Tumor Content Chart-Assisted HER2/CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens.
Evaluating HER2 gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining HER2 and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The HER2/CEP17 ratio is determined by three variables-TCR and absolute copy numbers of HER2 and CEP17-by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of HER2/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of HER2 status.
Division of Diagnostic Pathology, the University of Tokyo Hospital, Tokyo, Japan.|Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Keisuke
K
Matsusaka
Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.|Department of Genomic Pathology, Medical Research Institute Tokyo Medical and Dental University, Tokyo, Japan.
Shumpei
S
Ishikawa
Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.
Atsuhito
A
Nakayama
Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.
Tetsuo
T
Ushiku
Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.
Aiko
A
Nishimoto
Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.|Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.|Department of Gastrointestinal Surgery, the University of Tokyo Hospital, Tokyo, Japan.
Masayuki
M
Urabe
Division of Diagnostic Pathology, the University of Tokyo Hospital, Tokyo, Japan.
Nobuyuki
N
Kaneko
Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.
Akiko
A
Kunita
Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Atsushi
A
Kaneda
Genome Science Division, Research Center for Advanced Science and Technology, the University of Tokyo, Tokyo, Japan.
Hiroyuki
H
Aburatani
Department of Endoscopy and Endoscopic Surgery, the University of Tokyo Hospital, Tokyo, Japan.
Mitsuhiro
M
Fujishiro
Department of Gastrointestinal Surgery, the University of Tokyo Hospital, Tokyo, Japan.
Yasuyuki
Y
Seto
Division of Diagnostic Pathology, the University of Tokyo Hospital, Tokyo, Japan.|Department of Pathology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.
Masashi
M
Fukayama
D057205:Automation, Laboratory; D001706:Biopsy; D045744:Cell Line, Tumor; D002886:Chromosomes, Human, Pair 17; D003627:Data Interpretation, Statistical; D005557:Formaldehyde; D018628:Gene Dosage; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D013274:Stomach Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D016610:Tissue Embedding; D016707:Tissue Fixation
D005557:Formaldehyde; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human