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EasyPGX ready EGFR

User manual – version 2022/05

The “EasyPGX® ready EGFR” kit detects 86 mutations of the oncogene EGFR by Real-Time PCR.

For in vitro diagnostic use

RT023

8057289090606

48

2022/05

Diatech Pharmacogenetics S.r.l

Via Ignazio Silone, 1b - 60035 Jesi (AN) Italy
C.F./P.Iva/R.Imprese di Ancona n. 02483840423
Tel. +39 0731 213243 Fax +39 0731 213239
info@diatechpharmacogenetics.com www.diatechpharmacogenetics.com
 Changes made since the previous version 2021/11:

▪ Update of EasyPGX EGFR strips related to the ex20ins assay

▪ Update of detectable mutations
▪ Update of “Scientific validity” section
▪ Addition of AriaDx real-time instrument
▪ Addition of sample handling and storage details
▪ Section “Data Analysis”: update of EasyPGX® analysis software vers.4.0.12
▪ Update of “Performance validation” section

For further details contact the technical support of the Diatech Pharmacogenetics
(support@diatechpharmacogenetics.com).

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Index page

INTENDED USE 4

PRINCIPLE OF THE ASSAY 6

SCIENTIFIC VALIDITY 7

KIT CONTENTS 9

DOCUMENTS AVAILABLE ON-LINE 10

MATERIALS REQUIRED BUT NOT PROVIDED 10

STABILITY AND STORAGE 11

SYMBOLS 11

PRODUCT USE LIMITATIONS 12

QUALITY ASSURANCE 12

WARNINGS AND PRECAUTIONS 13

ANALYTICAL PROCEDURE 14
DNA EXTRACTION 14
Use of EasyPGX® Extraction reagents 15
Use of the other recommended kits (see “Materials Required but Not Provided”) 15
INSTRUMENT SETUP 15
Plate set-up 15
AMPLIFICATION AND MUTATION DETECTION 16
DATA ANALYSIS 17

TROUBLESHOOTING 19

PERFORMANCE EVALUATION 21

OPERATOR NOTES 28

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INTENDED USE

The in vitro diagnostic “EasyPGX® ready EGFR” kit is intended for the qualitative detection by Real-Time PCR of 86 EGFR somatic
mutations in the genomic DNA isolated from tumor tissue (fresh, frozen or formalin fixed paraffin-embedded (FFPE)) or in circulating
tumor DNA (ctDNA) extracted from EDTA blood derived plasma in patients with non-small cell lung cancer (NSCLC).

The “EasyPGX® ready EGFR” kit is validated for the use in combination with the following instrument:

▪ EasyPGX® qPCR instrument 96 - Diatech Pharmacogenetics (96-well plate) and the equivalent “AriaDx Real-time PCR
instrument” - Agilent Technologies

and accessories:
▪ EasyPGX® Analysis software - Diatech Pharmacogenetics

The device is not automated.

The device is intended only for laboratory professional use.

List of detectable mutations:

LEGACY GENOMIC
TRANSCRIPT EX HGVS AMINO ACID HGVS NUCLEOTIDE
MUTATION ID MUTATION ID
LRG_304 t1 18 COSM6252 COSV51767289 p.Gly719Ser c.2155G>A
LRG_304 t1 18 COSM6253 COSV51766606 p.Gly719Cys c.2155G>T
LRG_304 t1 18 COSM6239 COSV51769339 p.Gly719Ala c.2156G>C
LRG_304 t1 18 COSM20848 COSV51771914 p.Gly719AlafsTer29** c.2156del

LRG_304 t1 19 COSM26443 COSV51783839 p.I740_K745dup** c.2217_2234dup

LRG_304 t1 19 COSM255152 COSV51807771 p.K745_E746insTPVAIK** c.2234_2235insAACTCCCGTCGCTATCAA
LRG_304 t1 19 COSM9869973 COSV105110148 p.E746Ifs*16** c.2235_2236del
LRG_304 t1 19 COSM4386694 COSV51834173 p.I740_K745dup** c.2218_2235dup
LRG_304 t1 19 COSM18420 COSV51840492 p.E746del** c.2235_2237del
LRG_304 t1 19 COSM28517 COSV51769556 p.E746_E749del c.2235_2246del
LRG_304 t1 19 COSM13550 COSV51817953 p.E746_A750delinsIP** c.2235_2248delinsAATTC
LRG_304 t1 19 COSM6223 COSV51765119 p.E746_A750del c.2235_2249del
LRG_304 t1 19 COSM13552 COSV51782151 p.E746_T751delinsIP** c.2235_2251delinsAATTC
LRG_304 t1 19 COSM6506513 COSV51827045 p.E746_T751delinsFPS** c.2235_2251delinsATTCCCGT
LRG_304 t1 19 COSM13549 COSV51860456 p.E746_T751delinsA** c.2235_2251delinsAG
LRG_304 t1 19 COSM24869 COSV51810835 p.E746_T751del** c.2235_2252del
LRG_304 t1 19 COSM13551 COSV51850034 p.E746_T751delinsI c.2235_2252delinsAAT
LRG_304 t1 19 COSM12385 COSV51784563 p.E746_S752delinsI** c.2235_2255delinsAAT
LRG_304 t1 19 COSM26444 COSV51786996 p.K745_E746insVPVAIK** c.2219_2236dup
LRG_304 t1 19 COSM6225 COSV51765066 p.E746_A750del c.2236_2250del
LRG_304 t1 19 COSM26513 COSV51811932 p.E746_T751delinsS** c.2236_2251delinsT
LRG_304 t1 19 COSM12728 COSV51811139 p.E746_T751del c.2236_2253del
LRG_304 t1 19 COSM51497 COSV51829458 p.E746V** c.2237_2238inv
LRG_304 t1 19 COSM144207 COSV51777590 p.E746_A750delinsAP** c.2237_2248delinsCAC
LRG_304 t1 19 COSM6980200 COSV51766481 p.E746_T751delinsVS** c.2237_2251delinsTTT
LRG_304 t1 19 COSM12678 COSV51769364 p.E746_T751delinsA c.2237_2251del
LRG_304 t1 19 COSM6924852 COSV51779850 p.E746_T751delinsAPS** c.2237_2251delinsCACCAT
LRG_304 t1 19 COSM18421 COSV51859776 p.E746_T751delinsVP** c.2237_2251delinsTTC
LRG_304 t1 19 COSM12416 COSV51771891 p.E746_T751delinsVA** c.2237_2253delinsTTGCT
LRG_304 t1 19 COSM52935 COSV51826919 p.E746_T751delinsVP** c.2237_2253delinsTTCCT

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 LEGACY GENOMIC
TRANSCRIPT EX HGVS AMINO ACID HGVS NUCLEOTIDE
MUTATION ID MUTATION ID
LRG_304 t1 19 COSM12367 COSV51769704 p.E746_S752delinsA c.2237_2254del
LRG_304 t1 19 COSM12384 COSV51765862 p.E746_S752delinsV c.2237_2255delinsT
LRG_304 t1 19 COSM51524 COSV51793797 p.E746_P753delinsVQ** c.2237_2258delinsTTCA
LRG_304 t1 19 COSM6978341 COSV51857882 p.L747Qfs*16** c.2238_2247del
LRG_304 t1 19 COSM12422 COSV51782279 p.L747_A750delinsP c.2238_2248delinsGC
LRG_304 t1 19 COSM18428 COSV51868232 p.E746_A750delinsDP** c.2238_2248delinsTC
LRG_304 t1 19 COSM22944 COSV51803491 p.L747_T751delinsP** c.2238_2251delinsGC
LRG_304 t1 19 COSM12419 COSV51863059 p.L747_T751delinsQ c.2238_2252delinsGCA
LRG_304 t1 19 COSM6220 COSV51772418 p.E746_S752delinsD c.2238_2255del
LRG_304 t1 19 COSM12421 COSV51859811 p.L747_S752delinsQH** c.2238_2255delinsGCAACA
LRG_304 t1 19 COSM26441 COSV51838924 p.L747_S752delinsQ** c.2238_2256delinsGCAA
LRG_304 t1 19 COSM24267 COSV51782925 p.L747P** c.2239_2240delinsCC
LRG_304 t1 19 COSM6218 COSV51780076 p.L747_E749del c.2239_2247del
LRG_304 t1 19 COSM12382 COSV51765099 p.L747_A750delinsP c.2239_2248delinsC
LRG_304 t1 19 COSM4170220 COSV51852099 p.L747_A750delinsP** c.2239_2250delinsCCG
LRG_304 t1 19 COSM12383 COSV51765856 p.L747_T751delinsP c.2239_2251delinsC
LRG_304 t1 19 COSM12420 COSV51800510 p.L747_T751delinsQ** c.2239_2252delinsCA
LRG_304 t1 19 COSM23572 COSV51775848 p.L747_T751delinsA** c.2239_2253delinsGCT
LRG_304 t1 19 COSM133196 COSV51858239 p.L747_S752delinsQH** c.2239_2255delinsCAACA
LRG_304 t1 19 COSM6255 COSV51767308 p.L747_S752del c.2239_2256del
LRG_304 t1 19 COSM12403 COSV51778874 p.L747_S752delinsQ** c.2239_2256delinsCAA
LRG_304 t1 19 COSM12387 COSV51785746 p.L747_P753delinsQ c.2239_2258delinsCA
LRG_304 t1 19 COSM51501 COSV51847828 p.L747_P753delinsQ** c.2239_2259delinsCAA
LRG_304 t1 19 COSM1667023 COSV51835919 p.L747_K754delinsANKG** c.2239_2261delinsGCCAACAAGGG
LRG_304 t1 19 COSM24970 COSV51774751 p.L747_K754del** c.2239_2262del
LRG_304 t1 19 COSM10001753 COSV105864664 p.L747Qfs*7** c.2239_2264del
LRG_304 t1 19 COSM85891 COSV51778643 p.L747_A755delinsAN** c.2239_2264delinsGCCAA
LRG_304 t1 19 COSM26704 COSV51772009 p.L747S** c.2240T>C
LRG_304 t1 19 COSM4170221 COSV51810296 p.L747_A750delinsS** c.2240_2248del
LRG_304 t1 19 COSM6210 COSV51768180 p.L747_T751delinsS c.2240_2251del
LRG_304 t1 19 COSM12369 COSV51766247 p.L747_T751del c.2240_2254del
LRG_304 t1 19 COSM12370 COSV51767961 p.L747_P753delinsS c.2240_2257del
LRG_304 t1 19 COSM20883 COSV51820251 p.L747_K754delinsST** c.2240_2261delinsCGAC
LRG_304 t1 19 COSM6933365 COSV51834473 p.L747_A755delinsSMS** c.2240_2263delinsCGATGT
LRG_304 t1 19 COSM1667026 COSV51813655 p.L747_A755delinsSKG** c.2240_2264delinsCGAAAGG

LRG_304 t1 20 COSM6240 COSV51765492 p.Thr790Met c.2369C>T

LRG_304 t1 20 COSM6241 COSV51768106 p.Ser768Ile c.2303G>T
LRG_304 t1 20 COSM20884 COSV51850427 p.A767_V769dup c.2303_2304insTGTGGCCAG
LRG_304 t1 20 COSM12376 COSV51766549 p.A767_V769dup c.2300_2308dup
LRG_304 t1 20 COSM13558 COSV51775806 p.A767_V769dup c.2309_2310delinsCCAGCGTGGAT
LRG_304 t1 20 COSM12737 COSV51768016 p.D770_N771delinsAGG** c.2309_2312delinsCTGGTGG
LRG_304 t1 20 COSM12378 COSV51769298 p.D770_N771insG c.2310_2311insGGT
LRG_304 t1 20 COSM53189 COSV51822959 p.N771delinsGY** c.2311delinsGGTT

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 LEGACY GENOMIC
TRANSCRIPT EX HGVS AMINO ACID HGVS NUCLEOTIDE
MUTATION ID MUTATION ID
LRG_304 t1 20 COSM13428 COSV51774688 p.S768_D770dup c.2303_2311dup
LRG_304 t1 20 COSM1651743 COSV51790591 p.D770_N771insSVE** c.2311_2312insGCGTCGAAA
LRG_304 t1 20 COSM24434 COSV51828355 p.N771delinsSH** c.2311_2312insGTC
LRG_304 t1 20 COSM1651744 COSV51841320 p.N771delinsSGH** c.2311_2312insGTGGCC
LRG_304 t1 20 COSM13554 COSV51781121 p.N771_P772delinsSVDNR** c.2312_2315delinsGCGTGGACAACCG
LRG_304 t1 20 COSM12377 COSV51781591 p.H773dup c.2317_2319dup
LRG_304 t1 20 COSM12380 COSV51810066 p.P772_H773dup c.2314_2319dup
LRG_304 t1 20 COSM18432 COSV51787185 p.H773_V774dup c.2316_2321dup
LRG_304 t1 20 COSM22948 COSV51771118 p.H773_V774dup c.2317_2322dup

LRG_304 t1 21 COSM6224 COSV51765161 p.Leu858Arg c.2573T>G

LRG_304 t1 21 COSM133630 COSV51767322 p.Leu858Arg** c.2573_2574delinsGA
LRG_304 t1 21 COSM12429 COSV51804843 p.Leu858Arg** c.2573_2574delinsGT
LRG_304 t1 21 COSM6213 COSV51766344 p.Leu861Gln c.2582T>A

** mutation coverage determined by in silico analysis.

PRINCIPLE OF THE ASSAY

The “EasyPGX® ready EGFR” kit is delivered in 8-well strips preloaded with a complete amplification mix in a dry, room temperature
(+2/+25°C) stable format and it contains reagents for DNA extraction from formalin-fixed paraffin-embedded (FFPE) samples.

The “EasyPGX® ready EGFR” kit is designed to selectively amplify mutant specific sequences in samples that contain a mixture of
wild-type and mutated DNA. The detection is achieved using fluorescent probes labelled with FAM and HEX.

The “EasyPGX® ready EGFR” kit is composed of seven assays for the detection of the EGFR mutations and a control assay for the
assessment of DNA content in the sample.
Each assay contains primers and probes for the detection of the target (FAM) as well as an endogenous control gene (HEX).
The amplification of the endogenous control gene enables to verify the amplification procedure and the possible presence of
inhibitors, which may cause false negative results.

1. EGFR G719x: the assay detects the mutations of codon 719 but does not distinguish between them
2. EGFR T790M: the assay detects the T790M mutation
3. EGFR S768I: the assay detects the S768I mutation
4. EGFR ex20ins: the assay detects mutations/insertions of the exon 20 of EGFR but does not distinguish between them
5. EGFR L858R: the assay detects the L858R mutation
6. EGFR L861Q: the assay detects the L861Q mutation
7. EGFR ex19del: the assay detects the most frequent deletions of the exon 19 of EGFR but does not distinguish between them
8. EGFR ctrl: the assay detects a region of EGFR without any known polymorphism/mutation

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SCIENTIFIC VALIDITY

Lung cancer is the second most commonly diagnosed cancer and the leading cause of global cancer mortality, accounting for an
estimated 2 million diagnoses and 1.8 million deaths. Non small cell lung cancer (NSCLC) represents almost 85% of all lung cancer
cases and it can be further divided into 3 major histological subtypes: non-squamous (70%), squamous (25%) and not otherwise
specified (5%). Lung cancer is a heterogeneous neoplasm characterized by different clonal sub-populations that have different
molecular characteristics.

Increased understanding of disease etiology over the last twenty years has led to the development of new therapies directed against
specific biomarkers, starting the era of precision medicine. The most frequent genetic alterations in NSCLC are associated with the
epidermal growth factor (EGF) signaling pathway, which contains genes such as EGFR, K-RAS and B-RAF. Mutations in these
genes are all potential targets for cancer therapies and current guidelines recommend mutation testing and identification of EGFR,
K-RAS and B-RAF mutations in all patients with a de-novo diagnosis of advanced lung carcinoma.

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor with tyrosine kinase activity that transduces growth
factor signals from the extracellular environment to the cell. EGFR mutations are the most common druggable genetic alterations in
lung adenocarcinomas. EGFR exon 19 deletions and the L858R point mutation comprise almost 85% of all EGFR somatic mutations
and they can be used to predict response to EGFR tyrosine kinase inhibitors (TKIs). These mutations confer sensitivity to
first- (gefitinib, erlotinib), second- (afatinib, dacomitinib) and third-generation (osimertinib) EGFR TKIs. Osimertinib is a second-line
tyrosine kinase inhibitor that has been approved for relapsed patients with non-small cell lung cancer with the EGFR resistance
mutation T790M.

Other EGFR mutations are termed uncommon EGFR mutations, of which L861Q and exon 20 insertions are the most frequent. In
particular the L861Q point mutation results in sensitivity to first- and second-generation TKIs; whereas exon 20 insertions confer
resistance to clinically approved TKIs. Recently a clinical study has reported the efficacy of amivantamab for the treatment of
patients with EGFR Exon 20 insertion positive NSCLC. Amivantamab is a bispecific antibody for EGFR and MET with immune
cell-directing activity that binds to each receptor's extracellular domain overcoming resistance to TKI. EMA has granted a conditional
marketing authorization for amivantamab in non-small-cell lung cancer (NSCLC) patients with an activating EGFR exon 20 insertion
mutation after the failure of platinum-based chemotherapy.

References
▪ International Agency for Research on Cancer. Global Cancer Observatory: cancer today. World Health Organization.
https://gco. iarc.fr/today (accessed Jan 19, 2020).
▪ Thai AA, Solomon BJ, Sequist LV, et al. Lung cancer. Lancet. 2021 Aug 7;398(10299):535-554.
▪ Zhang J, Fujimoto J, Zhang J et al. Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion
sequencing. Science. 2014 Oct 10;346(6206):256-9.
▪ da Cunha Santos G, Shepherd FA, Tsao MS. EGFR mutations and lung cancer. Annu Rev Pathol. 2011;6:49-69.
▪ Takeda M, Nakagawa K. First- and Second-Generation EGFR-TKIs Are All Replaced to Osimertinib in Chemo-Naive EGFR
Mutation-Positive Non-Small Cell Lung Cancer? Int J Mol Sci. 2019 Jan 3;20(1):146.
▪ Zhang T, Wan B, Zhao Y et al. Treatment of uncommon EGFR mutations in non-small cell lung cancer: new evidence and
treatment. Transl Lung Cancer Res. 2019 Jun;8(3):302-316.
▪ Park K, Haura EB, Leighl NB et al. Amivantamab in EGFR Exon 20 Insertion-Mutated Non-Small-Cell Lung Cancer Progressing
on Platinum Chemotherapy: Initial Results From the CHRYSALIS Phase I Study. J Clin Oncol. 2021 Oct 20;39(30):3391-3402.
▪ Kalemkerian GP, Narula N, Kennedy EB, et al. Molecular Testing Guideline for the Selection of Patients With Lung Cancer for
Treatment With Targeted Tyrosine Kinase Inhibitors: American Society of Clinical Oncology Endorsement of the College of
American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology Clinical
Practice Guideline Update. J Clin Oncol. 2018 Mar 20;36(9):911-919.
▪ Lindeman NI, Cagle PT, Aisner DL, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for
Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International
Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn. 2018 Mar;20(2):129-159.
▪ Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK
tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung
Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med. 2013 Jun;137(6):828-60.
▪ Planchard D, Popat S, Kerr K, et al. on behalf of the ESMO Guidelines Committee. Metastatic non-small cell lung cancer:
ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Originally published in 2018 Ann Oncol (2018)
29(Suppl 4): iv192-iv237. Updated version published 15 September 2020 by the ESMO Guidelines Committee
▪ NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Non-Small Cell Lung Cancer. Version 2.2021 — December
15, 2020.
▪ AIOM. Linee guida – NEOPLASIE DEL POMONE. 30 Ottobre 2020
▪ Gruppo di Lavoro di AIOM e SIAPEC-IAP. Raccomandazioni AIOM e SIAPEC per l’analisi mutazionale del gene EGFR nel
carcinoma polmonare. Aggiornamento Marzo 2014.
▪ Paez JG, Jänne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.
Science. 2004 Jun 4;304(5676):1497-500.
▪ Lynch TJ, Bell DW, Sordella R et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of
non-small-cell lung cancer to gefitinib. N Engl J Med. 2004 May 20;350(21):2129-39.

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▪ Hirsch FR, Varella-Garcia M, Bunn PA Jr et al. Molecular predictors of outcome with gefitinib in a phase III placebo-controlled
study in advanced non-small-cell lung cancer (ISEL). J Clin Oncol. 2006 Nov 1;24(31):5034-42.
▪ Vyse S, Huang PH. Targeting EGFR exon 20 insertion mutations in non-small cell lung cancer. Signal Transduct Target Ther.
2019 8;4:5.
▪ Kosaka T, Yatabe Y, Endoh H et al. Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell
lung cancer and acquired resistance to gefitinib. Clin Cancer Res. 2006 Oct 1;12(19):5764-9.
▪ Mok TS, Wu YL, Thongprasert S et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma (IPASS). N Engl J Med.
2009 Sep 3;361(10):947-57.
▪ Han JY, Park K, Kim SW, et al. First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-
smokers with adenocarcinoma of the lung. J Clin Oncol. 2012 Apr 1;30(10):1122-8.
▪ Mitsudomi T, Morita S, Yatabe Y, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer
harbouring mutations of the epidermal growth factor receptor (WJTOG3405). Lancet Oncol. 2010 Feb;11(2):121-8.
▪ Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-
positive non-small-cell lung cancer (OPTIMAL, CTONG-0802). Lancet Oncol. 2011 Aug;12(8):735-42.
▪ Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients
with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC). Lancet Oncol. 2012 Mar;13(3):239-46.
▪ Yang JC, Wu YL, Schuler M, et al. Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung
adenocarcinoma (LUX-Lung 3 and LUX-Lung 6). Lancet Oncol. 2015 Feb;16(2):141-51.
▪ Su PL, Chen CW, Wu YL et al. First-line treatment with irreversible tyrosine kinase inhibitors associated with longer OS in
EGFR mutation-positive non-small cell lung cancer. Thorac Cancer. 2021 Feb;12(3):287-296.
▪ Wu YL, Cheng Y, Zhou X, et al. Dacomitinib versus gefitinib as firstline treatment for patients with EGFR-mutation-positive non-
smallcell lung cancer (ARCHER 1050). Lancet Oncol. 2017;18(11):1454–1466.
▪ Mok TS, Cheng Y, Zhou X, et al. Improvement in Overall Survival in a Randomized Study That Compared Dacomitinib With
Gefitinib in Patients With Advanced Non-Small-Cell Lung Cancer and EGFR-Activating Mutations (ARCHER 1050). J Clin
Oncol. 2018 Aug 1;36(22):2244-2250.
▪ Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in
155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013 Apr 15;19(8):2240-7.
▪ Mok TS, Wu Y-L, Ahn M-J, et al. Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer.(AURA3) N Engl J
Med. 2017 Feb 16;376(7):629-640.
▪ Ramalingam SS, Vansteenkiste J, Planchard D et al. Overall Survival with Osimertinib in Untreated, EGFR-Mutated Advanced
NSCLC (FLAURA). N Engl J Med. 2020 Jan 2;382(1):41-50.

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KIT CONTENTS

The “EasyPGX® ready EGFR” kit is delivered in 8-well strips preloaded with a complete amplification mix in a dry, room temperature
(+2/+25°C) stable format and it contains reagents for DNA extraction from formalin-fixed paraffin-embedded (FFPE) samples.
The kit contains sufficient reagents to carry out 48 tests.

Destination EXTRACTION AREA

Storage temperature +2/+25°C
EasyPGX® Extraction reagents
Active ingredient Description Safety Symbol
and Warning
EasyPGX Dep solution 1 x 6 ml Mixture of liquid Solution for deparaffinization of N/A
hydrocarbons formalin-fixed paraffin-embedded
(FFPE) samples
EasyPGX Buffer 1 x 6 ml EDTA Buffer for DNA extraction from N/A
Tween 20 formalin-fixed paraffin-embedded
Tris Buffer (FFPE) samples
EasyPGX Enzyme 1 x 600 µl 1 - < 3% Proteinase K Enzyme for DNA extraction from N/A
50 - < 80% Glycerol formalin-fixed paraffin-embedded
(FFPE) samples
Destination AMPLIFICATION AREA
Storage temperature +2/+25°C
Active ingredient Description Safety Symbol
and Warning
EasyPGX EGFR strips 4 x 12 strips Taq Polimerase WHITE 8-well strips: 8 dry N/A
Reaction Buffer complete mixtures containing
dNTPs specific primers and probes
MgCl2 targeting the following EGFR
Excipients mutations and the internal control:
< 2 % Upstream and Position 1: G719x
downstream EGFR Position 2: T790M
primers Position 3: S768I
< 0.1 % Fluorescent Position 4: exon 20
labelled EGFR probes Position 5: L858R
Position 6: L861Q
Position 7: exon 19
Position 8: control region
For the list of the detectable
mutation refer to “Intended Use”
8-strip flat optical caps 4 x 12 strips \ 0.2 ml 8-tube cap strips DNase-, N/A
RNase-free to be used to recap the
EasyPGX EGFR strips
EasyPGX EGFR pos ctrl * 5 tubes < 0.5 % Synthetic DNA DNA positive control in a dry format N/A
containing EGFR exon containing a mixture of synthetic
18, 19, 20 and 21 DNA sequences that corresponds
sequences to each mutation detected by this
< 3.5 % h-DNA kit in a background of wild-type
placenta genomic DNA
Excipients
WATER ** 8 x 1.5 ml \ DNase-, RNase-free water N/A

* Every aliquot must be resuspended with 700 µl of WATER before the use.
** 2 aliquots to be used exclusively to resuspend the dry positive controls, 2 aliquots to be used exclusively as negative control in the
PCR reaction and 4 aliquots to be used exclusively as samples diluent.

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DOCUMENTS AVAILABLE ON-LINE

The following documents are available at www.diatechpharmacogenetics.com/area-riservata:

▪ “EasyPGX® ready EGFR” - User Manual
▪ Template for EasyPGX® qPCR instrument 96: (xxxx indicates current version):
“RT800-96_RT023_template_xxxx.adxt”
▪ “EasyPGX® Analysis Software” (code RT800-SW) - User Manual
▪ “EasyPGX® qPCR instrument 96” (code RT800-96) - User Manual
▪ Safety Data Sheets (SDSs)

The following documents are available at www.agilent.com:

▪ “AriaDx Real-time PCR Instrument” – Agilent Technologies - User Manual
For further details please contact the Diatech Pharmacogenetics technical support:
email: support@diatechpharmacogenetics.com, tel. +39 0731 213243

MATERIALS REQUIRED BUT NOT PROVIDED

Genomic DNA extraction

The “EasyPGX® ready EGFR” kit contains reagents for DNA extraction from formalin-fixed paraffin-embedded (FFPE) samples.
Required accessories:
▪ EasyPGX® dry block (code RT801, Diatech Pharmacogenetics) or equivalent thermo-shaker compatible with 1.5 ml tubes
– Temperature 56 °C - 95 °C, mixing speed 1400 rpm.
▪ EasyPGX® centrifuge/vortex 1.5 ml (code RT802, Diatech Pharmacogenetics) or equivalent centrifuge/spinner and vortex
compatible with 1.5 ml tubes.
Other recommended options for DNA extraction and purification:
a. Tissue:
▪ “QIAamp DNA FFPE Tissue kit” (code 56404, Qiagen)
▪ “QIAamp DNA Mini kit” (code 51304, Qiagen)
▪ “Genomic DNA FFPE One-Step Kit” (code MGF-03, RBC); to be used with MagCore Automated Nucleic Acid Extractor
automatic systems (RBC Bioscience)
▪ “Genomic DNA Tissue Kit” (code MGT-02, RBC); to be used with MagCore Automated Nucleic Acid Extractor automatic
systems (RBC Bioscience)
▪ In case you are using FFPE tissues, you will also need:
o Xilene (e.g:. “Xylenes, histological grade” – code 534056, Sigma Aldrich)
o Absolute Ethanol (quality of analytical degree)
b. Plasma:
▪ “Helix Circulating Nucleic Acid” (code H8040, Diatech Pharmacogenetics)
▪ “QIAamp® Circulating Nucleic Acid” (code 55114, Qiagen)

For each of the above options, DNA extraction and purification shall be done following the related user manual indications and
prescriptions.
In case you employ kits which are different from those recommended, it is the user's responsibility to use standardized samples
(e.g: VEQ – EQAS quality schemes, Horizon Diagnostics samples) to verify that this does not imply a reduction of the performance
of the system under analysis.

Amplification
Real-Time PCR instrument:
▪ EasyPGX® qPCR instrument 96 code RT800-96, Diatech Pharmacogenetics (Agilent Aria Software v1.4).
Detection channels for FAM and HEX fluorescence. Range of environmental temperature: 20-30°C
Required accessory:
▪ EasyPGX® centrifuge/vortex 8-well strips (code RT803, Diatech Pharmacogenetics) or equivalent centrifuge/spinner and
vortex compatible with 8-well strips
Materials:
▪ 1.5 ml polypropylene twist-lock tubes (DNase-, RNase-, DNA-, PCR inhibitor-free)
▪ Micropipettes (volumes from 1 to 1.000 µl)
▪ Sterile filter tips DNase-, RNase-free (volumes from 1 to 1.000 µl)
▪ Powder-free disposable gloves

Creation of sample grid and data analysis

▪ EasyPGX® Analysis Software version 4.0.12 or above (code RT800-SW, Diatech Pharmacogenetics)
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STABILITY AND STORAGE

Store all the reagents according to the instructions on the packages, in particular:

Store all the reagents at +2/+25°C in the original package. If in the storage environment there isn’t a temperature data-logger
for temperature monitoring, it is recommended to store all the reagents at +2/+8°C.

EasyPGX® Extraction reagents

▪ After the first use, store the EasyPGX Enzyme at +2/+8°C and use it within the expiration date.

EasyPGX® Amplification reagents

▪ Once a EasyPGX EGFR strips package is opened, store it at +2/+8°C and use the contained strips within 2 months and
within the expiration date.
▪ Once resuspended, store the EasyPGX EGFR pos ctrl at -20/-35°C and use it within the expiration date. Avoid thawing
and re-freezing more than four times, as this could lead to poor performance.
▪ Protect all the dry mixes from light to avoid degradation of the fluorescent dyes.
▪ If properly stored, the reagents remain stable until the expiration date displayed on the individual label.

SYMBOLS

Catalogue number (product code) Positive control

Global Trade Item Number Negative control

Batch code Consult the instruction for use

Content sufficient for <n> tests User manual (handbook)

For in vitro diagnostic use Use by date

Contents Temperature limits

Components Manifacturer

Number of aliquots Important Note

Quantity per aliquot Storage temperature

Caution Keep away from sunlight

Keep dry

The product fulfils the requirements of the Directive 98/79/EC on in vitro diagnostic (IVD) medical
devices, transposed in Italy in the D.Lgs No 332/2000 and subsequent legislative changes

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PRODUCT USE LIMITATIONS

▪ The “EasyPGX® ready EGFR” kit can only be used by specialized personnel, properly instructed and trained for Real-time PCR
technology and molecular biology.
▪ It is necessary to operate in compliance with the general guidelines of Good Laboratory Practice (GLP) and the instructions
contained in this manual.
▪ Do not use expired or incorrectly stored reagents.
▪ The “EasyPGX® ready EGFR” kit has been designed and validated for the use with the real-time qPCR instrument EasyPGX®
qPCR instrument 96 (code RT800-96) and with the accessory EasyPGX® analysis software (code RT800-SW). All these
items are manufactured and put on the market by Diatech Pharmacogenetics.
▪ The “EasyPGX® ready EGFR” is a qualitative test. The test is not for quantitative measurements of percent mutation.
▪ Diatech Pharmacogenetics can’t respond of results obtained using instruments or accessories other than those recommended
in this user manual.
▪ The reliability of the results also depends on the procedures carried out in the pre-amplification stages, including the selection of
starting biological specimens, the preservation of the samples and the DNA extraction.
▪ Samples called “wild-type” by EasyPGX® analysis software may harbor EGFR mutations not detectable by the kit. Detectable
mutations are listed in the section “Intended use”.
▪ Samples called “wild-type” by EasyPGX® analysis software may harbor mutations with an allele frequency below the Limit of
Detection (LoD) of the assays. LoD of each assay is reported in the section “Performance validation”.
▪ In presence of fluorescence artifacts, a sample may be improperly called “mutated” by EasyPGX® Analysis software. To
confirm mutations it is recommended to inspect the amplification curves. The fluorescence signal must come from a real
amplification reaction (sigmoidal curve), and not from an artifact (linear curve).
▪ All assays in the “EasyPGX® ready EGFR” kit amplify short DNA sequences. However, heavily fragmented DNA can generate
no amplification product.
▪ Any diagnostic results generated by this procedure must be interpreted with reference to other clinical or laboratory findings.
▪ The “EasyPGX® ready EGFR” kit is covered by the CE Mark in compliance with the European directive 98/79/EC on the in
vitro diagnostic (IVD) medical devices, only in those countries that accept the user manual translated in the languages available
on the website www.diatechpharmacogenetics.com/area-riservata.

QUALITY ASSURANCE

▪ The “EasyPGX® ready EGFR” kit has been designed, developed and validated in compliance with the Directive 98/79/EC on in
vitro diagnostic (IVD) medical devices, transposed in Italy in the D.Lgs No 332/2000 and subsequent legislative changes, and in
accordance with the procedures of the Company’s Quality System certified for conformance to the European regulatory
standards EN ISO 9001 and ISO 13485.
▪ The consistent quality of the “EasyPGX® ready EGFR” kit is guaranteed by the application of a tight process control on
materials and on operative procedures for product realization and its management till the Customer. The quality of each lot is
attested in the related Certificate of Analysis available upon request to the Customer Service
(support@diatechpharmacogenetics.com).

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WARNINGS AND PRECAUTIONS

1. The kit may only be used by specialist personnel, properly instructed and trained to perform in vitro laboratory techniques.
2. Carefully read this User Manual.
3. Check that the version of the User Manual in use corresponds to the one described on the “EasyPGX® ready EGFR” kit
box label.
4. Handle all samples as potentially infectious material inside a laminar flow hood (class II biological safety cabinet or higher).
5. Follow the laboratory safety procedures described in “Biosafety in Microbiological and Biomedical Laboratories” (Richmond,
JY and McKinney, RW (eds) - 5th edition (2009) and in the NCCLS (National Committee for Clinical Laboratory Standards)
Document M29-T. Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids and
Tissue. Tentative guidelines. – Villanova, PA:NCCLS, 1989).
6. Do not eat, drink or smoke in the laboratory. When handling biological samples, disposable gloves, gowns and goggles or
face masks should be worn to protect against biological agents.
7. Constantly check that the gloves are free from contamination by the biological material being treated. If not, replace them
immediately to avoid the possibility of cross-contamination between samples and contamination of the workplace. Wash
hands thoroughly after handling samples and reagents.
8. The Safety Data Sheet (SDS) is available in the reserved area of the web-site Diatech Pharmacogenetics
www.diatechpharmacogenetics.com, or can be requested to the the Diatech Pharmacogenetics technical support
support@diatechpharmacogenetics.com.
9. Perform the procedure in accordance with Good Laboratory Practice (GLP) general guidelines.
10. It is recommended to ensure that the laboratory work flow proceeds in a unidirectional manner, setting up two separate
working areas for:
o extraction of nucleic acids
o amplification reaction
11. Organize the laboratory so that dedicated pipettes, tips and materials are used for each activity.
12. Use sterile filter tips. Avoid aerosols.
13. Use tubes with twist-lock caps during the extraction of nucleic acids in order to avoid the leakage of the samples and
potential contamination.
14. During the procedures for nucleic acid extraction and amplification, avoid contamination of reagents with airborne microbes
by opening the reagents only within the hood.
15. Change the pipette tip before each pick up of reagents and every time you move from one sample to another in any stage
of the procedure.
16. The precision pipettes used should have an accuracy of within 3% of the set volume.
17. Periodically check the calibration status of the dispensing instruments.
18. Do not use reagents after the expiration date shown on each container.
19. All reagents supplied in the “EasyPGX® ready EGFR” kit are intended to be used solely with the other reagents included in
the same kit. Do not substitute or mix reagents from different batches, in order to maintain optimal performance.
20. Discard unused reagents and the expired kit and waste in accordance with current national laws and local regulations.
21. Extraction area: at the end of the procedure, decontaminate the pipettes and the laboratory surfaces on which work has
been carried out, by cleaning with appropriate products (e.g. FD 322, Dürr Dental, Germany) and UV irradiate the work
surface of the biological cabinet where the pipettes should be carefully placed after decontamination.
22. Amplification area: at the end of the procedure, decontaminate the pipettes and the laboratory surfaces on which work has
been carried out, by cleaning with appropriate products to eliminate nucleic acids and amplicons (e.g. “DNA Cleaner” -
code DC001, Diatech Pharmacogenetics) and subsequent UV irradiation, if available.
23. Avoid contamination of samples and reagents.
24. Store reagents and samples separately.
25. In order to avoid possible contamination from carry-over, do not open the reaction tubes after amplification.
26. Before use all reagents need to be mixed by inverting 10 times and centrifuged briefly.
27. All reagents contained in the kit are ready-to-use and don’t need to be diluted. The reagent dilution may result in a loss of
performance.
28. Include in each run at least 1 negative control (WATER) and 1 positive control (EasyPGX EGFR pos ctrl).
29. In order to avoid any mixing up of samples pay particular attention to samples dispensation, placement of strips into the
instrument, editing the sample name in the software.
30. The right to contest the kit before the expiration date becomes void if the product is used in violation of GLP guidelines and
the manufacturer’s recommendations.
31. The registered names and trademarks indicated in this document are to be considered protected by law, even when not
explicitly stated.

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ANALYTICAL PROCEDURE

DNA EXTRACTION

Perform this step in the area dedicated to DNA isolation and dilution, using dedicated materials and instruments.

For FFPE samples consider the following points:

▪ The good quality of the extracted DNA strictly depends on the conditions of paraffin-embedded tissue. In particular, 4-10% of
formalin fixation and a fixation period not longer than 14-24 hours are recommended.
▪ When cutting sections from paraffin-embedded blocks, the microtome's blade and tweezers should be cleaned between each
samples to avoid cross contamination of DNA.
▪ Extract DNA using 5 x 10 μm (or 1-6 x 5-10 μm) FFPE tissue sections. Sample should contain at least 50% of neoplastic cells.
It is not recommended to proceed with the extraction if the specimen contains less than 100 tumor cells as this may cause false
negative results due to the limit of detection (LoD) of the test, or false positive results due to artifacts.
▪ In the presence of a tumor cellularity lower than 50% or for specimens with less than 100 tumor cells, it is recommended to
perform a tissue microdissection.

For plasma samples consider the following points:

▪ Collect whole blood into EDTA-treated tubes and process it within 2-3 hours after sampling.
▪ Centrifuge at 2000xg for 10 minutes at +4°C.
▪ Transfer plasma in a 15ml tube without disturbing the leukocyte annular layer. To avoid cellular contamination aspirate plasma
from the region up to 5mm above the leukocyte layer.
▪ From 10ml of whole blood, approximately 4-5ml of plasma can be obtained.
▪ Centrifuge at maximum speed for 10 minutes at +4°C and transfer plasma into a conical tube without disturbing the pellet.
To avoid cellular contamination aspirate plasma from the region up to 7mm above the pellet.
▪ Store plasma at +2/+8°C for up to 24 hours, at 20°C for two weeks or at ≤-70°C for 45 days.

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Use of EasyPGX® Extraction reagents
▪ The EasyPGX® Extraction reagents, included in the kit, are intended for DNA extraction from FFPE tissues.
▪ Prepare a 1 x 10 μm (or 2 x 5 μm) thick paraffin-embedded tissue section of a surface area up to 250mm2 and add it into a
1.5 ml twist-lock tube (not provided) using a sterilized tweezer. Alternatively, transfer an equal amount of FFPE sample from an
histological slide into a 1.5 ml microcentrifuge tube (not provided) using a sterilized lancet.
▪ Set the EasyPGX® dry block at 56°C and wait the instrument to reach the temperature.
▪ Gently mix by invertion the EasyPGX Dep solution and add 100 μl into the 1.5 ml twist-lock tube (not provided).
▪ Gently mix by invertion the EasyPGX buffer and add 100 μl into the 1.5 ml twist-lock tube (not provided).
▪ Add 10 μl of EasyPGX Enzyme into the 1.5 ml twist-lock tube (not provided).
▪ Mix thoroughly by vortexing for 10 seconds and inverting the microcentrifuge tube 2-3 times, then centrifuge for 10 seconds
using the EasyPGX® centrifuge/vortex 1.5 ml.
▪ Verify that the material is completely included into the emulsion and incubate at 56°C for 1 hour at 1400 rpm in the EasyPGX®
dry block.
▪ Remove the 1.5 ml twist-lock tube, set the EasyPGX® dry block at 95°C and wait the instrument to reach the temperature.
▪ Mix 10 seconds by vortexing and incubate the 1.5 ml twist-lock tube at 95°C for 10 minutes (without shaking).
▪ Remove the 1.5 ml twist-lock tube and centrifuge it for 10 seconds.
▪ Transfer the lower phase into a new 1.5 ml twist-lock tube (not provided) paying attention to not withdraw the paraffin of the
supernatant and the tissue debris.
▪ Use 2 μl to do a 1:100 dilution with the provided WATER (2 μl extracted DNA + 220 μl WATER, the excess of water doesn’t
affect the final dilution) that will be use as the template for the PCR reaction or store the extracted DNA at -20°C, divided into
aliquots in order to maintain the experimental conditions constant in case of repetition.

Use of the other recommended kits (see “Materials Required but Not Provided”)
▪ The quantity of biological material required for the DNA extraction depends on protocols.
▪ Refer also to the extraction kit manual for selection and treatment of FFPE slides or for pre-processing of plasma samples.
▪ Perform the DNA extraction following the instructions of the extraction kit in use.
▪ If the extraction protocol involves the use of wash buffers containing ethanol, it is advisable to perform a further centrifugation
before final elution to remove any possible traces of ethanol. This will prevent inhibition of the reaction by the ethanol.
▪ After the extraction, proceed immediately with the quali-quantitative evaluation of the DNA and the amplification reaction, or
store the extracted DNA at -20°C, divided into aliquots in order to maintain the experimental conditions constant in case of
repetition.
▪ Just as an indication, for non-paraffin embedded samples like fresh/frozen tissue, plasma, blood, the recommended DNA
amount in each test tube is 5-10 ng; for paraffin embedded samples, the recommended DNA amount in each reaction tube is
15-30 ng.
▪ As absorbance reading cannot distinguish between fragmented and not fragmented DNA and therefore it can overestimate the
concentration of template, the suitability of the extracted DNA should be based on the EGFR ctrl mix (position 8) considering
that the optimal amount of 10-20 ng/reaction corresponds to Cq (Quantification Cycle) values FAM ~ 26 and HEX ~ 27.

INSTRUMENT SETUP

▪ Follow the instructions indicated in the instrument user manual to import the correct template with the following plate setup and
thermal profile:

Plate Setup
o All the 96 positions selected, Well type: “Unknown” and Add Dyes: “FAM” and “HEX”. Click Sync Plate.

Thermal Profile
Step
Hot Start (1 Cycle)
Amplification (40 Cycles)
Temperature/Time
95°C for 5 minutes
95°C for 15 seconds
56°C for 15 seconds
60°C for 45 seconds (Data Collection)

Plate set-up

▪ Use “EasyPGX® Analysis Software” for plate set-up. Refer to the software user manual for detailed procedure.
▪ Enter the number of clinical samples to be tested, to generate the Sample Grid.
▪ Enter unique pseudonymised or, if possible, anonymized sample names and export the Sample Grid.

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AMPLIFICATION AND MUTATION DETECTION
Perform this step in the area dedicated to PCR mixes preparation, using dedicated materials and instruments. Before starting
decontaminate pipettes, benches and hood in order to degrade any trace of DNA and possibly radiate with UV light for at least
30 minutes.
Each sample must be amplified with all the 8 different dry mixes contained in one EasyPGX EGFR strip: EGFR G719x
(position 1), EGFR T790M (position 2), EGFR S768I (position 3), EGFR ex20ins (position 4), EGFR L858R (position 5),
EGFR L861Q (position 6), EGFR ex19del (position 7), EGFR ctrl (position 8).
The number imprinted on the top and the small hole on the bottom of each strip indicate respectively the mix position 1 and the
mix position 8.

Mix 1 2 3 4 5 6 7 8

The kit content is optimized to analyze 10 clinical samples and 2 controls (EasyPGX EGFR pos ctrl and WATER) in each run.

BEFORE TO START:
▪ Centrifuge for 10 seconds the needed number of EasyPGX EGFR strips using the EasyPGX® centrifuge/vortex 8-well strips
considering that each run must include at least one amplification negative control (WATER) and one amplification positive
control (EasyPGX EGFR pos ctrl).
▪ Verify that the dry cakes are on the bottom of each well of the EasyPGX EGFR strips.
▪ Centrifuge for 10 seconds the EasyPGX EGFR pos ctrl and resuspend it by adding 700 μl of the provided WATER. Vortex
carefully for 10 seconds and then centrifuge for 10 seconds (perform this step in the area dedicated to DNA isolation and
dilution, using dedicated materials and instruments). To achieve a complete resuspension of the dry cake, after adding WATER,
store the liquid positive control at room temperature for 30 minutes before use.
▪ Identify uniquely each strip.
▪ Gently remove the seals from the strips paying attention to not get out the dry cakes.
▪ Add to the respective strip:

negative control 25 µl WATER

sample 25 µl DNA
positive control 25 µl of the resuspended
EasyPGX EGFR pos ctrl

It is strongly recommended to use as negative control the WATER provided with the kit and in particular those aliquots to be
used exclusively as negative control in the PCR reaction.

▪ Close carefully all the strips using the 8-strip flat optical caps. Verify that all caps are correctly closed.
▪ Mix by vortexing for 5 seconds holding the bottom of each strip on the side of the small hole.

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▪ Rotate all 8-well strips by 180 degrees and vortex again for 5 seconds holding the opposite side of the strip.
▪ Centrifuge the strips for 10 seconds.
▪ Check that the thermal profile is setted up correctly and start the run.

Before starting the run, check the right 8-well strip orientation on the instrument block: the number imprinted on the strip
must be on the top.

Before starting the run, if the instrument plate is not completely full, balance the plate with 8-well strips on columns 1 and 12.

Proceed with the analysis following the instructions of the section “Data Analysis”.

DATA ANALYSIS
General reccomandations
Data analysis must be performed automatically using the EasyPGX® analysis software version 4.0.12 or above
(code RT800-SW, Diatech Pharmacogenetics).

Refer to the EasyPGX® analysis software user manual for the export of the raw data in excel format and its import in the
analysis software.

▪ Launch EasyPGX® Analysis Software version 4.0.12 or above and import raw data in excel format. Refer to software user
manual for details.
▪ Data analysis with EasyPGX analysis software consists of three steps:

1. Analysis of reaction controls (run validity criteria): positive and negative controls must meet the acceptability criteria. Only if
control reactions fall within the acceptability ranges the analysis can proceed to step 2.
2. Analysis of sample quality (EGFR control assay): each sample must give Cq and ΔR values within the specified
acceptability range of EGFR control mix. The Cq for the control reaction reflects the total amount of amplifiable DNA
template in the sample and assay Cq range is set to ensure that there is sufficient amplifiable DNA to proceed with
analysis, but not so much as to overload the assay. Any samples that do not give Cq values within EGFR control mix range
are invalidated by the EasyPGX analysis software.
3. Determination of sample mutation status: only samples that are suitable for EGFR control assay can be analyzed to assess
the presence of mutation. For each sample must be calculated the difference between FAM Cq value of mutation specific
assay and FAM Cq of EGFR control assay:
ΔCq = [Mutation assay FAM Cq value] – [Control assay FAM Cq value]
Sample is classified as “Mut” if give a ΔCt less than or equal to the cut-off ΔCq value for that assay and a ΔR last more than
or equal to the cut-off ΔR last value for that assay.

▪ The “Summary” tab in the “Results” section allows to view a synthetic visualization of the obtained results for each sample.

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▪ The “Details” tab in the “Results” section allows to view in detail the obtained results for each sample.

In presence of errors, refer to the Troubleshooting section of this user manual.

To confirm the mutation, check that normalized fluorescence at cycle 40 derives from a real amplification reaction
(sigmoidal fluorescence curve) and not from an artifact (linear fluorescence curve).

If a sample fails for one or more assays, to confirm the results, it should be retested with all the mixes.

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TROUBLESHOOTING

Error: E01 - Possible error in the set up of the reaction /run: it is not possible to analyze the samples
Problem Possible reason
Low or absent amplification signal in the Incorrect selection of the fluorescence
channel “FAM” and/or in the channel acquisition channels.
“HEX” for both EasyPGX EGFR pos ctrl Incorrect setting of the thermal profile.
and samples.
Incorrect resuspension/storage of the
EasyPGX EGFR pos ctrl.
Reagents improperly stored or expired. ▪
Amplification signal weak or absent in Degradation of the EasyPGX EGFR
"FAM" and "HEX" only for the positive pos ctrl.
control EasyPGX EGFR pos ctrl. Incorrect or failure dispensation of the
EasyPGX EGFR pos ctrl.
Recommendation
▪ Check the fluorescence acquisition channels and repeat
amplification with the settings described in this manual.
▪ Check the temperature profile and repeat amplification
with the settings described in this manual.
▪ Add 700 μl of the provided WATER to a new aliquot.
Vortex and centrifuge for 10 seconds.
▪ Store the resuspended EasyPGX EGFR pos ctrl at
-35/-20°C and avoid thawing and refreezing more than four
times.
Protect the 8-well strips from light in their original package
with desiccant sachet.
▪ Store the 8-well strips at +2/+25°C.
▪ Once a EasyPGX EGFR PCR strips package is opened,
store it at +2/+8°C and use the contained strips within 2
months and within the expiration date.
▪ Once the dry mixes are resuspended, use immediately the
8-well strips in the PCR reaction.
▪ Do not use expired reagents.
▪ Repeat the amplification resuspending and testing a new
aliquot of EasyPGX EGFR pos ctrl.
▪ Repeat the amplification by pipetting the appropriate
volume of EasyPGX EGFR pos ctrl.

The positive control EasyPGX EGFR Wrong 8-well strip identification. ▪ Repeat the amplification after marking unambiguously the
pos ctrl shows no amplification signal in reaction strips for samples and controls.
the channel "FAM" for the assays of Incorrect dispensing of the samples. ▪ Repeat the amplification paying attention to the
mutations or the signal is detectable only dispensation of the DNA and the EasyPGX EGFR pos ctrl
for some mixes; while one or more in the 8-well reaction strips.
samples show a signal amplification in Incorrect samples names set-up in the ▪ Check samples names set-up.
all assays for mutations. software.
Error: E02 - Possible contamination: it is not possible to analyze the samples
Problem Possible reason Recommendation
The negative control WATER, shows an Contamination. ▪ The results shall be rejected and samples must be
amplification signal in both “FAM” and reamplified using new reagents.
"HEX" channel in one or more that one ▪ Prepare the PCR reaction in a dedicated area. Carefully
assay. decontaminate benches, pipettes and instruments.
Error: E03 - Suboptimal amount of starting template / PCR inhibition
Problem Possible reason Recommendation
Cq values for "FAM" and "HEX" ▪
Insufficient amount of starting DNA and When using EasyPGX® Extraction reagents contained in
channels ouside the acceptability range / or presence of PCR inhibitors. the kit:
for ctrl assay. EasyPGX EGFR pos ctrl 1) if it is assumed that the amount of starting DNA is
is within the expected values. insufficient repeat the amplification diluting the sample
1:50 with WATER or repeat the DNA extraction;
2) if you suspect the presence of inhibitors, repeat the
amplification diluting the sample 1:200 with WATER.
▪ When using other recommended reagents not included in
the kit for DNA extraction and purification:
1) check the quantity and quality of the extracted DNA e.g
with the spectrophotometer and, if not appropriate, repeat
the extraction faithfully following the instructions of the
extraction kit;
2) if the extraction protocol involves the use of washing
buffers containing ethanol, it is advisable to carry out a
further centrifugation prior to final elution to remove any
possible trace of alcohol;
3) if it is assumed that the amount of starting DNA is
insufficient, repeat the DNA extraction by reducing the
volume of elution or the diluition factor;
4) if you suspect the presence of inhibitors, repeat the
amplification diluting the sample 1:5 or 1:10 with WATER.
Error: E04 - Excess of template. Sample must be diluted with WATER so that Cq fall in the ranges indicated
Problem Possible reason Recommendation
Cq values for "FAM" and "HEX" Excess of DNA ▪ Samples must be diluted with WATER so that Cq fall in the
channels ouside the acceptability range ranges indicated in the section analysis of the ctrl mix.
for ctrl assay. EasyPGX EGFR pos ctrl Consider that the dilution 1:2 of the DNA increases the Cq
is within the expected values. of 1 unit.

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Error: E05 - Suboptimal amount of starting template / PCR inhibition / possible dispensing error of the sample
Problem Possible reason Recommendation
ΔCq, ΔR values for channel “HEX” Incorrect or no dispensation of the ▪ Repeat the amplification paying attention to the dispensation
ouside the acceptability range. samples. of the DNA in the 8-well reaction strips.
EasyPGX EGFR pos ctrl is within the
expected values.
One sample shows HEX Cq values Sample dispensation error. ▪ Repeat the amplification dispensing the correct volume of
different from each other for the DNA and including positive and negative controls.
assays.
Error: E06 – Probable excess of DNA. Proceed with the analysis of the samples
Problem Possible reason Recommendation
EasyPGX EGFR pos ctrl shows Cq Incorrect resuspension of the ▪ Add 700 μl of the provided WATER to a new aliquot. Vortex
and/or ΔR values ouside the EasyPGX EGFR pos ctrl. and centrifuge for 10 seconds.
acceptability range. Proceed with the
analysis of the sample. Incorrect dispensation of the EasyPGX ▪ Pipet the appropriate volume of EasyPGX EGFR pos ctrl.
EGFR pos ctrl.
Error E07: Contact the technical support
If the problems persist despite the implementation of the given recommendations and for any further questions or problems, please contact the
Diatech Pharmacogenetics technical support:
▪ e-mail support@diatechpharmacogenetics.com
▪ telephone +39 0731 213243
▪ fax +39 0731 213239

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PERFORMANCE EVALUATION
Performance evaluation has been performed using all the reagents included in the “EasyPGX® ready EGFR” kit.
The experiments have been performed according to the instructions reported in this user manual on the following instruments and
accessories:
▪ EasyPGX® qPCR instrument 96 - Diatech Pharmacogenetics
▪ EasyPGX® Analysis software vers.4.0.12 - Diatech Pharmacogenetics
▪ EasyPGX® dry block - Diatech Pharmacogenetics
▪ EasyPGX® centrifuge/vortex 1.5 ml - Diatech Pharmacogenetics
▪ EasyPGX® centrifuge/vortex 8-well strips - Diatech Pharmacogenetics

ANALYTICAL PERFORMANCE

Measuring range – EGFR control assay ranges

The objective of this study was to set the EGFR control assay range (Cq and ΔR last range) to assess DNA sample validity. The
control assay is used to assess the quality and quantity of DNA that can be amplified.
gDNA extracted from h-placenta or Horizon diagnostic standards with an input ranging from 250 to 0.5 ng/reaction were tested with
EGFR control assay for a total of 301 data points.

Both FAM and HEX Cq values have a non-gaussian distribution, so Cq range has been calculated using a nonparametric method:
Cq(1st percentile) ≤ Cq ≤ Cq(99th percentile)
Both FAM and HEX ΔR last values give a Gaussian distribution so ΔR last range has been defined as:
ΔR last ≥ mean ΔR last – 3 SD

EGFR control assay range has been set to:

Cq FAM ΔR last FAM Cq HEX ΔR last HEX

22 ≤ Cq ≤ 31 ≥ 200 22 ≤ Cq ≤ 32 ≥ 100

To validate the above ranges, 69 different clinical FFPE and plasma samples have been tested with EGFR control assay: 64 of 69
(93%) samples fall within the EGFR control assay defined range.

CUT-OFF definition

ΔCq and ΔR last cut-off values were developed using clinical FFPE and plasma specimens, cell line DNA, human placenta DNA,
Horizon diagnostic DNA standards and plasmidic DNA mixed with wild-type h-DNA.
The cut-offs were chosen, for each assay, to reduce as much as possible false positive and, false negative fraction and to improve
assay sensitivity.
For each assay ΔCq and ΔR last values of wild-type and mutant samples were plotted on a scatterplot to assess their distribution.
For wild-type samples all values from clinical and standard/commercial samples have been included;
Only samples that were within the range of EGFR control assay were included in the analysis.
Cut-offs for each assay and scatterplot graphs are reported below:

Assay FAM Cq ΔCq ΔR

G719x ≤8.5 ≥100
T790M ≤6 ≥400
S768I ≤10 ≥150
ex20ins ≤37 ≤12 \
L858R ≤10 ≥100
L861Q ≤10 ≥100
ex19del ≤9.5 ≥200

Limit of Blank (LoB)

To assess Limit of blank (LoB) the distribution of FAM ΔCq and FAM ΔR last values of wild-type samples has been evaluated.
FAM ΔCq and FAM ΔR last values of both clinical and commercial wild-type samples, suitable in terms of starting DNA amount
(EGFR control mix range), have been considered.
Wild-type samples harbouring mutations different from those detected by the specific assay have been included in the analysis of
the assay as wild-type sample.
Distribution have been evaluated using Shapiro test: for all assays ΔCq values and ΔR last values have a non-Gaussian distribution,
so LoB has been calculated as the upper 95th percentile.
ΔCut-off, LoB values and false positive rate for each assay are summarized below:

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 ΔCut-off LoB
ΔR last ΔR last False positive
Assay Cq FAM ΔCq FAM Cq FAM ΔCq FAM
FAM FAM call rate
\ ≤8.5 ≥100 \ 13.8 81.6 1/111 (0.89%) a
G719x
\ ≤6 ≥400 \ 7.5 521.6493 0/114 (0%)
T790M
\ ≤10 ≥150 \ 40 31.5 0/118 (0%)
S768I
≤37 ≤12 \ 40 12.5 56 0/89 (0%)
ex20ins
\ ≤10 ≥100 \ 40 45 0/179 (0%)
L858R
\ ≤10 ≥100 \ 15.4 91 1/124 (0.8%) b
L861Q
\ ≤9.5 ≥200 \ 11.4 79.2 0/155 (0%)
ex19del

a) One sample genotyped by the comparative method as wild-type, was called by RT023 as G719x. The sample has been tested
two times with RT023 resulting in 1 wt borderline call (ΔCq 8.6, ΔR 283) and 1 mutant G719x borderline call (ΔCq 7.7, ΔR 214).
Probably this sample is a real G719x mutant with a MAF% close to the LoD of the assay. The discrepant result may be due to a
different LoD between RT023 and comparative method.

b) One sample genotyped by the comparative method as wild-type, was called by RT023 as L861Q with a borderline call (ΔCq 7.3,
ΔR 147). The sample has been retested with RT023 resulting in a wt call. Probably this sample is a real L861Q mutant with a MAF%
close to the LoD of the assay. The discrepant result may be due to a different LoD between RT023 and comparative method.

Sensitivity and specificity

Sensitivity and specificity have been calculated according to CLSI EP12-A2 using Horizon diagnostic reference standards and
plasmids, whom genotype has been verified from the suppliers by sanger sequencing. Horizon diagnostic assess MAF% of its
reference standards by digital PCR:
- Mutant Horizon Diagnostic standards with a MAF% ≥LoD
- Plasmidic DNA mixed with wild-type DNA to simulate MAF% ≥LoD
- h-DNA placenta wild-type for EGFR somatic mutations
- Horizon diagnostic EGFR wild-type standard

Sensitivity and specificity have been calculated considering replicates of each sample tested.

Overall results demonstrated a specificity of 100% and a sensitivity ≥97%

Standards genotype
Assay RT023 result positive negative total sensitivity (CI 95%) specificity (CI 95%)
positive 127 0 127
100% 100%
ex19del negative 0 67 67
(97.1-100%) (94.6-100%)
total 127 67 194
positive 42 0 42
100% 100%
ex20ins negative 0 69 69
(91.6-100%) (94.7-100%)
total 42 69 111
positive 87 0 88
97% 100%
G719x negative 3(a) 31 34
(94.4-95.2%) (94.3-94.7%)
total 90 31 121
positive 186 0 186
100% 100%
L858R negative 0 74 74
(98.0-100%) (95.1-100%)
total 186 74 260
positive 103 0 103
100% 100%
L861Q negative 0 61 61
(96.4-100%) (94.1-100%)
total 103 61 164
positive 86 0 86
100% 100%
S768I negative 0 35 35
(97.8-97.9%) (94.9-95.2%)
total 86 35 121
positive 68 0 68
99% 100%
T790M negative 1(b) 31 32
(95.7-96.3%) (94.3-94.7%)
total 69 31 100
a) EGFR G719S Reference Standard Horizon Dx code HD253 MAF 10%, 7%, 5% one replicate each.
b) EGFR T790M Reference Standard 50% Horizon Dx code HD258 MAF 5%.

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Limit of detection (LoD)

LoD95 was defined as the minimum percentage of mutant DNA in a background of WT DNA that can be detected with a 95%
probability.
LoD100 was defined as the minimum percentage of mutant DNA in a background of WT DNA that can be detected with a 100%
probability.

The LoDs for each assay was assessed using Horizon diagnostic standards with a mutant allele frequency (MAF%) of 50%. Horizon
diagnostic standards were serially diluted using wild-type DNA:
▪ Ex19del (HD251; p.E746_A750del; c.2235_2249del)
▪ Ex20ins (HD260; p.EGFR V769_D770insASV, c.2300_2308dup CCAGCGTGG)
▪ T790M (HD258; p.T790M, c.2369C>T)
▪ G719x (HD253; p.G719S, c.2155G>A)
▪ L861Q (HD257; p.L861Q, c.2582T>A)
▪ S768I (HD261; p.S768I, c.2303G>T)
▪ L858R (HD254, p.L858R, c.2573T>G)

Three or four different MAF% series have been tested for each assay, generating at least 50 values for low MAF% mutant sample,
using three different lots of strips.
The LoD was determined at DNA input of 12.5ng/rection that corresponds to EGFR Control assay Cq of ~26.5.

Assay LoD100 LoD95

ex19del 2% 0.5%
ex20ins 10% 7%
G719x 7% 5%
L858R 2% 1%
L861Q 2% 1%
S768I 2% 0.5%
T790M 2% 1%

Cross-reactivity

To test cross-reactivity to other EGFR mutations, clinical samples harbouring different EGFR mutations have been tested with the 8
different assays of the kit. No clinical samples mutant for ex20ins were available.
Only samples that fitted in the EGFR control assay acceptability range were included in the analysis.
No cross-reactivity between EGFR mutations has been detected.

RT023 call
Assay Mutation status WT MUT
G719A 2 0
G719C + S768I 2 0
G719x + S768I 1 0
ex19del
L858R 8 0
L861Q 2 0
T790M + L858R 1 0
ex19del 14 0
ex20ins
L858R 5 0
ex19del 14 0
ex19del + T790M 3 0
G719x L858R 10 0
L861Q 2 0
T790M + L858R 1 0
ex19del 29 0
ex19del + T790M 3 0
G719A 2 0
G719C + S768I 2 0
L858R
G719x 1 0
G719x + S768I 1 0
L861Q 2 0
ex20ins 2 0

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 RT023 call
Assay Mutation status WT MUT
ex19del 19 0
ex19del + T790M 3 0
ex20ins 1 0
G719A 2 0
L861Q G719C + S768I 1 0
G719x 1 0
G719x + S768I 1 0
L858R 12 0
T790M + L858R 1 0
ex19del 15 0
ex19del + T790M 3 0
G719A 2 0
S768I G719x 1 0
L858R 10 0
L861Q 2 0
T790M + L858R 1 0
ex19del 15 0
G719A 2 0
G719C + S768I 2 0
T790M G719x 1 0
G719x + S768I 1 0
L858R 10 0
L861Q 2 0

Interference

Interference has been evaluated using patient clinical samples according to CLSI EP07 chapter 7.
Potentially interfering substances were added to a commercial plasma sample from healthy donors (NHP BioQ Control code P0002)
as follows:
• Hemoglobin 2 g/L
• Bilirubin, unconjugated 0.2 g/L
• Triglycerides 33 g/L

This contrived samples have been extracted with “Circulating DNA large volume kit (4ml)” (code MPD4000, RBC) used with
MagCore® Automated Nucleic Acid Extractor systems (RBC Bioscience).
Mutant DNA was also added to the extracted sample at a final concentration of 12.5 ng/reaction:

DNA Standard code Mutation Final MAF%

RM021 L861Q 12.50%
HD251 ex19del 12.50%
HD260 ex20ins 16%
HD254 L858R 16%

All samples were correctly genotyped, regardless of the presence of any type of interfering substance.

To assess the possible effect of interfering substances in DNA samples extracted using the “EasyPGX ready extraction reagents”,
the Horizon “Quantitative Multiplex Reference Standard FFPE” (code HD200) has been extracted using a double amount of each
extraction reagent (EasyPGX Buffer, EasyPGX Dep solution, EasyPGX Enzyme) and tested with ex19del, L858R, L861Q,
ex20ins and EGFR control assays. All samples were correctly genotyped, regardless of the presence any type of interfering
substance.

In addition Horizon reference standard with a different level of gDNA fragmentation due to formalin damage (Quantitative Multiplex
Formalin Compromised DNA Reference Standards (Mild, Moderate and Severe), codes HD701, HD798, HD799, HD803) have been
tested with ex19del, L858R, L861Q, ex20ins and EGFR control assays at a final concentration of 12.5 ng/reaction. All expected
mutations with a MAF% ≥LoD100 were correctly genotyped.

Finally to assess the possible effect of interfering substances present in clinical FFPE and plasma samples of NSCLC patients, ΔR
last fluorescence and Cq of EGFR control assay of different categories of samples have been compared. Plasma and FFPE
samples have been compared with samples free from interfering substances (gDNA placenta, Horizon diagnostic DNA standard,
plasmidic DNA).

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In the scatterplot ΔR last fluorescence and FAM EGFR control assay Cq values of test group (FFPE, plasma samples) and control
group (gDNA placenta, Horizon diagnostic DNA standard, plasmidic DNA) overlap, suggesting no interference by substances
present in NSCLC FFPE or plasma samples.

Reproducibility

The reproducibility was investigated by testing Horizon diagnostic and Genecopoeia reference standard DNA, harbouring a known
percentage of mutant allele, EGFR wild-type DNA and plasmid DNA mixed with wild-type DNA to obtain different percentage of
mutant allele.
Mutant samples were prepared to have a medium DNA input level of 12.5ng/reaction, corresponding to an EGFR control assay FAM
Cq value of approximately 26 (EGFR Control assay FAM range 22 ≤ Cq ≤ 31).
For each mutant sample percentages of mutant allele ranging from LoD to 1.5-2.5x LoD were tested.
Wild-type samples were prepared to have a DNA input range 250-0.5ng/reaction.
Reproducibility was evaluated using at least two different EasyPGX qPCR instruments (code RT800-96) and at least 3 lots of
EasyPGX EGFR strips.

All wild-type samples, for all assays produced 100% correct calls.
Overall results indicate that all assays of the kit are reproducible across instruments and EasyPGX EGFR strips lots in terms of
correct call.

To assess EasyPGX ready extraction reagents reproducibility 14 extraction session of sample HD141 (Horizon Diagnostic EGFR
wild-type FFPE standard) were performed. After extraction all replicates have been tested with RT023 or RT003 and resulted
suitable in terms of quantity and quality of amplifiable DNA according to EGFR control assay ranges.

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 Repeatability

The repeatability was investigated by testing Horizon diagnostic and Genecopoeia reference standard DNA, harbouring a known
percentage of mutant allele and human EGFR wild-type DNA.
Samples were prepared to have a medium DNA input level of 12.5ng/reaction, corresponding to an EGFR control assay FAM Cq
value of approximately 26 (EGFR Control assay FAM range 22 ≤ Cq ≤ 31). For each mutant sample percentage of mutant allele
equal to LoD100 was tested. Wild-type samples were prepared to have a DNA input range 250-0.5ng/reaction.
Repeatability was evaluated testing each sample at least in duplicate in at least two independent experiments.
All samples, both wild-type and mutant, for all assays produced 100% correct calls.
Overall results indicate that all assays of the kit are repeatably in terms of correct call.

In addition, to test repeatability intra-run of DNA extraction using EasyPGX extraction reagents, HD141 and HD200 (Quantitative
Multiplex Reference Standard FFPE) have been extracted in duplicates in two experiments for a total of 4 replicates for each
sample. All replicates have been tested with RT023 or RT003 (Easy EGFR kit code RT003, Diatech Pharmacogenetics) and
resulted suitable in terms of quantity and quality of amplifiable DNA according to EGFR control assay ranges.

Linearity

Effect of DNA input on Cq of EGFR control assay has been assessed.

Wild-type h-placenta DNA input from 250ng/reaction to 0.5ng/reaction (corresponding to EGFR control assay Cq range 21.5-30.5)
has been tested.
Amplification efficiency of the Control Assay was calculated using linear regression with Cq on the y-axis (response variable) and
log10(DNA input - ng/rxn) on the x-axis (explanatory variable).

Amplification Efficiency = [10^(-1 / Slope) -1] -1

Intercept Slope Amplification

R- Slope Amplification
Assay Channel Intercept Standar Slope Standard Efficiency
Square 95% CI Efficiency
d Error Error 95% CI
Human -3.640 0.883,
FAM 0.942 29.645 0.171 -3.361 0.138 0.984
Placental -3.083 1.110
Control
DNA (250- -3.609, 0.893,
HEX 0.936 30.206 0.177 -3.321 0.142 1.000
0.5 ng/rxn) -3.033 1.136

Primer and probe specificity

A specificity analysis was conducted using the Basic Local Alignment Search Tool (BLAST) to ensure that the primers used in the
EGFR Kit would amplify only human EGFR sequences. An analysis was also performed to verify that no known EGFR
polymorphisms were reported in the primers and probes regions.
Results confirm that primers and probes are free from known polymorphisms and amplify specifically only EGFR gene. The only
exception is polymorphism rs1050171 p.Gln787Gln c.2361G>A which underlines T790M forward primer. Laboratory tests conducted
on samples containing both T790M and Q797Q polymorphisms show that the Q797Q doesn’t interfere with T790M detection.
In detail, 6 clinical FFPE samples, harbouring both T790M and rs1050171 p.Gln787Gln c.2361G>A in homozygosity (A:100%) have
been tested with RT023. Samples have been previously analysed by “Myriapod NGS cancer panel DNA” (code NG033 Diatech
Pharmacogenetics). As expected, 6/6 samples generated a T790M call with RT023.

Inclusivity

An in silico analysis has been performed to assess the capability of primers and probes of the kit to detect secondary NSCLC EGFR
mutations with low prevalence.
NSCLC EGFR somatic mutations have been retrieved from Cosmic database (Catalog of somatic mutation in cancer database,
accessed on 2021/04/26). Primers and probes of RT023 kit have been aligned against EGFR mutant sequences to assess their
capability of detect each mutation.
In Cosmic database there are 93693 mutant NSCLC samples, but only for 6450 of them is known the nucleotide change; RT023 can
detect 87% (5582/6450) of NSCLC mutations.
All detectable mutations are listed in the section “Intended use” of this manual.

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CLINICAL PERFORMANCE

To assess clinical performances of RT023, DNA isolated from FFPE tumor tissue and from plasma of NSCLC clinical samples have
been tested.
Clinical samples were suitable for the presence of mutations detected by the kit, and have been already genotyped through one of
the following comparative methods:
▪ Pyrosequencing technology - (“EGFR TKI Response® (sensitivity)”, code UP034; “EGFR TKI response® (resistance)”,
code UP035 - Diatech Pharmacogenetics)
▪ MALDI-TOF Mass Spectrometry using MassArray® platform - (“Myriapod® Cancer Status”, code SQ020 and “Myriapod®
Lung Status” code SQ011 - Diatech Pharmacogenetics)
▪ Real-time PCR - Easy® EGFR (code RT003 - Diatech Pharmacogenetics)
▪ Sanger sequencing

According to CLSI EP12-A2, as the comparative methods are not the criterion for diagnostic accuracy, percentage of agreement
with comparative CE IVD methods has been assessed.
Overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) have been assessed
according to the agreement of mutation call between the RT023 kit and comparative methods.
The percentages, together with the corresponding 95% CI are summarized below.

Comparative method OPA PPA NPA

Assay RT023 result
positive negative total (95% CI) (95% CI) (95% CI)
positivo 28 0 28 99% 97% 100%
ex19del negativo 1a 75 76 94.8-99.8 80.4-96.5 95.1-100
totale 29 75 104
Positivo 4 1b 5 99% 100% 98%
G719X negativo 0 64 64 92.2-99.7 69.4-81.6 95.4-96.1
totale 4 65 69
positivo 2 0 2 100% 100% 100%
ex20ins negativo 0 41 41 91.8-100 34.2-100 91.4-100
totale 2 41 43
positivo 14 0 14 100% 100% 100%
L858R negativo 0 100 100 96.7-100 78.5-100 96.3-100
totale 14 100 114
positivo 3 1c 4 99% 100% 99%
L861Q negativo 0 75 75 93.2-99.8 43.9-100 92.9-99.8
totale 3 76 79
positivo 2 0 2 100% 100% 100%
S768I negativo 0 68 68 94.8-100 50.7-83.6 97.3-97.4
totale 2 68 70
positivo 3 0 3 100% 100% 100%
T790M negativo 0 67 67 94.8-100 62.6-81.3 97.2-97.3
totale 3 67 70

a) One sample genotyped by NGS as ex19del with low confidence, was called by RT023 as wild-type type (ΔCq 40, ΔR 40).
The sample has been retested using a second comparative method (“EasyPGX® Ready Lung Fast Track” code RT045 -
Diatech Pharmacogenetics) that confirmed the wild-type call. The discrepant result may be due to a different LoD between
RT023 and comparative method.
b) One sample genotyped by the comparative method as wild-type, was called by RT023 as G719x. The sample has been
tested two times with RT023 resulting in 1 WT borderline call (ΔCq 8.6, ΔR 283) and 1 mutant G719x borderline call (ΔCq
7.7, ΔR 214). Probably this sample is a real G719x mutant with a MAF% close to the LoD of the assay. The discrepant
result may be due to a different LoD between RT023 and comparative method.
c) One sample genotyped by the comparative method as wild-type, was called by RT023 as L861Q with a borderline call
(ΔCq 7.3, ΔR 147).
d) Un campione genotipizzato dal metodo comparativo come wild-type, è stato chiamato L861Q mutato da RT023, con una
chiamata al limite del range (ΔCq 7.3, ΔR 147). The sample has been retested with RT023 resulting in a wt call. Probably
this sample is a real L861Q mutant with a MAF% close to the LoD of the assay. The discrepant result may be due to a
different LoD between RT023 and comparative method.

Cited guidelines:
▪ CLSI. User protocol for evaluation of qualitative test performance; Approved guideline – Second edition. CLSI document EP12-
A2. Wayne, PA: clinical and laboratory standards institute; 2008.
▪ CLSI. Interference testing in clinical chemistry. 3rd ed. CLSI guideline EP07. Wayne, PA: clinical and laboratory standards
institute; 2018.

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OPERATOR NOTES

EasyPGX® ready EGFR

Code: RT023 28/30 2022/05
EasyPGX® ready EGFR
Code: RT023 29/30 2022/05
 Diatech Pharmacogenetics S.r.l
via Ignazio Silone, 1 b - 60035 Jesi AN Italy
Tel +39-0731-213243 Fax +39-0731-213239
info@diatechpharmacogenetics.com
www.diatechpharmacogenetics.com