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1.
Methods Mol Biol ; 1866: 163-171, 2019.
Article in English | MEDLINE | ID: mdl-30725415

ABSTRACT

This chapter reviews how total methionine (MET) restriction (MR) of a human brain tumor xenograft, effected by the combination of recombinant L-methionine-α-deamino-γ-lyase (rMETase) and a MET-free diet, greatly potentiates standard chemotherapy for brain tumors in mouse models. The growth of human brain tumor Daoy, SWB77, and D-54 xenografts in nude mice was arrested after the depletion of mouse plasma methionine (MET) with a combination of an MR diet and rMETase and homocysteine to rescue normal cells and tissues. MET was depleted to below 5 µm by this treatment. MR for 10-12 days inhibited tumor growth, but did not prevent tumor regrowth after treatment cessation. A single dose of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), which was ineffective alone, was administered at the end of the MR regimen, and caused a more than 80-day growth delay for Daoy and D-54 and a 20-day growth delay for SWB77. The total MR treatment regimens also increased the efficacy of temozolomide (TMZ) against the SWB77 xenograft when administered at the end of the MET regimen.


Subject(s)
Methionine/deficiency , Neoplasms/diet therapy , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Choline , Homocysteine/metabolism , Humans , Liver/drug effects , Liver/pathology , Methionine/blood , Mice, Nude , Neoplasms/drug therapy , Neoplasms/pathology , Temozolomide/pharmacology , Temozolomide/therapeutic use , Xenograft Model Antitumor Assays
2.
Neoplasia ; 19(10): 848-855, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28923537

ABSTRACT

This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT) response in metastatic melanoma patients. Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT-particularly with BRAF-wild-type disease. In preclinical studies, specific gut microbiota promotes regression of melanoma in mice. We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN), or pembrolizumab (P). IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease. ICT responders for all types of therapies were enriched for Bacteroides caccae. Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis. Among P responders, the microbiome was enriched for Dorea formicogenerans. Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders. Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.


Subject(s)
Gastrointestinal Microbiome , Melanoma/etiology , Melanoma/metabolism , Metabolomics , Metagenome , Metagenomics , Adult , Aged , Aged, 80 and over , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Biomarkers, Tumor , Chromatography, High Pressure Liquid , Cluster Analysis , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Melanoma/drug therapy , Melanoma/pathology , Metabolomics/methods , Metagenomics/methods , Middle Aged , Molecular Targeted Therapy , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Tandem Mass Spectrometry , Treatment Outcome
3.
Transfus Apher Sci ; 56(3): 431-433, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28512017

ABSTRACT

Von Willebrand disease (VWD) is the most common congenital bleeding disorder and is due to quantitative or qualitative defects of von Willebrand factor (VWF). Acquired defects of VWF, termed acquired von Willebrand syndrome (AVWS), are due to a host of different mechanisms. Autoantibody-mediated AVWS may be associated with lymphoproliferative or immunological disorders, such as systemic lupus erythematosus (SLE). A large majority of AVWS cases are type 1 or type 2A-like and patients tend to have a mild to moderate bleeding tendency. We report a case of severe autoimmune AVWS in a woman with SLE who presented with clinical and laboratory features of type 3 VWD (undetectable VWF antigen, ristocetin cofactor activity, and VWF multimers). A mixing study demonstrated an inhibitor to VWF (6BU/mL). Her bleeds were managed with antifibrinolytics, recombinant activated factor VII, and activated prothrombin complex concentrate. She was initially treated with steroids and intravenous immunoglobulin therapy. However, her bleeding symptoms continued until she was treated with rituximab, and her VWF parameters normalized. She relapsed two years later due to non-compliance with her immunosuppressive medications and expired another two years later secondary to complications of sepsis and uremic pericarditis. This case emphasizes the importance of aggressive initial therapy of SLE to reduce secondary complications, frequent patient monitoring, and continued treatment of the underlying autoimmune disorder in patients with AVWS.


Subject(s)
Autoimmune Diseases/diagnosis , von Willebrand Diseases/diagnosis , Adult , Autoimmune Diseases/mortality , Autoimmune Diseases/pathology , Female , Humans , Survival Analysis , Young Adult , von Willebrand Diseases/mortality , von Willebrand Diseases/pathology
4.
Oncologist ; 22(4): 369-374, 2017 04.
Article in English | MEDLINE | ID: mdl-28314841

ABSTRACT

Multiple factors critical to the effectiveness of academic phase I cancer programs were assessed among 16 academic centers in the U.S. Successful cancer centers were defined as having broad phase I and I/II clinical trial portfolios, multiple investigator-initiated studies, and correlative science. The most significant elements were institutional philanthropic support, experienced clinical research managers, robust institutional basic research, institutional administrative efforts to reduce bureaucratic regulatory delays, phase I navigators to inform patients and physicians of new studies, and a large cancer center patient base. New programs may benefit from a separate stand-alone operation, but mature phase I programs work well when many of the activities are transferred to disease-oriented teams. The metrics may be useful as a rubric for new and established academic phase I programs. The Oncologist 2017;22:369-374.


Subject(s)
Academic Medical Centers , Neoplasms/epidemiology , Clinical Trials as Topic , Humans , Neoplasms/genetics , Program Development , United States
5.
Nature ; 539(7627): 112-117, 2016 11 03.
Article in English | MEDLINE | ID: mdl-27595394

ABSTRACT

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/metabolism , Indans/pharmacology , Indans/therapeutic use , Kidney Neoplasms/drug therapy , Kidney Neoplasms/metabolism , Sulfones/pharmacology , Sulfones/therapeutic use , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic , Drug Resistance, Neoplasm/drug effects , Erythropoietin/antagonists & inhibitors , Erythropoietin/blood , Female , Gene Expression Regulation, Neoplastic , Humans , Indans/administration & dosage , Indoles/pharmacology , Indoles/therapeutic use , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , Mutation , Pyrroles/pharmacology , Pyrroles/therapeutic use , Reproducibility of Results , Sulfones/administration & dosage , Sunitinib , Xenograft Model Antitumor Assays
6.
PLoS One ; 11(5): e0155326, 2016.
Article in English | MEDLINE | ID: mdl-27176603

ABSTRACT

Ideally, thrombophilia testing should be tailored to the type of thrombotic event without the influence of anticoagulation therapy or acute phase effects which can give false positive results that may result in long term anticoagulation. However, thrombophilia testing is often performed routinely in unselected patients. We analyzed all consecutive thrombophilia testing orders during the months of October and November 2009 at an academic teaching institution. Information was extracted from electronic medical records for the following: indication, timing, comprehensiveness of tests, anticoagulation therapy at the time of testing, and confirmatory repeat testing, if any. Based on the findings of this analysis, we established local guidelines in May 2013 for appropriate thrombophilia testing, primarily to prevent testing during the acute thrombotic event or while the patient is on anticoagulation. We then evaluated ordering practices 22 months after guideline implementation. One hundred seventy-three patients were included in the study. Only 34% (58/173) had appropriate indications (unprovoked venous or arterial thrombosis or pregnancy losses). 51% (61/119) with an index clinical event were tested within one week of the event. Although 46% (79/173) were found to have abnormal results, only 46% of these had the abnormal tests repeated for confirmation with 54% potentially carrying a wrong diagnosis with long term anticoagulation. Twenty-two months after guideline implementation, there was an 84% reduction in ordered tests. Thus, this study revealed that a significant proportion of thrombophilia testing was inappropriately performed. We implemented local guidelines for thrombophilia testing for clinicians, resulting in a reduction in healthcare costs and improved patient care.


Subject(s)
Academic Medical Centers , Blood Coagulation Tests , Practice Patterns, Physicians'/statistics & numerical data , Thrombophilia/diagnosis , Thrombophilia/epidemiology , Academic Medical Centers/standards , Academic Medical Centers/statistics & numerical data , Adult , Aged , Aged, 80 and over , Blood Coagulation Tests/adverse effects , Blood Coagulation Tests/economics , Blood Coagulation Tests/methods , Blood Coagulation Tests/standards , Comorbidity , Female , Follow-Up Studies , Health Care Costs , Humans , Male , Middle Aged , Practice Patterns, Physicians'/standards , Retrospective Studies , Thrombophilia/etiology , Young Adult
7.
J Urol ; 195(1): 180-7, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26300218

ABSTRACT

PURPOSE: In clear cell renal cell carcinoma BAP1 and PBRM1 are 2 of the most commonly mutated genes (10% to 15% and 40% to 50%, respectively). We sought to determine the prognostic significance of PBRM1 and BAP1 expression in clear cell renal cell carcinoma. MATERIALS AND METHODS: We used immunohistochemistry to assess PBRM1 protein expression in 1,479 primary clear cell renal cell carcinoma tumors that were previously stained for BAP1. A centralized pathologist reviewed all cases and categorized tumors as positive or deficient for PBRM1 and BAP1. Kaplan-Meier and Cox regression models were used to evaluate association of PBRM1 and BAP1 expression with the risk of death from renal cell carcinoma and the risk of metastasis after adjustment for age and the Mayo Clinic SSIGN (stage, size, grade and necrosis) score. RESULTS: PBRM1 and BAP1 expression was PBRM1+ BAP1+ in 40.1% of tumors, PBRM1- BAP1+ in 48.6%, PBRM1+ BAP1- in 8.7% and PBRM1- BAP1- in 1.8%. The incidence of PBRM1 and BAP1 loss in the same tumor was significantly lower than expected (actual 1.8% vs expected 5.3%, p <0.0001). Compared to patients with PBRM1+ BAP1+ tumors those with PBRM1- BAP1+ lesions were more likely to die of renal cell carcinoma (HR 1.39, p = 0.035), followed by those with PBRM1+ BAP1- and PBRM1- BAP1- tumors (HR 3.25 and 5.2, respectively, each p <0.001). PBRM1 and BAP1 expression did not add independent prognostic information to the SSIGN score. CONCLUSIONS: PBRM1 and BAP1 expression identified 4 clinical subgroups of patients with clear cell renal cell carcinoma who had divergent clinical outcomes. The clinical value of these biomarkers will be fully realized when therapies targeting pathways downstream of PBRM1 and BAP1 are developed.


Subject(s)
Carcinoma, Renal Cell/classification , Carcinoma, Renal Cell/genetics , Kidney Neoplasms/classification , Kidney Neoplasms/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Adult , Aged , Aged, 80 and over , DNA-Binding Proteins , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Prognosis , Tumor Cells, Cultured , Young Adult
8.
Sci Rep ; 5: 16047, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26538094

ABSTRACT

Immunomagnetic assay has been developed to detect rare circulating tumor cells (CTCs), which shows clinical significance in cancer diagnosis and prognosis. The generation and fine-tuning of the magnetic field play essential roles in such assay toward effective single-cell-based analyses of target cells. However, the current assay has a limited range of field gradient, potentially leading to aggregation of cells and nanoparticles. Consequently, quenching of the fluorescence signal and mechanical damage to the cells may occur, which lower the system sensitivity and specificity. We develop a micromagnet-integrated microfluidic system for enhanced CTC detection. The ferromagnetic micromagnets, after being magnetized, generate localized magnetic field up to 8-fold stronger than that without the micromagnets, and strengthen the interactions between CTCs and the magnetic field. The system is demonstrated with four cancer cell lines with over 97% capture rate, as well as with clinical samples from breast, prostate, lung, and colorectal cancer patients. The system captures target CTCs from patient blood samples on a standard glass slide that can be examined using the fluorescence in-situ hybridization method for the single-cell profiling. All cells showed clear hybridization signals, indicating the efficacy of the compact system in providing retrievable cells for molecular studies.


Subject(s)
Neoplastic Cells, Circulating/pathology , Cell Count/methods , Cell Line, Tumor , Cell Separation/methods , Humans , MCF-7 Cells , Magnetic Fields , Magnets , Microfluidics/methods , Prognosis , Sensitivity and Specificity , Single-Cell Analysis/methods
9.
Article in English | MEDLINE | ID: mdl-26316815

ABSTRACT

PURPOSE: This study interrogates the molecular status of individual cells in patients with triple-negative breast cancers and explores the molecular identification and characterization of these tumors to consider the exploitation of a potential-targeted therapeutic approach. PATIENTS AND METHODS: Hyperspectral immunologic cell by cell analysis was applied to touch imprint smears obtained from fresh tumors of breast cancer patients. RESULTS: Cell by cell analysis confirms significant intratumoral molecular heterogeneity in cancer markers with differences from polymerase chain reaction marker reporting. The individual cell heterogeneity was recognized in adjacent cells examined with panels of ten molecular markers in each single cell and included some markers that are considered to express "stem-cell" character. In addition, heterogeneity did not relate either to the size or stage of the primary tumor or to the site from within the cancer. CONCLUSION: There is a very significant molecular heterogeneity when "adjacent cells" are examined in triple-negative breast cancer, thereby making a successful targeted approach unlikely. In addition, it is not reasonable to consider that these changes will provide an answer to tumor dormancy.

10.
Mol Cancer Res ; 13(4): 784-94, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25573953

ABSTRACT

UNLABELLED: Protein S-palmitoylation is a widespread and dynamic posttranslational modification that regulates protein-membrane interactions, protein-protein interactions, and protein stability. A large family of palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif, catalyzes S-palmitoylation; the role of these enzymes in cancer is largely unexplored. In this study, an RNAi-based screen targeting all 23 members of the DHHC family was conducted to examine the effects on the growth in non-small cell lung cancer (NSCLC). Interestingly, siRNAs directed against DHHC5 broadly inhibited the growth of multiple NSCLC lines but not normal human bronchial epithelial cell (HBEC) lines. Silencing of DHHC5 by lentivirus-mediated expression of DHHC5 shRNAs dramatically reduced in vitro cell proliferation, colony formation, and cell invasion in a subset of cell lines that were examined in further detail. The phenotypes were restored by transfection of a wild-type DHHC5 plasmid but not by a plasmid expressing a catalytically inactive DHHC5. Tumor xenograft formation was severely inhibited by DHHC5 knockdown and rescued by DHHC5 expression, using both a conventional and tetracycline-inducible shRNA. These data indicate that DHHC5 has oncogenic capacity and contributes to tumor formation in NSCLC, thus representing a potential novel therapeutic target. IMPLICATIONS: Inhibitors of DHHC5 enzyme activity may inhibit non-small cell lung cancer growth.


Subject(s)
Acyltransferases/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA, Small Interfering/metabolism , Acyltransferases/antagonists & inhibitors , Acyltransferases/genetics , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Membrane Proteins/antagonists & inhibitors , Mice , Neoplasm Transplantation
11.
Urol Oncol ; 33(1): 23.e9-23.e14, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25465300

ABSTRACT

BACKGROUND: Recurrent mutations in polybromo-1 (PBRM1, ~40%) and BRCA1-associated protein-1 (BAP1, ~10%) occur in clear cell renal cell carcinoma (ccRCC), but their prevalence in non-ccRCC or renal oncocytoma (RO) is unknown. We evaluated loss of PBRM1 and BAP1 staining in ccRCC, papillary RCC (pRCC), chromophobe RCC (chRCC), and RO tumors using an immunohistochemistry assay in which negative staining was associated with loss-of-function mutations. METHODS: We identified 458 patients treated surgically for ccRCC, pRCC, chRCC, and RO between 2004 and 2012. We performed immunohistochemistry assays to evaluate PBRM1 and BAP1 protein expression to classify tumors as PBRM1 or BAP1 negative. We compared loss of staining of these 2 proteins in ccRCC and non-ccRCC using the Fisher exact test. RESULTS: For the total cohort of 458 patients, we successfully stained both PBRM1 and BAP1 in 408 tumor samples. Consistent with the mutation rate, loss of PBRM1 and BAP1 staining occurred in 43% (80/187) and 10% (18/187) of ccRCC cases, respectively. However, loss of PBRM1 staining occurred in only 3% (2/59), 6% (1/17), and 0% (0/34) of pRCC, chRCC, and RO tumors, respectively (P<0.0001). BAP1 loss was not observed in any of the pRCC (n = 61), chRCC (n = 17), or RO (n = 34) tumors, (P = 0.00021). CONCLUSION: Our data suggest that biallelic inactivation of PBRM1 or BAP1 is less common in non-ccRCC when compared with ccRCC tumors. These findings suggest that loss of PBRM1 or BAP1 are key events in ccRCC, whereas other pathways may support tumorigenesis in non-ccRCC subtypes.


Subject(s)
Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/metabolism , Nuclear Proteins/biosynthesis , Transcription Factors/biosynthesis , Tumor Suppressor Proteins/biosynthesis , Ubiquitin Thiolesterase/biosynthesis , Adult , Aged , Aged, 80 and over , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Carcinoma, Renal Cell/surgery , Cohort Studies , DNA-Binding Proteins , Female , Humans , Immunohistochemistry , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Kidney Neoplasms/surgery , Male , Middle Aged , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/deficiency , Ubiquitin Thiolesterase/genetics
12.
Melanoma Res ; 24(6): 556-67, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25304236

ABSTRACT

Metastatic melanoma is a deadly form of cancer with few therapeutic options and the cause of more than 9480 deaths annually in the USA alone. Novel treatment options for this disease are urgently needed. Here we test the efficacy of a novel melanoma drug, the human recombinant Co-arginase (CoArgIPEG), against an aggressive A375 melanoma mouse model. CoArgIPEG is a modification of the naturally occurring human enzyme with improved stability, catalytic activity, and potentially lower immunogenicity compared with current amino acid-depleting drugs. Marked tumor growth reductions (mean P=0.0057) with apoptosis induction and proliferation inhibition are noted with CoArgIPEG treatment, both in the presence and in the absence of supplemental citrulline. Further, improved therapeutic efficacy has been noted against A375 xenografts relative to the naturally occurring human recombinant arginase enzyme at lower doses of CoArgIPEG. Unfortunately, after 1 month, half of the relapsing tumors showed argininosuccinate synthase induction, which correlated with Ser62-phosphorylated cMyc. Although argininosuccinate synthase induction could not be induced in vitro, a drug targeting pathway previously demonstrated to be associated with Ser62 cMyc phosphorylation - U0126 - in combination with CoArgIPEG demonstrated an in-vitro synergistic response (combination indices 0.13±0.10 and 0.14±0.10 with or without citrulline, respectively). Overall, favorable efficacy and potential synergy with other antimelanoma drugs support CoArgIPEG as a potent, novel cancer therapeutic.


Subject(s)
Antineoplastic Agents/therapeutic use , Arginase/therapeutic use , Melanoma/drug therapy , Recombinant Proteins/therapeutic use , Skin Neoplasms/drug therapy , Animals , Cobalt/chemistry , Cobalt/therapeutic use , Female , Humans , Hydrolases/chemistry , Hydrolases/therapeutic use , Jurkat Cells , Melanoma/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Polyethylene Glycols/chemistry , Polyethylene Glycols/therapeutic use , Skin Neoplasms/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
13.
Cancer Sci ; 105(8): 1040-8, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24837299

ABSTRACT

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are validated molecular targets in cancer therapy. Dual blockade has been explored and one such agent, lapatinib, is in clinical practice but with modest activity. Through chemical screening, we discovered a novel EGFR and HER2 inhibitor, S-222611, that selectively inhibited both kinases with IC50 s below 10 nmol/L. S-222611 also inhibited intracellular kinase activity and the growth of EGFR-expressing and HER2-expressing cancer cells. In addition, S-222611 showed potent antitumor activity over lapatinib in a variety of xenograft models. In evaluations with two patient-oriented models, the intrafemoral implantation model and the intracranial implantation model, S-222611 exhibited excellent activity and could be effective against bone and brain metastasis. Compared to neratinib and afatinib, irreversible EGFR/HER2 inhibitors, S-222611 showed equivalent or slightly weaker antitumor activity but a safer profile. These results indicated that S-222611 is a potent EGFR and HER2 inhibitor with substantially better antitumor activity than lapatinib at clinically relevant doses. Considering the safer profile than for irreversible inhibitors, S-222611 could be an important option in future cancer therapy.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Animals , Cell Line, Tumor , Disease Models, Animal , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Nude , Mice, SCID , Xenograft Model Antitumor Assays
14.
Cancer ; 120(7): 1059-67, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24382589

ABSTRACT

BACKGROUND: The majority of patients diagnosed with clear cell renal cell carcinoma (ccRCC) have low-risk disease with a < 10% chance of ccRCC-specific death. DNA sequencing revealed that mutations in BAP1 (BRCA1 associated protein-1) occur in 5% to 15% of ccRCC cases and are associated with poor outcomes. The vast majority of BAP1 mutations abolish protein expression. In this study, we used a highly sensitive and specific immunohistochemistry (IHC) assay to test whether BAP1 expression is an independent marker of ccRCC-specific survival, particularly in patients with low-risk disease. METHODS: BAP1 expression was assessed, using IHC, in 1479 patients who underwent nephrectomy to treat clinically localized ccRCC. A centralized pathologist dichotomized patients as either BAP1-positive or BAP1-negative. The authors employed Kaplan-Meier and Cox regression models to associate BAP1 expression with cancer-specific survival. RESULTS: A total of 10.5% of tumors were BAP1-negative, 84.8% of tumors were BAP1-positive, and 4.6% of tumors had ambiguous staining for BAP1. Patients with BAP1-negative tumors have an increased risk of ccRCC-related death (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 2.28-4.10; P = 6.77 × 10(-14) ). BAP1 expression remained an independent marker of prognosis after adjusting for the UCLA integrated staging system (UISS) (HR = 1.67; 95% CI = 1.24-2.25; P < .001). Finally, BAP1 was an independent prognostic marker in low-risk patients with a Mayo Clinic stage, size, grade, and necrosis (SSIGN) score of ≤ 3 (HR = 3.24; 95% CI = 1.26-8.33; P = .015). CONCLUSIONS: This study used a large patient cohort to demonstrate that BAP1 expression is an independent marker of prognosis in patients with low-risk (SSIGN≤ 3) ccRCC.


Subject(s)
Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/metabolism , Tumor Suppressor Proteins/biosynthesis , Ubiquitin Thiolesterase/biosynthesis , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Carcinoma, Renal Cell/genetics , Cohort Studies , Female , Humans , Immunohistochemistry , Kidney Neoplasms/genetics , Male , Middle Aged , Prognosis , Risk Factors , Survival Analysis , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics
15.
ACS Nano ; 7(10): 8816-23, 2013 Oct 22.
Article in English | MEDLINE | ID: mdl-24016305

ABSTRACT

Sensitive and quantitative assessment of changes in circulating tumor cells (CTCs) can help in cancer prognosis and in the evaluation of therapeutics efficacy. However, extremely low occurrence of CTCs in the peripheral blood (approximately one CTC per billion blood cells) and potential changes in molecular biomarkers during the process of epithelial to mesenchymal transition create technical hurdles to the enrichment and enumeration of CTCs. Recently, efforts have been directed toward development of antibody-capture assays based on the expression of the common biomarker-the epithelial cell adhesion molecule (EpCAM) of epithelium-derived cancer cells. Despite some promising results, the assays relying on EpCAM capture have shown inconsistent sensitivity in clinical settings and often fail to detect CTCs in patients with metastatic cancer. We have addressed this problem by the development of an assay based on hybrid magnetic/plasmonic nanocarriers and a microfluidic channel. In this assay, cancer cells are specifically targeted by antibody-conjugated magnetic nanocarriers and are separated from normal blood cells by a magnetic force in a microfluidic chamber. Subsequently, immunofluorescence staining is used to differentiate CTCs from normal blood cells. We demonstrated in cell models of colon, breast, and skin cancers that this platform can be easily adapted to a variety of biomarkers, targeting both surface receptor molecules and intracellular biomarkers of epithelial-derived cancer cells. Experiments in whole blood showed capture efficiency greater than 90% when two cancer biomarkers are used for cell capture. Thus, the combination of immunotargeted magnetic nanocarriers with microfluidics provides an important platform that can improve the effectiveness of current CTC assays by overcoming the problem of heterogeneity of tumor cells in the circulation.


Subject(s)
Immunomagnetic Separation , Nanostructures , Neoplasms/pathology , Biomarkers, Tumor/metabolism , Humans , Microscopy, Electron, Transmission
16.
Neurology ; 81(3): 256-63, 2013 Jul 16.
Article in English | MEDLINE | ID: mdl-23771486

ABSTRACT

OBJECTIVE: The study goal was to assess the benefits and potential limitations in the use of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles in the MRI diagnosis of CNS inflammatory diseases and primary CNS lymphoma. METHODS: Twenty patients with presumptive or known CNS lesions underwent MRI study. Eighteen patients received both gadolinium-based contrast agents (GBCAs) and 1 of 2 USPIO contrast agents (ferumoxytol and ferumoxtran-10) 24 hours apart, which allowed direct comparative analysis. The remaining 2 patients had only USPIO-enhanced MRI because of a renal contraindication to GBCA. Conventional T1- and T2-weighted MRI were acquired before and after contrast administration in all patients, and perfusion MRI for relative cerebral blood volume (rCBV) assessment was obtained in all 9 patients receiving ferumoxytol. RESULTS: USPIO-enhanced MRI showed an equal number of enhancing brain lesions in 9 of 18 patients (50%), more enhancing lesions in 2 of 18 patients (11%), and fewer enhancing lesions in 3 of 18 patients (17%) compared with GBCA-enhanced MRI. Four of 18 patients (22%) showed no MRI enhancement. Dynamic susceptibility-weighted contrast-enhanced perfusion MRI using ferumoxytol showed low rCBV (ratio <1.0) in 3 cases of demyelination or inflammation, modestly elevated rCBV in 5 cases of CNS lymphoma or lymphoproliferative disorder (range: 1.3-4.1), and no measurable disease in one case. CONCLUSIONS: This study showed that USPIO-enhanced brain MRI can be useful in the diagnosis of CNS inflammatory disorders and lymphoma, and is also useful for patients with renal compromise at risk of nephrogenic systemic fibrosis who are unable to receive GBCA.


Subject(s)
Brain Diseases/diagnosis , Brain/pathology , Central Nervous System Neoplasms/diagnosis , Ferric Compounds , Lymphoma/diagnosis , Nanoparticles , Adult , Aged , Brain Diseases/pathology , Central Nervous System Neoplasms/pathology , Female , Humans , Inflammation/diagnosis , Inflammation/pathology , Lymphoma/pathology , Magnetic Resonance Imaging , Male , Middle Aged
17.
Blood ; 121(14): 2669-77, 2013 Apr 04.
Article in English | MEDLINE | ID: mdl-23365458

ABSTRACT

Mutations in the transcription factor GATA2 underlie the syndrome of monocytopenia and B- and natural killer (NK)-cell lymphopenia associated with opportunistic infections and cancers. In addition, patients have recurrent and severe viral infections. NK cells play a critical role in mediating antiviral immunity. Human NK cells are thought to mature in a linear fashion, with the CD56(bright) stage preceding terminal maturation to the CD56(dim) stage, considered the most enabled for cytotoxicity. Here we report an NK cell functional defect in GATA2-deficient patients and extend this genetic lesion to what is considered to be the original NK cell-deficient patient. In most cases, GATA2 deficiency is accompanied by a severe reduction in peripheral blood NK cells and marked functional impairment. The NK cells detected in peripheral blood of some GATA2-deficient patients are exclusively of the CD56(dim) subset, which is recapitulated on in vitro NK cell differentiation. In vivo, interferon α treatment increased NK cell number and partially restored function but did not correct the paucity of CD56(bright) cells. Thus, GATA2 is required for the maturation of human NK cells and the maintenance of the CD56(bright) pool in the periphery. Defects in GATA2 are a novel cause of profound NK cell dysfunction.


Subject(s)
CD56 Antigen/immunology , Cell Differentiation/immunology , GATA2 Transcription Factor/genetics , Killer Cells, Natural/immunology , Lymphopenia/genetics , Antigens, CD34/metabolism , CD56 Antigen/metabolism , Cytotoxicity, Immunologic/immunology , GATA2 Transcription Factor/immunology , GATA2 Transcription Factor/metabolism , Humans , Immunophenotyping , K562 Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Lymphocyte Activation/immunology , Lymphocyte Count , Lymphopenia/immunology , Lymphopenia/metabolism , Stromal Cells/cytology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
18.
Biomed Microdevices ; 15(4): 673-681, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23109037

ABSTRACT

Combining the power of immunomagnetic assay and microfluidic microchip operations, we successfully detected rare CTCs from clinical blood samples. The microfluidic system is operated in a flip-flop mode, where a computer-controlled rotational holder with an array of microfluidic chips inverts the microchannels. We have demonstrated both theoretically and experimentally that the direction of red blood cell (RBC) sedimentation with regards to the magnetic force required for cell separation is important for capture efficiency, throughput, and purity. The flip-flop operation reduces the stagnation of RBCs and non-specific binding on the capture surface by alternating the direction of the magnetic field with respect to gravity. The developed immunomagnetic microchip-based screening system exhibits high capture rates (more than 90%) for SkBr3, PC3, and Colo205 cell lines in spiked screening experiments and successfully isolates CTCs from patient blood samples. The proposed motion controlled microchip-based immunomagnetic system shows great promise as a clinical tool for cancer diagnosis and prognosis.


Subject(s)
Immunomagnetic Separation/instrumentation , Microfluidic Analytical Techniques/instrumentation , Motion , Nanotechnology/instrumentation , Neoplastic Cells, Circulating/pathology , Blood Sedimentation , Cell Line, Tumor , Erythrocytes/cytology , Humans , Magnetic Fields , Neoplasms/blood , Viscosity
19.
Transl Res ; 159(5): 366-75, 2012 May.
Article in English | MEDLINE | ID: mdl-22500509

ABSTRACT

We developed a hyperspectral microscopic imaging (HMI) platform that can precisely identify and quantify 10 molecular markers in individual cancer cells in a single pass. The exploitation of an improved separation of circulating tumor cells and the application of HMI provided an opportunity (1) to identify molecular changes in these cells, (2) to recognize the coexpression of these markers, (3) to pose an important opportunity for noninvasive diagnosis, and (4) to use targeted therapy. We balanced the intensity of 10 fluorochromes bound to 10 different antibodies, each specific to a particular tumor marker, so that the intensity of each fluorochrome can be discerned from overlapping emissions. Using 2 touch preps from each primary breast cancer, the average molecular marker intensities of 25 tumor cells gave a representative molecular signature for the tumor despite some cellular heterogeneity. The intensities determined by the HMI correlate well with the conventional 0-3+ analysis by experts in cellular pathology. Because additional multiplexes can be developed using the same fluorochromes but different antibodies, this analysis allows quantification of many molecular markers on a population of tumor cells. HMI can be automated completely, and eventually, it could allow the standardization of protein biomarkers and improve reproducibility among clinical pathology laboratories.


Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Fluorescent Dyes , Genes, erbB-2 , Humans , Microscopy
20.
Expert Opin Biol Ther ; 12(1): 53-61, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22171665

ABSTRACT

INTRODUCTION: Amino acid auxotrophy or the metabolic defect which renders cancer incapable of surviving under amino acid depleted conditions is being exploited and explored as a therapeutic against cancer. Early clinical data on asparagine- and arginine-depleting drugs have demonstrated low toxicity and efficacy in melanoma, hepatocellular carcinoma and acute lymphoblastic leukemia. Methionine auxotrophy is a novel niche currently under exploration for targeting certain cancers. AREAS COVERED: In this review we explore the discovery of methionine auxotrophy followed by in vitro, in vivo and patient data on targeting cancer with methionine depletion. We end with a small discussion on bioengineering, pegylation and red blood cell encapsulation as mechanisms for decreasing immunogenicity of methionine-depleting drugs. We hope to provide a platform for future pharmacology, toxicology and cytotoxicity studies with methionine depletion therapy and drugs. EXPERT OPINION: Although methionine auxotrophy seems as a viable target, extensive research addressing normal versus cancer cell toxicity needs to be conducted. Further research also needs to be conducted into the molecular mechanism associated with methionine depletion therapy. Finally, novel methods need to be developed to decrease the immunogenicity of methionine-depleting drugs, a current issue with protein therapeutics.


Subject(s)
Methionine/metabolism , Neoplasms/drug therapy , Animals , Antineoplastic Agents/therapeutic use , Bioengineering/methods , Cell Line, Tumor , Erythrocytes/cytology , Humans , Immune System , Medical Oncology/methods , Methionine/therapeutic use , Mice , Models, Biological , Neoplasm Transplantation , Neoplasms/diet therapy , Polyethylene Glycols/chemistry , Pseudomonas putida/metabolism , Rats
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