 38.4%; log-rank p = 0.433). There were no differences in major adverse cardiac and cerebrovascular events (MACCE) at either 30 days (4.6% vs. 9.3%; log-rank p = .14) or 1 year (21.1% vs. 21.5%; log-rank p = .653). Similar findings were seen among patients who received newer-generation SAPIEN-3 valves. CONCLUSION: Utilizing MAC and omitting intraprocedural transesophageal echocardiography during TAVR seems to be more efficient without compromising safety. Better TAVR outcomes can be achieved with newer generation valves without needing GA.

15.
Anal Bioanal Chem ; 413(10): 2709-2719, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33206215

RESUMO

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications. Graphical Abstract.

16.
J Thorac Cardiovasc Surg ; 161(5): 1724-1730, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-31924356

RESUMO

OBJECTIVE: Redo sternotomy in patients with arterial cardiac structures adherent to the sternum carries a risk of catastrophic bleeding. In some of those cases, particularly if they have undergone multiple previous operations, deep hypothermic circulatory arrest alone may not provide sufficient time for a controlled dissection. METHODS: We present a series of 6 cases at risk for exsanguination during sternal re-entry successfully reoperated using percutaneous cardioplegic cardiac arrest induced before completed sternal re-entry to avoid or minimize the hypothermic circulatory arrest time. RESULTS: All patients survived their complex operations. CONCLUSIONS: Percutaneous cardioplegic arrest allows safer repeat sternotomy in patients with arterial cardiac structures adherent to the sternum.

18.
Mol Cell Proteomics ; 2020 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-33234532

RESUMO

The early detection of pancreatic ductal adenocarcinoma is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers CA19-9 and sTRA are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and non-cancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry approach was utilized to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of PDAC patients represented by tissue microarrays and whole tissue sections. Orthogonally, these same tissues were characterized by multi-round immunofluorescence which defined expression of CA19-9 and sTRA as well as other lectins towards carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated bi-antennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9 expressing tissues tended to be bi-, tri- and tetra-antennary structures with both core and terminal fucose residues and bisecting N-acetylglucosamines. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored tri- and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-IHC and IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

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