Testing for EGFR Mutations and ALK Rearrangements in Advanced Non-Small-Cell Lung Cancer: Considerations for Countries in Emerging Markets

Figures & data

Table 1 Mutation-Detection Assays (Eg EGFR, BRAF)

Assay	Advantage	Disadvantage
Sanger sequencing (direct DNA sequencing)	Identification of all known and previously unknown mutations within the studied region	High tumor content required (mutation detected when allele frequency >25% total DNA [50% tumor content]) Low sensitivity
Allele-specific real-time PCR (targeted assays)	Allows rapid multiplex genotyping of specific known hotspot mutations More sensitive than Sanger (requires 5–10% of the starting tumor DNA)	Designed only to detect most frequent mutations Unable to detect mutations different from those included in the assay (low frequency or novel mutations)
NGS (massive parallel sequencing technology)	Detection of multiple genetic alterations (mutations, gene fusions, CNV), allowing the sequencing of large regions of the genome with higher sensitivity Can be performed by FFPE extraction and freshly collected tissue specimens	Effective implementation of NGS requires good-quality DNA and RNA (not always present in FFPE samples) Validation of panels can be expensive and difficult for some laboratories in low- or mid-income countries

Note: Data from Mok et al.⁹⁸ Abbreviations: CNV, copy-number variation; FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing; PCR, polymerase chain reaction; TAT, turnaround time.

Table 2 Rearrangement-Detection Assays (Eg ALK, ROS1 Fusion)

Assay	Advantage	Disadvantage
RT-PCR	High sensitivity High specificity Can be used on mRNA/cDNA to directly detect fusion genes	Not applicable for unknown partners High-quality of RNA is required (difficult to apply in long-term stored tissue and FFPE samples) Multiplexed assays are required to cover the large variety of fusion transcripts^Citation185,Citation186
FISH	Sensitive and specific Detects fusions irrespective of the fusion partner Break Apart assay Vysis CDx was established as a ‘gold standard’ to detect ALK fusion in NSCLC Allows use of archived FFPE tissue samples and all cytology samples^Citation187^–^Citation189	Unable to identify the specific gene fusion partner Interpretation requires specialized training The ‘break apart’ can be difficult to identify due to small physical separation of ALK or ROS1 fragments Testing is relatively costly and time-consuming Samples may not contain enough assessable cells to be properly interpreted The 15% cutoff and potential false-positive and false-negative signaling profiles may challenge interpretation
IHC	Widely available, relatively inexpensive Rapid TAT For ALK rearrangement, IHC is valid with any quantity and percentage of positive tumor cells (useful in biopsy) or cytology samples with a small amount of tumor cells^Citation190,Citation191	ROS1-positive IHC requires confirmation by FISH (≥15% tumor cells) LDT IHC tests require careful validation
NGS	Same advantages as detailed for mutation studies There are some commercial lung cancer-specific fusion panels^Citation153,Citation192	Same disadvantages as detailed for mutation studies Good quality RNA is required

Note: Data from Tsao et al.¹⁰⁵Abbreviations: ALK, anaplastic lymphoma kinase; cDNA, circulating DNA; CDx, companion diagnostic test; FFPE, formalin-fixed paraffin-embedded; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; LDT, laboratory-developed test; mRNA, messenger RNA; NGS, next-generation sequencing; NSCLC, non-small-cell lung cancer; RT PCR, reverse transcriptase polymerase chain reaction; TAT, turnaround time.

Table 3 US FDA-Approved Drugs and Companion Diagnostics for Advanced NSCLC

Targeted Agent	Companion Diagnostic Test
EGFR
Erlotinib	Cobas^® EGFR Mutation Test v2
Gefitinib	Therascreen EGFR RGQ PCR Kit
Afatinib	Therascreen EGFR RGQ PCR Kit
Osimertinib	Cobas^® EGFR Mutation Test v2
ALK
Crizotinib	Vysis ALK Break Apart FISH Probe Kit
Ceritinib	Ventana ALK (D5F3) CDx Assay
Alectinib	Ventana ALK (D5F3) CDx Assay
Lorlatinib	Ventana ALK (D5F3) CDx Assay

Note: Adapted with permission from Schwartzberg L, Kim ES, Liu D, Schrag D. Precision oncology: who, how, what, when, and when not? Am Soc Clin Oncol Educ Book. 2017;37:160–169⁸⁸ with data from these studies.^106,193

Abbreviations: ALK, anaplastic lymphoma kinase; CDx, companion diagnostic test; EGFR, epidermal growth factor receptor; FDA, Food and Drug Administration; FISH, fluorescence in situ hybridization; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; RGQ, Rotor-Gene Q.

Table 4 Testing Frequency for EGFR Mutations and/or ALK Rearrangements in Patients with Advanced NSCLC

					Testing Frequency
Author	Period	Location	Method	No. Centers/HCPs	n	Overall, n (%)	EGFR, n (%)	ALK, n (%)	Dx Tests
North America
Pan^Citation125	2007–2011	USA	RR	iKnowMedTMdatabase^a	1168		128 (11%) 2% <2010; 32% in 2011	28 (2%)
Enewold^Citation117	2010	USA	RR	SEER Database^b	1358		228 (17%) 157 (21%) in AdC
MacLean^Citation123	2010–2012	USA	RR	Humana Research Database	2623	1579 (61%)
Lim^Citation122	2010–2013	CAN	RR	1 center	258^c		150 (58%)	50 (19%)	IHC/FISH/NGS
Shen^Citation126	2013–2014	USA	RR	Marketscan Database^d	5842	1039 (18%)	1039 (18%)
Gutierrez^Citation118	2013–2015	USA	RR	15 centers/89 oncol.	814	479 (59%)			PCR/FISH/NGS
Schink^Citation135	2014	USA	S	57 centers	NA	57 (100%)	57 (100%)	57 (100%)	EGFR – PCR 44%; ALK – FISH 64%
Illei^Citation119	2011–2017	USA	RR	Flatiron Health Database	31,483			16,726 (53%) 32% 2011; 62% 2016
South America
Palacio^Citation124	2011–2016	BRA	RR	All public and private settings	11,684		4440 (38%) 13% 2011; 42% 2016		SS
Europe
Sluga^Citation127	2008–2014	NL	RR	4 centers	2206	879 (40%)^e 11% 2008; 75% 2014
Ess^Citation42	2008–2014	SUI	RR	1 center	718		447 (62%)	265 (37%)
Don-Carolis^Citation130	2014	UK	S	15 centers	23,131^f	12,140 (53%)	12,140 (53%) includes both ALK and EGFR testing
Gobbini^Citation138	2014–2015	ITL	OS	38 centers	1787	1388 (78%)	1353 (76%)	942 (53%)	EGFR-direct sequencing; ALK-IHC and/or break apart FISH; NGS in some centers
Ryska^Citation136	2014–2015	Central, Eastern Europe^g	S	42 oncol.	NA		75–100%	ALK testing – all except BG	EGFR: RT-PCR; ALK-IHC + FISH and/or FISH alone. ISR, NGS
Middle East
Bar^Citation137	2013	ISR	S	24 oncol.			19 (79.2%)
Asia-Pacific
McKeage^Citation139	2010–2016	NZ	OS	All non-SCC NSCLC from Auckland, Manukau, Waitemata and Northland	3130			407 (13%)	Vysis break apart FISH
Yatabe^Citation128	2011–2012	Asia-Pac	RR	40 centers/11 countries^h	22,193		18% (CHN) – 65% (JPN)^i		DNA sequencing. 75% – IHC (poorly differentiated samples)
Hotta^Citation131	2012	JPN	S	871 oncol.	NA		775 (89%)	525 (60%)
Isobe^Citation120	2011–2013	JPN	RR	5 centers	129^c	105 (81%)	105 (81%)	25 (19%)
Zhou^Citation129	2015–2016	CHN	RR	12 centers	932		665 (71%)	416 (45%)
Prabhash^Citation134	2015–2016	IND	INTV	111 oncol.	NA	105 (95%)	105 (95%)
International
Lee^Citation121	2011–2013	INT	RR	78 centers/8 countries^j	1440	43% (BRA) – 85% (TW)	41% (DE) – 97% (TW)^j	3% (TW) – 27% (ITL)
Peters^Citation133	2014–2016 2016–2016	INT	2 surveys^k	S1: 562 oncol.^l S2: 707 oncol.^m	NA		450 (80%)
Jankovic^Citation132	2017–2018	INT	S	36 oncol./18 countries^n	NA	90% guided by test results	85%	58%	PCR 60%; SS 26%; NGS 26%; FISH 26%; IHC 16%

Notes: ^aCaptures demographic, clinical, laboratory and treatment data for patients receiving care within US Oncology’s network of approximately 1200 community-based oncologists. ^bRandom selection of patients with NSCLC in the SEER Database. ^cData are reported for patients with non-SCC only. ^dMarketscan Database contains health insurance claims for 50 million individuals from >100 large- or medium-sized US-based employers. ^e853 patients had NSCLC-NOS or AdC, 26 had SCC. ^fPatients eligible for an EGFR diagnostic test. ^gBulgaria, Croatia, Czech Republic, Israel, Slovakia, Slovenia, Poland, Hungary, Turkey. ^hChina, Hong Kong, Indonesia, Japan, South Korea, Malaysia, Philippines, Singapore, Taiwan, Thailand and Vietnam. ⁱData not provided by Indonesia, Malaysia, Singapore, the Philippines or Vietnam. ^jItaly (69%), Spain (56%), Germany (41%), Australia (64%), Japan (81%), South Korea (44%), Taiwan (97%) and Brazil (81%). ^kTwo surveys, 18 months apart. ^lCanada, France, Germany, Italy, Japan, South Korea, Spain, Taiwan, UK and USA. ^mSurvey 2 included physicians from China. ⁿPortugal, Serbia, Morocco, UK, Azerbaijan, Uzbekistan, Peru, Brazil, Kuwait, France, Spain, Austria, Estonia, Belgium, Montenegro, Nigeria, USA and Poland.

Abbreviations: AdC, adenocarcinoma; ALK, anaplastic lymphoma kinase; BRA, Brazil; CAN, Canada; CHN, China; DE, Germany; Dx, diagnostic; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; HCP, healthcare professional; IHC, immunohistochemistry; IND, India; INT, international; INTV, interview; ITL, Italy; ISR, Israel; JPN, Japan; NA, not available; NGS, next-generation sequencing; NL, Netherlands; NOS, not otherwise specified; NR, not reported; NSCLC, non-small-cell lung cancer; NZ, New Zealand; oncol, oncologist; OS, observational study; Ref, reference; RR, retrospective review; RT PCR, reverse transcriptase polymerase chain reaction; S, survey; SCC, squamous cell carcinoma; SD, standard deviation; SEER, Surveillance Epidemiology and End Results; SS, Sanger sequencing; SUI, Switzerland; TAT, turnaround time; TW, Taiwan; UK, United Kingdom; USA, United States of America.

Table 5 Potential Barriers to Biomarker Testing

Access to targeted therapies^Citation118,Citation135,Citation137^–^Citation139,Citation143

Reimbursement for testing^Citation133,Citation138

Tissue sample quality^Citation98,Citation143

TAT for test results^Citation42,Citation120,Citation131

Coordination among multiple specialist groups^Citation126

Accurate interpretation of results/physician education^Citation118

Abbreviation: TAT, turnaround time.

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