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1.
J Thorac Oncol ; 16(2): 259-268, 2021 02.
Article in English | MEDLINE | ID: mdl-33334571

ABSTRACT

INTRODUCTION: We retrospectively examined progression-free survival (PFS) and response by ALK fluorescence in situ hybridization (FISH) status in patients with advanced ALK immunohistochemistry (IHC)-positive NSCLC in the ALEX study. METHODS: A total of 303 treatment-naive patients were randomized to receive twice-daily alectinib 600 mg or crizotinib 250 mg. ALK status was assessed centrally using Ventana ALK (D5F3) CDx IHC and Vysis ALK Break Apart FISH Probe Kit. Primary end point is investigator-assessed PFS. Secondary end points of interest are objective response rate and duration. RESULTS: Investigator-assessed PFS was significantly prolonged with alectinib versus crizotinib in ALK IHC-positive and FISH-positive tumors (n = 203, 67%) (hazard ratio [HR] = 0.37, 95% confidence interval [CI]: 0.25-0.56; p < 0.0001) and ALK IHC-positive and FISH-uninformative tumors (n = 61, 20%) (HR = 0.39, 95% CI: 0.20-0.78) but not in ALK IHC-positive and FISH-negative tumors (n = 39, 13%) (HR = 1.33, 95% CI: 0.6-3.2). Objective response rates were higher with alectinib versus crizotinib in ALK IHC-positive and FISH-positive tumors (90.6% versus 81.4%; stratified OR = 2.22, 95% CI: 0.97-5.07) and ALK IHC-positive and FISH-uninformative tumors (96.0% versus 75.0%; OR = 9.29, 95% CI: 1.05-81.88) but not in ALK IHC-positive and FISH-negative tumors (28.6% versus 44.4%; OR = 0.45, 95% CI: 0.12-1.74). Next-generation sequencing was performed in 35 of 39 patients with ALK IHC-positive and FISH-negative tumors; no ALK fusion was identified in 20 of 35 patients (57.1%) by next-generation sequencing, but 10 of 20 (50.0%) had partial response or stable disease. CONCLUSIONS: Outcomes of patients with ALK IHC-positive and FISH-positive and ALK IHC-positive and FISH-uninformative NSCLC were similar to those of the overall ALEX population. These results suggest that Ventana ALK IHC is a standard testing method for selecting patients for treatment with alectinib.


Subject(s)
Lung Neoplasms , Anaplastic Lymphoma Kinase/genetics , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Retrospective Studies
2.
J Thorac Oncol ; 2020 Oct 24.
Article in English | MEDLINE | ID: mdl-34756882

ABSTRACT

INTRODUCTION: We retrospectively examined progression-free survival (PFS) and response by ALK fluorescence in-situ hybridization (FISH) status in patients with advanced ALK immunohistochemistry (IHC)-positive non-small-cell lung cancer (NSCLC) in the ALEX study. METHODS: 303 treatment-naïve patients were randomized to receive twice-daily alectinib 600 mg or crizotinib 250 mg. ALK status was assessed centrally using Ventana ALK (D5F3) CDx IHC and Vysis ALK Break Apart FISH Probe Kit. Primary endpoint: investigator-assessed PFS. Secondary endpoints of interest: objective response rate (ORR) and duration. RESULTS: Investigator-assessed PFS was significantly prolonged with alectinib versus crizotinib in ALK IHC-positive/FISH-positive tumors (n = 203, 67%) (HR 0.37, 95% CI: 0.25-0.56) and ALK IHC-positive/FISH-uninformative tumors (n = 61, 20%) (HR 0.39, 95% CI: 0.20-0.78), but not ALK IHC-positive/FISH-negative tumors (n = 39, 13%) (HR 1.33, 95% CI: 0.6-3.2). ORRs were higher with alectinib versus crizotinib in ALK IHC-positive/FISH-positive tumors 90.6% versus 81.4%; stratified odds ratio [OR] 2.22, 95% CI: 0.97-5.07) and ALK IHC-positive/FISH-uninformative tumors (96.0% versus 75.0%; OR 9.29, 95% CI: 1.05-81.88), but not ALK IHC-positive/FISH-negative tumors (28.6% versus 44.4%; OR 0.45, 95% CI: 0.12-1.74). Next-generation sequencing (NGS) was performed in 35/39 patients with ALK IHC-positive/FISH-negative tumors; no ALK fusion was identified in 20/35 (57.1%) patients by NGS, but 10/20 (50.0%) had partial response/stable disease. CONCLUSION: Outcomes of patients with ALK IHC-positive/FISH-positive and ALK IHC-positive/FISH-uninformative NSCLC were similar to the overall ALEX population. These results suggest that Ventana ALK IHC is a standard testing method for selecting patients for treatment with alectinib.

3.
Appl Immunohistochem Mol Morphol ; 28(5): 331-338, 2020.
Article in English | MEDLINE | ID: mdl-30724750

ABSTRACT

Diagnostic assays for molecular alterations highly correlated with prognosis, predictive efficacy or safety of therapeutics are valuable clinical tools and in some cases approved as companion diagnostics (CDx) by the Federal Food and Drug Administration. For example, assays that determine echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) translocation status have been approved as CDx assay for therapies that target this molecular alteration. Characterizing the parameters that may compromise diagnostic accuracy for molecular biomarkers is critical for optimal patient care. To investigate the impact of pre-analytical handling and processing of tumor tissue on commonly used diagnostic immunohistochemistry-based assays for ALK and mesenchymal epithelial transition protein [c-mesenchymal epithelial transition (c-MET)], we investigated the effects of cold ischemia, fixative type, fixation time, and cut-slide age on staining consistency and intensity using human lung xenograft tumor tissue. Cold ischemia times for up to 5 to 6 hours for c-MET or ALK, respectively had minimal impact on staining. The optimal fixation conditions for both assays were found to be at least 6 hours and up to 48 hours for c-MET or 72 hours for ALK, in 10% neutral buffered formalin and Zinc formalin. The ALK antigen demonstrated marked staining intensity differences across non-neutral buffered formalin fixative types and times. Finally, cut-slide age influenced assay performance for both ALK and c-MET, with maximum stability observed when cut slides were stored at ambient temperatures (30°C) for no longer than 3, and 5 months, respectively. This study highlights the potential for pre-analytical factors to confound diagnostic test result interpretation.


Subject(s)
Anaplastic Lymphoma Kinase/metabolism , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Proto-Oncogene Proteins c-met/metabolism , Tissue Fixation/methods , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Female , Fixatives/chemistry , Humans , Immunohistochemistry , Lung , Lung Neoplasms/pathology , Mice , Mice, SCID , Prognosis , Xenograft Model Antitumor Assays
4.
J Thorac Oncol ; 12(5): 804-813, 2017 05.
Article in English | MEDLINE | ID: mdl-28147239

ABSTRACT

INTRODUCTION: The availability of high-quality, rigorously validated diagnostic tests that can be broadly implemented is necessary to efficiently identify patients with anaplastic lymphoma kinase (ALK)-positive NSCLC who can potentially benefit from treatment with crizotinib. Here we present data on the recently approved Ventana ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ), the only immunohistochemistry (IHC)-based assay linked to treatment outcome. METHODS: NSCLC specimens prospectively tested for anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by flourescent in situ hybridization (FISH) in the PROFILE 1014 clinical trial of crizotinib versus chemotherapy (N = 1018, including 179 ALK-positive and 754 ALK-negative specimens) were evaluated using the ALK (D5F3) CDx assay. Hazard ratios for progression-free survival comparing crizotinib and chemotherapy for ALK IHC-positive patients and ALK FISH-positive patients, as well as for concordance with the enrollment ALK FISH assay, were determined. RESULTS: Results from both assays were obtained for 933 cases. Percent positive, negative, and overall agreement rates were 86.0% , 96.3%, and 94.3%, respectively. There were 53 discrepant cases, of which 25 were ALK FISH-positive/ALK IHC-negative and 28 were ALK FISH-negative/ALK IHC-positive. The hazard ratios using observed outcomes were 0.401 for ALK FISH-positive/ALK IHC-positive cases and 0.407 for all ALK FISH-positive cases tested with ALK IHC versus 0.454 for all ALK FISH-positive cases enrolled in the trial. Outcome data for ALK FISH-negative/ALK IHC-positive cases were not available for analysis. Between-reader agreement rates for ALK IHC involving three independent laboratories exceeded 98%. CONCLUSIONS: The ALK (D5F3) CDx assay is a stand-alone companion diagnostic test for identification of patients for treatment with crizotinib. This automated assay provides an effective option to accurately and rapidly identify patients with ALK-positive NSCLC. The simple binary scoring algorithm results in high reader-to-reader precision.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Immunohistochemistry/methods , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Pyrazoles/therapeutic use , Pyridines/therapeutic use , Receptor Protein-Tyrosine Kinases/metabolism , Anaplastic Lymphoma Kinase , Crizotinib , Disease-Free Survival , False Negative Reactions , False Positive Reactions , Female , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Observer Variation , Patient Selection , Reproducibility of Results , Treatment Outcome
5.
Hum Pathol ; 56: 194-203, 2016 10.
Article in English | MEDLINE | ID: mdl-27349303

ABSTRACT

Companion diagnostics assay interpretation can select patients with the greatest targeted therapy benefits. We present the results from a prospective study demonstrating that pathologists can effectively learn immunohistochemical assay-interpretation skills from digital image-based electronic training (e-training). In this study, e-training was used to train board-certified pathologists to evaluate non-small cell lung carcinoma for eligibility for treatment with onartuzumab, a MET-inhibiting agent. The training program mimicked the live training that was previously validated in clinical trials for onartuzumab. A digital interface was developed for pathologists to review high-resolution, static images of stained slides. Sixty-four pathologists practicing in the United States enrolled while blinded to the type of training. After training, both groups completed a mandatory final test using glass slides. The results indicated both training modalities to be effective. Overall, 80.6% of e-trainees and 72.7% of live trainees achieved passing scores (at least 85%) on the final test. All study participants reported that their training experience was "good" and that they had received sufficient information to determine the adequacy of case slide staining to score each case. This study established that an e-training program conducted under highly controlled conditions can provide pathologists with the skills necessary to interpret a complex assay and that these skills can be equivalent to those achieved with face-to-face training using conventional microscopy. Programs of this type are scalable for global distribution and offer pathologists the potential for readily accessible and robust training in new companion diagnostic assays linked to novel, targeted, adjuvant therapies for cancer patients.


Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Non-Small-Cell Lung/enzymology , Computer-Assisted Instruction , Education, Medical, Continuing/methods , Immunohistochemistry , Inservice Training/methods , Lung Neoplasms/enzymology , Microscopy , Pathology, Clinical/education , Proto-Oncogene Proteins c-met/analysis , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/antagonists & inhibitors , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Clinical Competence , Clinical Decision-Making , Computer Graphics , Curriculum , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Patient Selection , Predictive Value of Tests , Program Evaluation , Prospective Studies , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Reproducibility of Results , United States , Workflow
6.
Methods ; 52(4): 287-300, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20807574

ABSTRACT

With the advent of personalized medicine, anatomic pathology-based molecular assays, including in situ hybridization (ISH) and mRNA detection tests, are performed routinely in many laboratories and have increased in their clinical importance and complexity. These assays require appropriately fixed tissue samples that preserve both nucleic acid targets and histomorphology to ensure reliable test results for determining patient treatment options. However, all aspects of tissue processing, including time until tissue fixation, type of fixative, duration of fixation, post-fixation treatments, and sectioning of the sample, impact the staining results. ASCO/CAP has issued pre-analytical guidelines to standardize tissue processing for HER2 testing in breast carcinoma specimens: 10% neutral-buffered formalin (NBF) with a fixation time from at least 6 to 48h [1]. Often, this recommendation is not followed to the detriment of staining results [2]. In this paper, we used a human breast carcinoma cell line (MCF7) generated as xenograft tumors as a model system to analyze the effects of different pre-analytical conditions on ISH staining. We performed H&E, FISH and dual colorimetric HER2 ISH assays using specimens fixed across a range of times in six different commonly used fixatives. Additionally, we investigated the effects of varying tissue section thickness, which also impacted the quality of ISH staining. Finally, we evaluated the effects of three different decalcifying solutions on human breast specimens, typically a treatment that occurs post-fixation for evaluating metastases to bone. The results indicate that time and type of fixation treatment, as well as appropriate tissue thickness and post-fixation treatment, all contribute to the quality of ISH staining results. Our data support the ASCO/CAP recommendations for standardized tissue processing (at least 6h in formalin-based fixatives and 4µm section thickness) and indicate that certain fixatives and post-fixative treatments are detrimental to molecular staining results.


Subject(s)
Breast Neoplasms/chemistry , In Situ Hybridization, Fluorescence/methods , In Situ Hybridization/methods , Tissue Fixation/methods , Animals , Cell Line, Tumor , Eosine Yellowish-(YS) , Female , Fixatives , Haptens , Hematoxylin , Humans , Mice , Receptor, ErbB-2/analysis , Staining and Labeling , Transplantation, Heterologous
7.
J Biol Inorg Chem ; 14(6): 905-12, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19381697

ABSTRACT

The periplasmic metallochaperone CusF coordinates Cu(I) and Ag(I) through a unique site consisting of a Met(2)His motif as well as a Cu(I)-pi interaction between a nearby tryptophan, W44, and the metal ion. Through mutational analyses we investigate here the role that W44 in CusF plays in metal coordination. Nuclear magnetic resonance spectra show that the specificity of CusF for Cu(I) and Ag(I) is not altered by mutation of W44. X-ray absorption spectroscopy studies reveal that W44 protects the bound Cu(I) from oxidation as well as from adventitious ligands. Competition assays demonstrate that W44 does not significantly contribute to the affinity of CusF for metal, but that substitution of W44 by methionine, which forms a fourth Cu(I) ligand, substantially increases the affinity. These studies indicate that W44 is important in maintaining a moderate-affinity and solvent-shielded three-coordinate environment for Cu(I), which has implications for the function of CusF as a metallochaperone.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Copper/metabolism , Escherichia coli , Tryptophan/metabolism , Absorption , Binding Sites , Binding, Competitive , Cation Transport Proteins/genetics , Copper Transport Proteins , Escherichia coli Proteins , Indoles/metabolism , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Mutation , Oxidation-Reduction , Protein Conformation , Spectrum Analysis , Substrate Specificity , X-Rays
8.
Protein Sci ; 16(10): 2287-93, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17893365

ABSTRACT

Elevated levels of copper or silver ions in the environment are an immediate threat to many organisms. Escherichia coli is able to resist the toxic effects of these ions through strictly limiting intracellular levels of Cu(I) and Ag(I). The CusCFBA system is one system in E. coli responsible for copper/silver tolerance. A key component of this system is the periplasmic copper/silver-binding protein, CusF. Here the X-ray structure and XAS data on the CusF-Ag(I) and CusF-Cu(I) complexes, respectively, are reported. In the CusF-Ag(I) structure, Ag(I) is coordinated by two methionines and a histidine, with a nearby tryptophan capping the metal site. EXAFS measurements on the CusF-Cu(I) complex show a similar environment for Cu(I). The arrangement of ligands effectively sequesters the metal from its periplasmic environment and thus may play a role in protecting the cell from the toxic ion.


Subject(s)
Cation Transport Proteins/chemistry , Copper/chemistry , Silver/chemistry , Binding Sites , Copper Transport Proteins , Crystallography, X-Ray , Escherichia coli Proteins , Models, Molecular , Spectrum Analysis , X-Rays
9.
Biochemistry ; 45(37): 11096-102, 2006 Sep 19.
Article in English | MEDLINE | ID: mdl-16964970

ABSTRACT

The periplasmic protein CusF, as a part of the CusCFBA efflux complex, plays a role in resistance to elevated levels of copper and silver in Escherichia coli. Although homologues have been identified in other Gram-negative bacteria, the substrate of CusF and its precise role in metal resistance have not been described. Here, isothermal titration calorimetry (ITC) was used to demonstrate that CusF binds with high affinity to both Cu(I) and Ag(I) but not Cu(II). The affinity of CusF for Ag(I) was higher than that for Cu(I), which could reflect more efficient detoxification of Ag(I) given the lack of a cellular need for Ag(I). The chemical shifts in the nuclear magnetic resonance (NMR) spectra of CusF-Ag(I) as compared to apo-CusF show that the region of CusF most affected by Ag(I) binding encompasses three absolutely conserved residues: H36, M47, and M49. This suggests that these residues may play a role in Ag(I) coordination. The NMR spectra of CusF in the presence of Cu(II) do not indicate specific binding, which is in agreement with the ITC data. We conclude that Cu(I) and Ag(I) are the likely physiological substrates.


Subject(s)
Cation Transport Proteins/metabolism , Copper/metabolism , Iodides/metabolism , Silver Compounds/metabolism , Binding Sites , Calorimetry/methods , Cation Transport Proteins/chemistry , Copper/chemistry , Copper Transport Proteins , Escherichia coli/metabolism , Escherichia coli Proteins , Iodides/chemistry , Magnetic Resonance Spectroscopy , Protein Conformation , Silver Compounds/chemistry , Substrate Specificity
10.
Biochemistry ; 44(31): 10533-40, 2005 Aug 09.
Article in English | MEDLINE | ID: mdl-16060662

ABSTRACT

We have determined the crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance in Escherichia coli. The protein forms a five-stranded beta-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in beta-strands 2 and 3. These residues are clustered at one end of the beta-barrel, and their side chains are oriented toward the interior of the barrel. Cu(I) can be modeled into the apo-CusF structure with only minimal structural changes using H36, M47, and M49 as ligands. The unique structure and metal binding site of CusF are distinct from those of previously characterized copper-binding proteins.


Subject(s)
Carrier Proteins/chemistry , Cation Transport Proteins/chemistry , Copper/chemistry , Escherichia coli Proteins/chemistry , Periplasm/chemistry , Protein Folding , Amino Acid Sequence , Apoproteins/chemistry , Copper Transport Proteins , Crystallography, X-Ray , Drug Resistance, Bacterial , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Sequence Homology, Amino Acid
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