1 Lung Cancer /EML4-ALK Screening KitYuan Yuan

2 Crizotinib for ALK Positive Advanced NSCLCSado and Mano et al, Nature 2007

3 Sample preparation and staining for FISH: Vysis ALK Break Apart FISH Probe Kit, Abbott

4 Vysis ALK Break Apart FISH Probe Kit, Abbott“Break-apart” assay used, in which orange and green probes are separated after inversion and ALK rearrangement

5 ALK FISH scoring

6 FISH based assay Require special equipment, technique and expertise for interpretation High false negative for intrachromosomal deletion or inversion involving very few DNA base pairs Cost issues, more expensive Tissue issues, FISH requires at least 50 tumor cells

7 How Yin-Yang Probes work?Em Ex Em Ex F Q F Q + _ F _ Q + Hybrid: +/Target + _ Target Yin-Yang Probe (+/- strand) F: Fluorophore Q: Quencher QuanDx has the world-wide ownership of Yin-Yang Probes

8 RT-qPCR based assay Sensitive, less expensive and rapidCan detect multiple biomarkers simultaneously by multiplex RT-PCR May require high-quality RNA Can not detect unknown fusion partners

9 QuanDx Lung cancer Screening KitTUBE FUSION GENES TOTAL NUMBER OF FUSION VARIENTS A KIF5B-RET 7 KIF5B-ALK 3 B CD74-ROS1 TFG-ALK C SLC34A2-ROS1 2 EML4-ALK 15 D

10 Lung cancer screening cDNA synthesis Real-time qPCRInternal control included 20 testes / kit six fusion genes including 35 fusion varients 3-channel real time detection at annealing temperature

11 Volume(μL) per 20 μL reactioncDNA synthesis Make the 20 μL RT reaction mix on ice as below, dilute to 100 μL after reverse transcription. Reagents Volume(μL) per 20 μL reaction RT buffer 4.8 RT Enzyme 1 Total RNA x DEPC H2O 14.2-x 25 oC 5 min 37 oC 30 min 85 oC

12 60℃ 45 sec (detection channels: FAM and HEX)Real-time qPCR Make four master mixes separately as below: PCR mix A~D: 5*n Supermix: 10*n Pipette 15 μL of each of the 4-PCR master mixes A~D for each well. Load 5 μL templates into each well. 1hr 21 min Cycle # Preheating 95℃ 3 min 1 PCR Cycle 95℃ 20 sec 40 60℃ 45 sec (detection channels: FAM and HEX)

13 EML4-ALK Screening Kit Green for FAM; Blue for HEX Fluorescence SignalResults FAM+HEX EML4-ALK Positive FAM Only EML4-ALK Negative Green for FAM; Blue for HEX

14 QuanDx EML4-ALK Screening KitMultiplexing: simultaneous detection of 15 fusion variants Compatible with current real-time qPCR instruments Rapid results within 2 hours Easy to perform for routine screening