Imaging MS (IMS) has emerged as a powerful tool for biomarker discovery. A key advantage of this ... more Imaging MS (IMS) has emerged as a powerful tool for biomarker discovery. A key advantage of this technique is its ability to probe the proteome directly from a tissue section with preservation of the spatial relationships of the sample and minimal sample preparation. This allows for direct correlation of protein expression with histology. Here, we present the latest developments in imaging MS and their relevance to clinical mass spectral analysis. IMS allows for high throughput analysis of tissue samples and is fully compatible with biostatistical analysis without prior knowledge of protein expression. Several studies are presented of applications in which direct tissue mass spectral analysis has provided insight into clinical questions not readily available by other means. Examples include the determination of lymph node status from investigation of primary breast tumors, prediction of response of breast tumors to chemotherapy, classification and prediction of progression of lung lesions, and exploration of ‘molecular’ margins in invasive disease.
Scandinavian Journal of Rheumatology, May 29, 2012
Objective-To identify and image protein biomarker candidates in the synovial tissue of patients w... more Objective-To identify and image protein biomarker candidates in the synovial tissue of patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). Methods-A novel matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) technique was applied to the analysis of synovial tissue. Patients were classified according to the American College of Rheumatology (ACR) criteria for RA. Frozen sections were stained to obtain morphological data. Serial sections were desiccated, and spotted with matrix for MALDI analysis. Ions generated by laser irradiation of the tissue were separated in time, based on their m/z ratio, and were subsequently detected. IMS was used in a 'profiling' mode to detect discrete spots for rapid evaluation of proteomic patterns in various tissue compartments. Photomicrographs of the stained tissue images were reviewed by a pathologist. Areas of interest (10 discrete areas/compartment) were marked digitally and the histologyannotated images were merged to form a photomicrograph of the section taken before the MALDI measurement. Pixel coordinates of these areas were transferred to a robotic spotter, the matrix was spotted, and the coordinates of the spots were transferred to a mass spectrometer for spectral acquisition. The data generated were then subjected to biocomputation analysis to reveal the biomarker candidates. Results-Several peaks (m/z) consistent in mass with calgranulins, defensins, and thymosins were detected and their distribution in various synovial compartments (synovial lining and sublining layer) was demonstrated.
6000 Background: Our previous study has shown that a matrix-assisted laser desorption ionization ... more 6000 Background: Our previous study has shown that a matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) profile in serum or plasma can predict lung cancer patient survival after treatment with epidermal growth factor receptor inhibitors (EGFRI). We examined the ability of this same MALDI-MS profile in plasma or sera to predict for survival benefit of EGFRIs in patients with head and neck squamous cell carcinoma (HNSCC). Methods: Spectra were obtained in triplicate using MALDI-MS from 314 samples obtained from five HNSCC cohorts treated with 1) gefitinib (G, n = 100); 2) erlotinib and bevacizumab (E/B, n = 81); 3) cetuximab (C, n = 21); 4) surgery (S, n = 78); and 5) palliative chemotherapy (PC, n = 34). Each sample was classified into “good” or “poor” outcome groups and overall survival was examined using this MALDI-MS classifier (VeriStrat, Biodesix, Steamboat Springs, CO). Results: Successful classification could be achieved in 311/314 (98%) of the samples. In all EGFRI-treated cohorts, the classifier predicted survival benefit while the cohorts without EGFRI-treatment showed no survival difference by log-rank testing (G: p = 0.007, HR 0.41, 95%-CI 0.22–0.79; E/B: p = 0.02, HR 0.20, 95%-CI 0.05–0.78; C: p=0.06, HR 0.26, 95%-CI 0.06–1.06; and PC: p = 0.76, HR 0.88, 95%-CI 0.4–1.97), independent of performance status, age, gender and smoking history. Conclusions: This study suggests that the same predictive algorithm for MALDI-MS generated from patients with lung cancer treated with EGFRIs is also predictive of survival outcome in HNSCC patients treated with both TKIs and cetuximab, and may allow rational selection of patients most likely to benefit from an EGFRI monotherapy. [Table: see text]
Background: Pathological grade is a useful prognostic factor for stratifying breast cancer patien... more Background: Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable (well-differentiated tumors) and less favorable (poorly-differentiated tumors) outcome groups. The current system of tumor grading, however, is highly subjective and a large proportion of tumors are characterized as intermediate-grade, making determination of optimal treatments difficult.Methods: Primary breast tumor specimens from patients diagnosed with well- (n=27) and poorly-differentiated (n=51) invasive ductal carcinoma were obtained from patients enrolled in the Clinical Breast Care Project. Frozen tissues were sectioned and mounted on gold coated MALDI target plates for protein expression profiling. Hematoxylin and eosin (H&E) stained slides were prepared from serial sections for histological characterization. MALDI matrix was deposited as individual spots on the tissue sections in a histology directed manner to assay specific areas and tissue types of interest. Mass spectral data were then acquired from multiple sites across each tissue section.Results: 129 features were observed in well-differentiated and 132 in poorly-differentiated tumors. While the majority of features detected were similar between the two groups, 6 protein features were expressed at significantly lower and 12 at significantly higher levels in the poorly-differentiated tumors, including increased expression of Calgranulin A and Calgizzarin.Conclusions: Protein expression differences detected here suggest that well- and poorly-differentiated invasive breast tumors are molecularly distinct diseases and that these protein changes may contribute to the structural integrity of the tumor cell. In particular, calgranulin A and calgizzarin are members of the S100 protein family, and function in processes such as cell proliferation and differentiation. Further refinement of this differentiation protein signature may not only improve our understanding of the biological processes involved with tumor grade but provide pathologists with new molecular tools to classify breast tumors and reduce the subjectivity associated with current grading criteria. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6126.
Background-Wilms tumor (WT) is thought to arise in children of black African ancestry with greate... more Background-Wilms tumor (WT) is thought to arise in children of black African ancestry with greater frequency than whites. To clarify the biological basis for race disparities in WT, we first verified that black children residing in Tennessee have an increased incidence of WT, and second, established molecular profiles in WT that are specific to race. Materials and Methods-To assess race disparities in WT epidemiology, the Tennessee Cancer Registry (TCR) was queried for all in-state patients less than 20 years of age and registered between 1999-2008. To explore race disparities in WT biology, six black and four white WT specimens acquired in Tennessee were analyzed using imaging mass spectrometry (IMS). Results-TCR data show that black children are overrepresented among WT patients (29%) relative to all other childhood cancers (18.5%; p=0.01). WT ranked the 5 th most common cancer diagnosis among blacks, but 9 th among whites. The diagnosis of WT occurred 79% more frequently among blacks (n=28) than whites (n=69; p=0.01), and proportionally more blacks tended to present with distant disease. Although overall survival from WT was not statistically different between blacks (92.9%) and whites (94.0%), black males showed the lowest survival (85%; p=0.21). IMS analysis identified peptide spectra from both WT blastema and stroma that independently classify specimens according to race with greater than 80% accuracy. Conclusions-In Tennessee, black children appear more susceptible than whites to develop WT. Race-specific molecular profiles can be determined that may help to clarify pathways of Wilms tumorigenesis and the biological basis for race disparities in WT incidence and biology.
Background-Differentiating Spitz nevus (SN) from Spitzoid malignant melanoma (SMM) is one the mos... more Background-Differentiating Spitz nevus (SN) from Spitzoid malignant melanoma (SMM) is one the most difficult problems in Dermatopathology. Specific Aim-To identify differences on proteomic level between SN and SMM. Methods-We performed Imaging Mass Spectrometry (IMS) analysis on formalin-fixed, paraffin embedded tissue samples (FFPE) to identify differences on proteomic level between SN and SMM. The diagnosis of SN and SMM was based on histopathologic criteria, clinical features, and follow up data, which confirmed that none of the lesions diagnosed as SN recurred or metastasized. The melanocytic component (tumor) and tumor microenvironment (dermis) from 114 cases of SN and SMM from the Yale Spitzoid Neoplasm Repository were analyzed. After obtaining mass spectra from each sample, classification models were built using a training set of biopsies from 26 SN and 25 SMM separately for tumor and for dermis. The classification algorithms developed on the training data set were validated on another set of 30 samples from SN and 33 from SMM. Results-We found proteomic differences between the melanocytic components of SN and SMM and identified five peptides that were differentially expressed in the two groups. From these data, 29 out of 30 SN and 26 out of 29 SMM were recognized correctly based on tumor analysis in the validation set. This method correctly classified SN with 97% sensitivity and 90% specificity in the validation cohort. Conclusions-IMS analysis can reliably differentiate SN from SMM in FFPE tissue based on proteomic differences.
Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, p... more Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions.
946 SHOTGUN PROTEOMICS IDENTIFIES SPECIFIC PROTEIN PROFILES IN APOPTOTIC BODIES FROM BILIARY EPIT... more 946 SHOTGUN PROTEOMICS IDENTIFIES SPECIFIC PROTEIN PROFILES IN APOPTOTIC BODIES FROM BILIARY EPITHELIAL CELLS A. Lleo, W. Zhang, W.H. McDonald, D. Friedman, E.H. Seeley, P.S. Leung, P. Invernizzi, M.E. Gershwin. Center for Autoimmune Liver Diseases, IRCCS Istituto Clinico Humanitas, Rozzano, Italy; Division of Rheumatology, Allergy, and Clinical Immunology, University of California Davis, Davis, CA, Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA E-mail: [email protected]
AD is a pruritic allergic inflammatory skin disease with increasing prevalence among pediatric an... more AD is a pruritic allergic inflammatory skin disease with increasing prevalence among pediatric and adolescent age groups. Current topical treatments are associated with a range of detrimental side effects. Isoprenylcysteine (IPC) small molecules represent a novel class of topically applied non-steroidal anti-inflammatory drugs, whose action is locally restricted. We demonstrate here the use of a novel IPC analog, SIG-1451 for AD in multiple cell-based assays targeting key pro-inflammatory cytokines that drive AD allergic pathogenesis. In human PBMCs, SIG-1451 inhibits IL-4 cytokine release elicited by CD3/CD28 (IC 50 ¼ 20 mM) and abrogates a Ni 2+-TLR4 response in endothelial cells by reducing IL-6 (IC 50 ¼ 0.02 mM). In NHEKs, SIG-1451 inhibits S. aureus-induced release of TSLP (IC 50 ¼ 3 mM). SIG-1451 activity in vitro is equal to or more potent than topical AD therapies, FK-506 and AN2728, with the exception of the induction of IL-4 by AN2728. Allergic responses are characterized by early and late phases, possibly representing different inflammatory pathways. Thus, we suggest SIG-1451's stronger inhibition of IL-6 production would predict a greater effect than AN2728 on the early inflammatory phase, while it would be less effective in targeting the late phase characterized by IL-4 production. Utilizing in vivo models, SIG-1451 exhibits anti-inflammatory activity in the TPA acute inflammation ear model. Moreover, in the delayed type hypersensitivity (DTH) oxazolone mouse model, which involves both early and late phases, SIG-1451 has higher potency than AN2728, reducing edema and has similar effect on blocking IL-4 production, possibly due to SIG-1451's greater effect on early phase pathways. Based on these findings, SIG-1451 represents a drug development candidate and we have commenced preclinical safety evaluations towards IND submission.
Mass spectrometry imaging studies the proteome and identifies molecular signatures comprising a u... more Mass spectrometry imaging studies the proteome and identifies molecular signatures comprising a unique combination of 5 to 20 proteins that enable a specific diagnosis of cancer or disease. There are proteomic differences between the melanocytic cells of benign nevi and malignant melanomas, which can be identified by mass spectrometry imaging. This technology may assist in the classification of diagnostically challenging melanocytic lesions. Mass spectrometry imaging diagnosis of atypical Spitzoid neoplasms seems to predict the clinical outcome better than histopathology and is strongly associated with aggressive clinical behavior. Mass spectrometry analysis using a proteomic signature may improve the diagnosis and prediction of outcome/risk stratification for patients with atypical Spitzoid neoplasms and other types of challenging melanocytic lesions.
Global internal standard technology (GIST) is being developed for the quantification of all prima... more Global internal standard technology (GIST) is being developed for the quantification of all primary structure and post-translational variants of proteins in a proteome. This paper is directed at an analysis of phosphorylation, primarily of serine and threonine. Quantification was achieved by acylation of primary amino groups in peptide cleavage fragments of proteins with isotopically coded derivatizing agents. Peptides from controls were globally coded with an isotopically "light" form of the reagent while those from experimental samples were coded with a "heavy" form of the reagent. The two types coding reagents used in this work were N-hydroxyl succinimide derivatives of acetate and 4-trimethylammoniumbutyrate. Heavy isotope forms were produced by deuteration of methyl groups. Subsequent to coding and mixing, the two samples were passed through a Ga(III) immobilized metal affinity chromatography (IMAC) column and the selected peptide fraction was further resolved by reversed-phase chromatography (RPC) and analyzed by mass spectrometry (MS). Relative differences in phosphopeptide concentration between samples were derived from isotope ratio measurements of the peptide isoforms observed in mass spectra. The method was validated with model peptides.
Purpose-Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious anti... more Purpose-Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrixassisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). Methods-BAL cells from 27 sarcoidosis, 14 PPD-controls, and 9 subjects with nontuberculosis mycobacterial (NTM) infections were analyzed for production of IFN-γ after stimulation with
Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions ... more Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions in an entire organ or animal through registration and stacking of serial tissue sections. Here we review the current state of 3D imaging mass spectrometry as well as provide insights and perspectives on the process of generating 3D mass spectral data along with a discussion of the process necessary to generate a 3D image volume.
Background: We hypothesized that a serum proteomic profile predictive of survival benefit in non–... more Background: We hypothesized that a serum proteomic profile predictive of survival benefit in non–small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) reflects tumor EGFR dependency regardless of site of origin or class of therapeutic agent.Methods: Pretreatment serum or plasma from 230 patients treated with cetuximab, EGFR-TKIs, or chemotherapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC) were analyzed by mass spectrometry. Each sample was classified into “good” or “poor” groups using VeriStrat, and survival analyses of each cohort were done based on this classification. For the CRC cohort, this classification was correlated with the tumor EGFR ligand levels and KRAS mutation status.Results: In the EGFR inhibitor–treated cohorts, the classification predicted survival (HNSCC: gefitinib, P = 0.007 and erlotinib/bevacizumab, P = 0.02; CRC: cetuximab, P = 0.0065) whereas the chemotherapy cohort showed no survival difference. For CRC patients, tumor EGFR ligand RNA levels were significantly associated with the proteomic classification, and combined KRAS and proteomic classification provided improved survival classification.Conclusions: Serum proteomic profiling can detect clinically significant tumor dependence on the EGFR pathway in non–small cell lung cancer, HNSCC, and CRC patients treated with either EGFR-TKIs or cetuximab. This classification is correlated with tumor EGFR ligand levels and provides a clinically practical way to identify patients with diverse cancer types most likely to benefit from EGFR inhibitors. Prospective studies are necessary to confirm these findings. Cancer Epidemiol Biomarkers Prev; 19(2); 358–65
Drug resistance emergence is a common problem that limits long term outcome benefits in the era o... more Drug resistance emergence is a common problem that limits long term outcome benefits in the era of precision cancer therapy. Recently, we identified an early precision drug escape mechanism with adaptive tumor cellular reprogramming emerging within days after drug initiation. Here we present a mass spectrometry imaging (MSI) approach to interrogate the biomolecular changes occurring within residual tumor cells under precision treatment with an ALK-specific kinase inhibitor TAE684 in EML4-ALK fusion (ALK+) lung adenocarcinoma xenograft. ALK+ H3122 lung adenocarcinoma murine xenograft model was established for in vivo treatment with TAE684, at a daily dose of 25mg/kg by orogastric gavage (n = 6). Diluent control was included as comparison (n = 6). Tumor measurement revealed expected remarkable tumor response with TAE684. Control tumors and drug-treated residual tumor tissues were harvested for MSI studies, at day 7 and day 14 during tumor response. MSI was carried out on formalin fixed, paraffin embedded tissues to compare peptide profiles between control tumors and 7- and 14-day ALK-TKI treated tumors using a histology guided mass spectrometry approach. Briefly, two sections were collected from each sample, one for mass spectrometry and one for histology. Mass spectrometry sections were deparaffinized, antigen retrieved, and subjected to on-tissue tryptic digestion. Tumoral areas of interest (100 μm diameter, ∼20 per sample) were annotated on digital microscopy images of the stained sections. The annotated images were merged with digital images of the unstained sections using Photoshop and this combined image was used to guide data acquisition from the areas of interest. Additionally, frozen control and day 14 TAE684 treated tumors were subjected to full section MSI to determine the ALK inhibitor drug distribution as well as the changing landscape of lipids and metabolites. Statistical analysis of the peptide data resulted in determination of 580 significant peaks using Wilcoxon rank sum test with a Bonferroni correction. A genetic algorithm classification model consisting of 24 peptide peaks was generated using a leave-20%-out cross validation over 10 iterations that resulted in an overall classification accuracy across the 3 groups of over 98%. Direct MS/MS fragmentation revealed that TAE684 was detected within the frozen dosed tumors, but was absent from the control tumors. Several lipids (notably, m/z 732.78, 744.67, and 770.72 increased, m/z 769.65 and 820.71 decreased) were found to undergo alterations in expression as a result of TAE684 treatment. MSI allowed for the direct in situ determination of biomolecules that are changing in expression landscape in ALK+ lung cancer as a result of TAE684 treatment. These results provide a rationale to advance our MSI studies to deepen our insights in mechanisms of adaptive precision drug resistance to improve treatment outcomes. Citation Format: Erin H. Seeley, Pamela S. Cantrell, Callee M. Walsh, Sijin Wen, Satoshi Komo, Xiaoliang Wu, Wei Zhang, Patrick C. Ma. Mass spectrometry imaging determines biomarkers of early adaptive precision drug resistance in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3874.
The present findings implicate deregulation of the oxygen-sensing pathway in PRCC, and provide a ... more The present findings implicate deregulation of the oxygen-sensing pathway in PRCC, and provide a compelling argument that a select group of PRCC patients exhibiting strong tumorspecific E2-EPF or hypoxic profile may represent responders to small molecule inhibitors designed to antagonize the HIF-signaling pathway. HIF-responsive gene. It is upregulated in the majority of pRCC.
Imaging MS (IMS) has emerged as a powerful tool for biomarker discovery. A key advantage of this ... more Imaging MS (IMS) has emerged as a powerful tool for biomarker discovery. A key advantage of this technique is its ability to probe the proteome directly from a tissue section with preservation of the spatial relationships of the sample and minimal sample preparation. This allows for direct correlation of protein expression with histology. Here, we present the latest developments in imaging MS and their relevance to clinical mass spectral analysis. IMS allows for high throughput analysis of tissue samples and is fully compatible with biostatistical analysis without prior knowledge of protein expression. Several studies are presented of applications in which direct tissue mass spectral analysis has provided insight into clinical questions not readily available by other means. Examples include the determination of lymph node status from investigation of primary breast tumors, prediction of response of breast tumors to chemotherapy, classification and prediction of progression of lung lesions, and exploration of ‘molecular’ margins in invasive disease.
Scandinavian Journal of Rheumatology, May 29, 2012
Objective-To identify and image protein biomarker candidates in the synovial tissue of patients w... more Objective-To identify and image protein biomarker candidates in the synovial tissue of patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). Methods-A novel matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) technique was applied to the analysis of synovial tissue. Patients were classified according to the American College of Rheumatology (ACR) criteria for RA. Frozen sections were stained to obtain morphological data. Serial sections were desiccated, and spotted with matrix for MALDI analysis. Ions generated by laser irradiation of the tissue were separated in time, based on their m/z ratio, and were subsequently detected. IMS was used in a 'profiling' mode to detect discrete spots for rapid evaluation of proteomic patterns in various tissue compartments. Photomicrographs of the stained tissue images were reviewed by a pathologist. Areas of interest (10 discrete areas/compartment) were marked digitally and the histologyannotated images were merged to form a photomicrograph of the section taken before the MALDI measurement. Pixel coordinates of these areas were transferred to a robotic spotter, the matrix was spotted, and the coordinates of the spots were transferred to a mass spectrometer for spectral acquisition. The data generated were then subjected to biocomputation analysis to reveal the biomarker candidates. Results-Several peaks (m/z) consistent in mass with calgranulins, defensins, and thymosins were detected and their distribution in various synovial compartments (synovial lining and sublining layer) was demonstrated.
6000 Background: Our previous study has shown that a matrix-assisted laser desorption ionization ... more 6000 Background: Our previous study has shown that a matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) profile in serum or plasma can predict lung cancer patient survival after treatment with epidermal growth factor receptor inhibitors (EGFRI). We examined the ability of this same MALDI-MS profile in plasma or sera to predict for survival benefit of EGFRIs in patients with head and neck squamous cell carcinoma (HNSCC). Methods: Spectra were obtained in triplicate using MALDI-MS from 314 samples obtained from five HNSCC cohorts treated with 1) gefitinib (G, n = 100); 2) erlotinib and bevacizumab (E/B, n = 81); 3) cetuximab (C, n = 21); 4) surgery (S, n = 78); and 5) palliative chemotherapy (PC, n = 34). Each sample was classified into “good” or “poor” outcome groups and overall survival was examined using this MALDI-MS classifier (VeriStrat, Biodesix, Steamboat Springs, CO). Results: Successful classification could be achieved in 311/314 (98%) of the samples. In all EGFRI-treated cohorts, the classifier predicted survival benefit while the cohorts without EGFRI-treatment showed no survival difference by log-rank testing (G: p = 0.007, HR 0.41, 95%-CI 0.22–0.79; E/B: p = 0.02, HR 0.20, 95%-CI 0.05–0.78; C: p=0.06, HR 0.26, 95%-CI 0.06–1.06; and PC: p = 0.76, HR 0.88, 95%-CI 0.4–1.97), independent of performance status, age, gender and smoking history. Conclusions: This study suggests that the same predictive algorithm for MALDI-MS generated from patients with lung cancer treated with EGFRIs is also predictive of survival outcome in HNSCC patients treated with both TKIs and cetuximab, and may allow rational selection of patients most likely to benefit from an EGFRI monotherapy. [Table: see text]
Background: Pathological grade is a useful prognostic factor for stratifying breast cancer patien... more Background: Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable (well-differentiated tumors) and less favorable (poorly-differentiated tumors) outcome groups. The current system of tumor grading, however, is highly subjective and a large proportion of tumors are characterized as intermediate-grade, making determination of optimal treatments difficult.Methods: Primary breast tumor specimens from patients diagnosed with well- (n=27) and poorly-differentiated (n=51) invasive ductal carcinoma were obtained from patients enrolled in the Clinical Breast Care Project. Frozen tissues were sectioned and mounted on gold coated MALDI target plates for protein expression profiling. Hematoxylin and eosin (H&E) stained slides were prepared from serial sections for histological characterization. MALDI matrix was deposited as individual spots on the tissue sections in a histology directed manner to assay specific areas and tissue types of interest. Mass spectral data were then acquired from multiple sites across each tissue section.Results: 129 features were observed in well-differentiated and 132 in poorly-differentiated tumors. While the majority of features detected were similar between the two groups, 6 protein features were expressed at significantly lower and 12 at significantly higher levels in the poorly-differentiated tumors, including increased expression of Calgranulin A and Calgizzarin.Conclusions: Protein expression differences detected here suggest that well- and poorly-differentiated invasive breast tumors are molecularly distinct diseases and that these protein changes may contribute to the structural integrity of the tumor cell. In particular, calgranulin A and calgizzarin are members of the S100 protein family, and function in processes such as cell proliferation and differentiation. Further refinement of this differentiation protein signature may not only improve our understanding of the biological processes involved with tumor grade but provide pathologists with new molecular tools to classify breast tumors and reduce the subjectivity associated with current grading criteria. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6126.
Background-Wilms tumor (WT) is thought to arise in children of black African ancestry with greate... more Background-Wilms tumor (WT) is thought to arise in children of black African ancestry with greater frequency than whites. To clarify the biological basis for race disparities in WT, we first verified that black children residing in Tennessee have an increased incidence of WT, and second, established molecular profiles in WT that are specific to race. Materials and Methods-To assess race disparities in WT epidemiology, the Tennessee Cancer Registry (TCR) was queried for all in-state patients less than 20 years of age and registered between 1999-2008. To explore race disparities in WT biology, six black and four white WT specimens acquired in Tennessee were analyzed using imaging mass spectrometry (IMS). Results-TCR data show that black children are overrepresented among WT patients (29%) relative to all other childhood cancers (18.5%; p=0.01). WT ranked the 5 th most common cancer diagnosis among blacks, but 9 th among whites. The diagnosis of WT occurred 79% more frequently among blacks (n=28) than whites (n=69; p=0.01), and proportionally more blacks tended to present with distant disease. Although overall survival from WT was not statistically different between blacks (92.9%) and whites (94.0%), black males showed the lowest survival (85%; p=0.21). IMS analysis identified peptide spectra from both WT blastema and stroma that independently classify specimens according to race with greater than 80% accuracy. Conclusions-In Tennessee, black children appear more susceptible than whites to develop WT. Race-specific molecular profiles can be determined that may help to clarify pathways of Wilms tumorigenesis and the biological basis for race disparities in WT incidence and biology.
Background-Differentiating Spitz nevus (SN) from Spitzoid malignant melanoma (SMM) is one the mos... more Background-Differentiating Spitz nevus (SN) from Spitzoid malignant melanoma (SMM) is one the most difficult problems in Dermatopathology. Specific Aim-To identify differences on proteomic level between SN and SMM. Methods-We performed Imaging Mass Spectrometry (IMS) analysis on formalin-fixed, paraffin embedded tissue samples (FFPE) to identify differences on proteomic level between SN and SMM. The diagnosis of SN and SMM was based on histopathologic criteria, clinical features, and follow up data, which confirmed that none of the lesions diagnosed as SN recurred or metastasized. The melanocytic component (tumor) and tumor microenvironment (dermis) from 114 cases of SN and SMM from the Yale Spitzoid Neoplasm Repository were analyzed. After obtaining mass spectra from each sample, classification models were built using a training set of biopsies from 26 SN and 25 SMM separately for tumor and for dermis. The classification algorithms developed on the training data set were validated on another set of 30 samples from SN and 33 from SMM. Results-We found proteomic differences between the melanocytic components of SN and SMM and identified five peptides that were differentially expressed in the two groups. From these data, 29 out of 30 SN and 26 out of 29 SMM were recognized correctly based on tumor analysis in the validation set. This method correctly classified SN with 97% sensitivity and 90% specificity in the validation cohort. Conclusions-IMS analysis can reliably differentiate SN from SMM in FFPE tissue based on proteomic differences.
Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, p... more Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions.
946 SHOTGUN PROTEOMICS IDENTIFIES SPECIFIC PROTEIN PROFILES IN APOPTOTIC BODIES FROM BILIARY EPIT... more 946 SHOTGUN PROTEOMICS IDENTIFIES SPECIFIC PROTEIN PROFILES IN APOPTOTIC BODIES FROM BILIARY EPITHELIAL CELLS A. Lleo, W. Zhang, W.H. McDonald, D. Friedman, E.H. Seeley, P.S. Leung, P. Invernizzi, M.E. Gershwin. Center for Autoimmune Liver Diseases, IRCCS Istituto Clinico Humanitas, Rozzano, Italy; Division of Rheumatology, Allergy, and Clinical Immunology, University of California Davis, Davis, CA, Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA E-mail: [email protected]
AD is a pruritic allergic inflammatory skin disease with increasing prevalence among pediatric an... more AD is a pruritic allergic inflammatory skin disease with increasing prevalence among pediatric and adolescent age groups. Current topical treatments are associated with a range of detrimental side effects. Isoprenylcysteine (IPC) small molecules represent a novel class of topically applied non-steroidal anti-inflammatory drugs, whose action is locally restricted. We demonstrate here the use of a novel IPC analog, SIG-1451 for AD in multiple cell-based assays targeting key pro-inflammatory cytokines that drive AD allergic pathogenesis. In human PBMCs, SIG-1451 inhibits IL-4 cytokine release elicited by CD3/CD28 (IC 50 ¼ 20 mM) and abrogates a Ni 2+-TLR4 response in endothelial cells by reducing IL-6 (IC 50 ¼ 0.02 mM). In NHEKs, SIG-1451 inhibits S. aureus-induced release of TSLP (IC 50 ¼ 3 mM). SIG-1451 activity in vitro is equal to or more potent than topical AD therapies, FK-506 and AN2728, with the exception of the induction of IL-4 by AN2728. Allergic responses are characterized by early and late phases, possibly representing different inflammatory pathways. Thus, we suggest SIG-1451's stronger inhibition of IL-6 production would predict a greater effect than AN2728 on the early inflammatory phase, while it would be less effective in targeting the late phase characterized by IL-4 production. Utilizing in vivo models, SIG-1451 exhibits anti-inflammatory activity in the TPA acute inflammation ear model. Moreover, in the delayed type hypersensitivity (DTH) oxazolone mouse model, which involves both early and late phases, SIG-1451 has higher potency than AN2728, reducing edema and has similar effect on blocking IL-4 production, possibly due to SIG-1451's greater effect on early phase pathways. Based on these findings, SIG-1451 represents a drug development candidate and we have commenced preclinical safety evaluations towards IND submission.
Mass spectrometry imaging studies the proteome and identifies molecular signatures comprising a u... more Mass spectrometry imaging studies the proteome and identifies molecular signatures comprising a unique combination of 5 to 20 proteins that enable a specific diagnosis of cancer or disease. There are proteomic differences between the melanocytic cells of benign nevi and malignant melanomas, which can be identified by mass spectrometry imaging. This technology may assist in the classification of diagnostically challenging melanocytic lesions. Mass spectrometry imaging diagnosis of atypical Spitzoid neoplasms seems to predict the clinical outcome better than histopathology and is strongly associated with aggressive clinical behavior. Mass spectrometry analysis using a proteomic signature may improve the diagnosis and prediction of outcome/risk stratification for patients with atypical Spitzoid neoplasms and other types of challenging melanocytic lesions.
Global internal standard technology (GIST) is being developed for the quantification of all prima... more Global internal standard technology (GIST) is being developed for the quantification of all primary structure and post-translational variants of proteins in a proteome. This paper is directed at an analysis of phosphorylation, primarily of serine and threonine. Quantification was achieved by acylation of primary amino groups in peptide cleavage fragments of proteins with isotopically coded derivatizing agents. Peptides from controls were globally coded with an isotopically "light" form of the reagent while those from experimental samples were coded with a "heavy" form of the reagent. The two types coding reagents used in this work were N-hydroxyl succinimide derivatives of acetate and 4-trimethylammoniumbutyrate. Heavy isotope forms were produced by deuteration of methyl groups. Subsequent to coding and mixing, the two samples were passed through a Ga(III) immobilized metal affinity chromatography (IMAC) column and the selected peptide fraction was further resolved by reversed-phase chromatography (RPC) and analyzed by mass spectrometry (MS). Relative differences in phosphopeptide concentration between samples were derived from isotope ratio measurements of the peptide isoforms observed in mass spectra. The method was validated with model peptides.
Purpose-Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious anti... more Purpose-Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrixassisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). Methods-BAL cells from 27 sarcoidosis, 14 PPD-controls, and 9 subjects with nontuberculosis mycobacterial (NTM) infections were analyzed for production of IFN-γ after stimulation with
Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions ... more Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions in an entire organ or animal through registration and stacking of serial tissue sections. Here we review the current state of 3D imaging mass spectrometry as well as provide insights and perspectives on the process of generating 3D mass spectral data along with a discussion of the process necessary to generate a 3D image volume.
Background: We hypothesized that a serum proteomic profile predictive of survival benefit in non–... more Background: We hypothesized that a serum proteomic profile predictive of survival benefit in non–small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) reflects tumor EGFR dependency regardless of site of origin or class of therapeutic agent.Methods: Pretreatment serum or plasma from 230 patients treated with cetuximab, EGFR-TKIs, or chemotherapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC) were analyzed by mass spectrometry. Each sample was classified into “good” or “poor” groups using VeriStrat, and survival analyses of each cohort were done based on this classification. For the CRC cohort, this classification was correlated with the tumor EGFR ligand levels and KRAS mutation status.Results: In the EGFR inhibitor–treated cohorts, the classification predicted survival (HNSCC: gefitinib, P = 0.007 and erlotinib/bevacizumab, P = 0.02; CRC: cetuximab, P = 0.0065) whereas the chemotherapy cohort showed no survival difference. For CRC patients, tumor EGFR ligand RNA levels were significantly associated with the proteomic classification, and combined KRAS and proteomic classification provided improved survival classification.Conclusions: Serum proteomic profiling can detect clinically significant tumor dependence on the EGFR pathway in non–small cell lung cancer, HNSCC, and CRC patients treated with either EGFR-TKIs or cetuximab. This classification is correlated with tumor EGFR ligand levels and provides a clinically practical way to identify patients with diverse cancer types most likely to benefit from EGFR inhibitors. Prospective studies are necessary to confirm these findings. Cancer Epidemiol Biomarkers Prev; 19(2); 358–65
Drug resistance emergence is a common problem that limits long term outcome benefits in the era o... more Drug resistance emergence is a common problem that limits long term outcome benefits in the era of precision cancer therapy. Recently, we identified an early precision drug escape mechanism with adaptive tumor cellular reprogramming emerging within days after drug initiation. Here we present a mass spectrometry imaging (MSI) approach to interrogate the biomolecular changes occurring within residual tumor cells under precision treatment with an ALK-specific kinase inhibitor TAE684 in EML4-ALK fusion (ALK+) lung adenocarcinoma xenograft. ALK+ H3122 lung adenocarcinoma murine xenograft model was established for in vivo treatment with TAE684, at a daily dose of 25mg/kg by orogastric gavage (n = 6). Diluent control was included as comparison (n = 6). Tumor measurement revealed expected remarkable tumor response with TAE684. Control tumors and drug-treated residual tumor tissues were harvested for MSI studies, at day 7 and day 14 during tumor response. MSI was carried out on formalin fixed, paraffin embedded tissues to compare peptide profiles between control tumors and 7- and 14-day ALK-TKI treated tumors using a histology guided mass spectrometry approach. Briefly, two sections were collected from each sample, one for mass spectrometry and one for histology. Mass spectrometry sections were deparaffinized, antigen retrieved, and subjected to on-tissue tryptic digestion. Tumoral areas of interest (100 μm diameter, ∼20 per sample) were annotated on digital microscopy images of the stained sections. The annotated images were merged with digital images of the unstained sections using Photoshop and this combined image was used to guide data acquisition from the areas of interest. Additionally, frozen control and day 14 TAE684 treated tumors were subjected to full section MSI to determine the ALK inhibitor drug distribution as well as the changing landscape of lipids and metabolites. Statistical analysis of the peptide data resulted in determination of 580 significant peaks using Wilcoxon rank sum test with a Bonferroni correction. A genetic algorithm classification model consisting of 24 peptide peaks was generated using a leave-20%-out cross validation over 10 iterations that resulted in an overall classification accuracy across the 3 groups of over 98%. Direct MS/MS fragmentation revealed that TAE684 was detected within the frozen dosed tumors, but was absent from the control tumors. Several lipids (notably, m/z 732.78, 744.67, and 770.72 increased, m/z 769.65 and 820.71 decreased) were found to undergo alterations in expression as a result of TAE684 treatment. MSI allowed for the direct in situ determination of biomolecules that are changing in expression landscape in ALK+ lung cancer as a result of TAE684 treatment. These results provide a rationale to advance our MSI studies to deepen our insights in mechanisms of adaptive precision drug resistance to improve treatment outcomes. Citation Format: Erin H. Seeley, Pamela S. Cantrell, Callee M. Walsh, Sijin Wen, Satoshi Komo, Xiaoliang Wu, Wei Zhang, Patrick C. Ma. Mass spectrometry imaging determines biomarkers of early adaptive precision drug resistance in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3874.
The present findings implicate deregulation of the oxygen-sensing pathway in PRCC, and provide a ... more The present findings implicate deregulation of the oxygen-sensing pathway in PRCC, and provide a compelling argument that a select group of PRCC patients exhibiting strong tumorspecific E2-EPF or hypoxic profile may represent responders to small molecule inhibitors designed to antagonize the HIF-signaling pathway. HIF-responsive gene. It is upregulated in the majority of pRCC.
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Papers by Erin Seeley