Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA  Wenjun Zhang, Antony Hubbard, Leslie Baca-Parkinson, Stacey Stanislaw, Frank Vladich, Mark D. Robida, James G. Grille, Daniel Maxwell, Tsu-Shuen Tsao, William Carroll, Tracie Gardner, June Clements, Shalini Singh, Lei Tang  The Journal of Molecular Diagnostics  Volume 17, Issue 5, Pages 496-504 (September 2015) DOI: 10.1016/j.jmoldx.2015.04.007 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 PIK3CA oligonucleotide probe coverage map. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: Specific signals (red) in normal lung tissue stained with the chromosome 3 oligonucleotide probe in the absence of human placental DNA. B: Cross-reactive signals (red) in normal lung tissue stained with the pHS05 plasmid probe in the absence of human placental DNA. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Comparison of chromosome 3 (CHR3) oligonucleotide and pHS05 plasmid probe staining in two lung cases: case 1 (A and B) and case 2 (C and D). CHR3 oligonucleotide probe staining produces discrete red CHR3 signals with uniform shape and size (A and C). In contrast, CHR3 red signals stained with the pHS05 plasmid probe have irregular shapes and different sizes (B and D). The black signals are PIK3CA probe staining. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 PIK3CA/chromosome 3 oligonucleotide dual in situ hybridization staining on metaphase chromosome spreads. Black signals, PIK3CA; red signals, chromosome 3. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 PIK3CA/chromosome 3 oligonucleotide dual in situ hybridization staining on MCF-7 (A) and Calu-3 (B) cell line xenografts. Black signals, PIK3CA; red signals, chromosome 3. Unlike MCF-7, Calu-3 cells are known to have PIK3CA gene amplification. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Study of the pretreatment combination of CC1 time (64 and 92 minutes), CC2 time (24 minutes, 36 minutes, and no CC2), and in situ hybridization protease 3 (P3) incubation time (20 and 28 minutes) on five lung tumor cases. Main effects plots for PIK3CA (A) and chromosome 3 (CHR3) (B) signal intensities. PIK3CA (C) and CHR3 (D) signal intensities when stained under different assay conditions. Table 2 shows the signal intensity scoring criteria. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Effect of probe hybridization (hyb) time on PIK3CA and chromosome 3 (CHR3) signal intensity, staining coverage, and background. Table 2 shows the signal intensity and background scoring criteria. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 8 Representative images of PIK3CA/chromosome 3 oligonucleotide dual in situ hybridization staining on three squamous cell carcinoma (SCC) resection tissue cases (B, D, and F) and their corresponding hematoxylin and eosin–stained slides (A, C, and E). The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 9 Representative images of PIK3CA/chromosome 3 oligonucleotide dual in situ hybridization staining on fine-needle aspirate (FNA) specimens (B, D, and E) and their corresponding hematoxylin and eosin–stained slides (A and C). Case FNA 2 has two tumor areas as shown in panel C. D: Dual in situ hybridization staining of area 1. E: Dual in situ hybridization staining of area 2. The Journal of Molecular Diagnostics 2015 17, 496-504DOI: (10.1016/j.jmoldx.2015.04.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions