ABSTRACT
Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.
Subject(s)
Biomarkers/analysis , Chromogenic Compounds/chemistry , Coloring Agents/chemistry , Immunohistochemistry/methods , In Situ Hybridization/methods , 3,3'-Diaminobenzidine/chemistry , Biomarkers/chemistry , Chromogenic Compounds/chemical synthesis , Coloring Agents/chemical synthesis , Humans , Models, Chemical , Molecular Structure , Reproducibility of Results , Tyramine/chemistryABSTRACT
The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors.
Subject(s)
Chromosomes, Human, Pair 3/genetics , In Situ Hybridization, Fluorescence/methods , Oligonucleotides/chemistry , Phosphatidylinositol 3-Kinases/genetics , Automation, Laboratory , Base Sequence , Biopsy, Fine-Needle , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Class I Phosphatidylinositol 3-Kinases , DNA Probes/chemistry , Gene Dosage , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , MCF-7 Cells , Molecular Sequence Data , Nucleic Acid Hybridization , Signal Processing, Computer-Assisted , Tumor Cells, CulturedABSTRACT
INTRODUCTION: The use of targeted therapies toward specific oncogenic driver mutations has become a critical factor in the treatment of patients with lung cancer. It is therefore essential to utilize tests with high performance characteristics. Fluorescence in situ hybridization (FISH) is the standard method for detecting anaplastic lymphoma kinase (ALK) and ROS1 rearrangements in non-small-cell lung cancer but the utility of other methods such as immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) is unclear. METHODS: Three hundred and sixty-two lung cancer patients were tested with FISH, CISH, and IHC using three ALK antibodies (ALK1, 5A4, D5F3) and one ROS1 antibody in the detection of ALK and ROS1 rearrangements. RESULTS: There was a 97.4% concordance (298 of 306) between FISH and CISH for detection of ALK rearrangements. The ROS1 rearrangement status had a 97% (291 of 300) concordance between CISH and FISH. ALK protein expression was observed in 6 of 341 samples with the ALK1 and 5A4 antibodies and 5 of 341 samples with D5F3. All three antibodies stained each of the ALK FISH-positive samples (100% sensitivity). ROS1 protein expression was observed in 2 of 322 samples. One of three samples with a ROS1 rearrangement by FISH showed ROS1 protein expression (33.3% sensitivity). CONCLUSION: Our findings show good correlation between FISH versus CISH in the detection of ALK and ROS1 rearrangements. FISH versus IHC showed good correlation in the detection of ALK rearrangements but showed weak correlation in the detection of ROS1 rearrangements. These results suggest CISH and IHC could be complimentary detection methods to FISH in the detection of ALK and ROS1 rearrangements.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Mutation , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Female , Gene Rearrangement , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Middle Aged , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Retrospective StudiesABSTRACT
OBJECTIVES: Insulin-Like Growth Factor-1 is a potent growth-promoting peptide that is present in mammalian milk. Previous studies have suggested that milk-borne IGF-1 may be absorbed intact from the gastrointestinal tract of the suckling but the mechanism responsible for such transport is not well documented. The present study was designed to investigate in an in vivo suckling rat model whether or not intestinal absorption of IGF-1 is a saturable phenomenon. METHODS: Suckling rats (10-12 days postnatal age) were studied under anesthesia. A jejunal loop from each rat pup was isolated and injected intraluminally with 1-2 x 10 cpm of rh I-IGF-I. Injections were performed in paired littermates either with or without a preceding injection of unlabeled IGF-I of 20, 500, or 1000 ng/ml concentration. After flushing, the loops and livers were homogenized and counted in a gamma counter. In addition, homogenates of jejunum and liver were precipitated with trichloroacetic acid (TCA) and the precipitates also counted. In selected instances (jejunum), acid gel chromatography of homogenates was also performed. RESULTS: Retention of radioactivity was observed in all jejunal specimens, but the pre-incubation of jejunal loops with unlabeled IGF-1 was associated with a biphasic response, i.e. at low dose (20 ng/ml) pre-incubation limited retention of radioactivity, but at a high dose (1000 ng/ml), retention was enhanced (P < 0.05). Linear regression analysis confirmed this inverse relationship. Liver radioactivity followed a similar pattern. Between 40 and 49% of the radioactivity in jejunal and liver homogenates was TCA precipitable. Chromatography of jejunal homogenates showed that approximately 40% of cpm migrated in a position identical with that of intact IGF-1. CONCLUSIONS: The intestinal uptake of IGF-1 in the suckling is nonsaturable, confirming previous in vitro studies and suggesting that a nonreceptor-dependent method of transepithelial transport is important in this process.