PMID- 14696059
OWN - NLM
STAT- MEDLINE
DCOM- 20040819
LR  - 20091211
IS  - 1552-4949 (Print)
IS  - 1552-4949 (Linking)
VI  - 57
IP  - 1
DP  - 2004 Jan
TI  - Automatic quantification of gene amplification in clinical samples by IQ-FISH.
PG  - 15-22
AB  - BACKGROUND: The reliable detection and quantification of gene amplifications is 
      crucial to clinical practice. Although there are different detection techniques, 
      the fluorescence in situ hybridization (FISH) method has become highly accepted 
      over past years because it is a reliable, robust, and quick method. 
      Unfortunately, automatic quantification of gene amplification based on 
      fluorescence intensities has not been possible thus far. Because current spot 
      counting methods are reliable only when analyzing low amplification rates, we 
      attempted to establish another method, i.e., to quantify the intensity of 
      different FISH signals using an automatic fluorescence microscopical device on 
      interphase nuclei: interphase quantitative FISH (IQ-FISH). METHODS: We quantified 
      the fluorescence intensities of the differently labeled FISH probes (MYCN and 
      D2Z) hybridized to three different neuroblastoma cell lines, six peripheral blood 
      (PB) samples, 10 spiked PB samples, and nine neuroblastoma samples using the 
      Metafer4 system (MetaSystems, Altlussheim, Germany). To obtain the MYCN copy 
      number per cell, the ratio between the fluorescence intensities of the MYCN gene 
      and reference sequence (D2Z) was calculated. For automatic analysis of the 
      HER-2/neu status in tumor cells, labeled FISH probes specific for HER-2/neu and a 
      chromosome 17-specific probe were hybridized to peripheral blood and tumor 
      specimens and analyzed using the automatic device. RESULTS: When measuring the 
      fluorescence intensity per cell for both probe pairs (MYCN/D2Z and HER-2/17p), 
      amplified and non-amplified cells, showed distinct peaks with only little 
      overlap. Whereas normal cells showed a fluorescence ratio peak for MYCN/D2Z 
      between 200 and 800, cells with MYCN amplification clearly exceeded this ratio 
      value (1000 to 25,000). When mixing a varying number of MYCN amplified cells 
      (range 9-91%) to normal PB, the spiked tumor cells could be identified. Even one 
      neuroblastoma tumor cell in 1000 mononucleated cells could reliably be detected 
      using our device. In neuroblastoma patient samples, non-amplified cells were 
      distinguished from amplified cells. Automatically and manually counted signals 
      gave matching results in amplified and non-amplified samples. HER-2/neu-amplified 
      cells were automatically detected in the breast cancer samples analyzed. 
      CONCLUSION: The automatic measurement of fluorescence signal intensities not only 
      allows a reliable discrimination between non-amplified and amplified cells but 
      also exact quantification of amplified sequences. This is the prerequisite for 
      the following applications: detection of amplified cells in the bone marrow and 
      second-look specimens; comparison between primary and relapse or pre- and 
      post-chemotherapeutic specimens; detection of tumors with focal gene 
      amplification; and quantification of elimination of amplified gene sequences.
CI  - Copyright 2003 Wiley-Liss, Inc.
FAU - Narath, R
AU  - Narath R
AD  - CCRI, Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, 
      Austria.
FAU - Lorch, T
AU  - Lorch T
FAU - Rudas, M
AU  - Rudas M
FAU - Ambros, P F
AU  - Ambros PF
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Cytometry B Clin Cytom
JT  - Cytometry. Part B, Clinical cytometry
JID - 101235690
SB  - IM
MH  - Automation/methods
MH  - Cell Line, Tumor
MH  - *Gene Amplification
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Neuroblastoma
EDAT- 2003/12/30 05:00
MHDA- 2004/08/20 05:00
CRDT- 2003/12/30 05:00
PHST- 2003/12/30 05:00 [pubmed]
PHST- 2004/08/20 05:00 [medline]
PHST- 2003/12/30 05:00 [entrez]
AID - 10.1002/cyto.b.10058 [doi]
PST - ppublish
SO  - Cytometry B Clin Cytom. 2004 Jan;57(1):15-22. doi: 10.1002/cyto.b.10058.