Excellent agreement was observed between IHC and ADx-ARMS when detecting EGFR mutations ( em ?? /em =0.826; em P /em 0.01). Conclusion QDs-IHC is a simple and standardized method to detect EGFR mutations with its high sensitivity and specificity, as compared with real-time polymerase chain reaction. between IHC and ADx-ARMS when detecting EGFR mutations ( em ?? /em =0.826; em P /em 0.01). Conclusion QDs-IHC is a simple and standardized method to detect EGFR mutations with its high sensitivity and specificity, as compared with real-time polymerase chain reaction. In addition, the development of specific antibodies against EGFR mutation proteins might be useful for the diagnosis and treatment of lung cancer. strong class=”kwd-title” Keywords: quantum dots, lung cancer, EGFR, gene mutation, real-time PCR, immunohistochemistry Introduction With the aggravation of environmental pollution, lung cancer is almost the most malignant tumor in the world with its high morbidity and mortality.1 The most common histologic subtype is non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers.2 Although much progress has been made in the treatment of lung cancer, early diagnosis is difficult and the Luliconazole majority of patients has progressed to an advanced stage when diagnosed. The median survival rate for these patients is only 8C11 months.3 In 2004, a landmark discovery had been made in that somatic mutations in the epidermal growth factor receptor (EGFR) were associated with sensitivity to EGFR tyrosine kinase (TK) inhibitors (TKIs) (EGFR-TKI).4,5 In subsequent large-scale randomized clinical trials, the relationship between EGFR mutation status and efficacy of the EGFR-TKI drug was clearly explained.6C8 Based on these findings, EGFR mutation status in the TK domain can determine the treatment of advanced NSCLC. Patients with EGFR-activating mutations can benefit from EGFR-TKI treatment. Mutations associated with enhanced sensitivity to EGFR-TKIs are found in exons Luliconazole 18C21 of the TK domain of EGFR; in particular, del E746-A750 in exon 19 and the L858R point mutation in exon 21 account for nearly 90% of all the mutations in EGFR in lung cancer.7,9,10 Nowadays, the detection of EGFR mutation status in NSCLC patients has become an expert consensus.11 Different methodologies have been developed for molecular testing, such as the amplified refractory mutation system (ARMS), high-resolution melting, DNA direct sequencing, and next-generation sequencing (NGS). DNA direct sequencing is considered the gold standard for the assessment of EGFR mutation status in NSCLC; however, it is time consuming and laborious. The ARMS method is widely used in the clinical testing; however, the commercial assay kit for EGFR is very expensive, and the experiment needs to be done under good experimental Luliconazole conditions with sophisticated real-time polymerase chain reaction (PCR) instruments.12 Immunohistochemistry (IHC) is a well-established method that is widely applied in conventional pathological diagnosis. EGFR mutation-specific rabbit monoclonal antibodies against E746-A750 deletion and L858R (Cell Signaling Technology, Inc., Danvers, MA, USA) have been applied in IHC application. This provides a simple and rapid screening method for assessing EGFR mutation status.13C15 A nanofluorescent material, fluorescent semiconductor nanocrystal quantum dots (QDs), have been widely used in labeling some molecules, such as streptavidin and antibodies, through carbodiimide chemistry, optionally using EDAC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide). QD (605 nm)-labeled streptavidin emits bright red fluorescence with 605 nm as the maximum emission wavelength while being stimulated by an excitation light source 580 nm, which is different from green background autofluorescence. Those labeled materials can be successfully applied to biological imaging and IHC detection of gene mutations, such as with human epidermal growth factor receptor 2 (HER 2) amplification in breast cancer.16,17 Moreover, multiple markers can be visualized on one cell for in vitro multiplexed imaging; for example, clinically significant tumor biomarkers including HER2, EGFR, progesterone receptor, estrogen receptor, and mammalian target of rapamycin can be detected quantitatively and simultaneously in breast cancer cells using multicolor QDs.18 QD-based immunofluorescence histochemistry (QDs-IHC) is an established method; it has been validated in many published papers.17,19,20 The staining signal detected by QDs-IHC is much stronger with a lower background when compared with IHC.20 Based on the application of EGFR mutation-specific antibodies, this study was designed to develop a QD-based immunofluorescent approach for EGFR mutation detection, which is a simple, quick, and highly sensitive molecular method Hhex for diagnosing Luliconazole EGFR mutation status in NSCLC samples. Materials and methods Patients and samples Fifty-five cases of formalin-fixed, paraffin-embedded (FFPE) NSCLC specimens and ten cases of pleural effusion cell.