COBAS EGFR MUTATION TEST
Device Facts
| Record ID | P120019 |
|---|---|
| Device Name | COBAS EGFR MUTATION TEST |
| Applicant | Roche |
| Product Code | OWD · Pathology |
| Decision Date | May 14, 2013 |
| Decision | APPR |
| Device Class | Class 3 |
| Attributes | Expedited Review |
Intended Use
The cobas® EGFR Mutation Test is a real-time PCR test for the qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the epidermal growth factor receptor (EGFR) gene in DNA derived from formalin-fixed paraffin-embedded (FFPET) human non-small cell lung cancer (NSCLC) tumor tissue. The test is intended to be used as an aid in selecting patients with NSCLC for whom Tarceva® (erlotinib), an EGFR tyrosine kinase inhibitor (TKI), is indicated.
Device Story
The cobas® EGFR Mutation Test is an in vitro diagnostic device used to identify specific EGFR mutations in NSCLC tumor tissue. The process begins with manual DNA isolation from FFPET tissue sections using the cobas® DNA Sample Preparation Kit. The extracted DNA is then processed on the cobas z 480 analyzer, which performs automated real-time PCR amplification and detection. The system uses allele-specific PCR (AS-PCR) chemistry with fluorescently labeled probes to detect targeted mutations. The cobas® 4800 system software controls the analyzer, processes fluorescent signals, and applies interpretation logic to determine mutation status. Results are provided to clinicians to guide the selection of patients for treatment with Tarceva® (erlotinib). By identifying patients with EGFR-activating mutations, the test helps ensure that patients receive targeted therapy, potentially improving progression-free survival compared to standard chemotherapy.
Clinical Evidence
Clinical evidence based on retrospective analysis of the Phase 3 EURTAC trial (n=174). Bridging study compared cobas® EGFR Mutation Test results to the clinical trial assay (CTA). In the cobas-positive population (n=116), patients treated with erlotinib showed significantly improved median PFS (10.4 vs 5.4 months; HR=0.34; p<0.0001) compared to chemotherapy. Analytical performance showed 5% mutation sensitivity. Agreement with Sanger sequencing: PPA 96.6%, NPA 88.3%. Agreement with MPS: PPA 94.0%, NPA 97.7%.
Technological Characteristics
Real-time PCR assay using allele-specific PCR (AS-PCR) chemistry. Utilizes Thermus species Z05-AS1 DNA polymerase and AmpErase (uracil-N-glycosylase) for carryover prevention. Analyzes DNA from FFPET tissue. Automated amplification and detection on cobas z 480 analyzer. Software-based result interpretation using Ct and CtR values. Requires 50 ng DNA input per reaction. Connectivity to LIS supported.
Indications for Use
Indicated for qualitative detection of EGFR exon 19 deletions and exon 21 (L858R) mutations in DNA from FFPET NSCLC tumor tissue to aid in selecting patients for Tarceva (erlotinib) therapy.
Regulatory Classification
Identification
The somatic gene mutation detection system is an in vitro diagnostic nucleic acid amplification and real-time detection system for the identification of genetic mutations in DNA extracted from human tumor tissues.
Related Devices
- P150047 — cobas EGFR MUTATION TEST v2 · Roche Molecular Systems, Inc. · Jun 1, 2016
- P150044 — cobas EGFR MUTATION TEST v2 · Roche Molecular Systems, Inc. · Sep 28, 2016
- P120022 — THERASCREEN EGFR RGQ PCR KIT · QIAGEN GmbH · Jul 12, 2013
- P140023 — COBAS KRAS MUTATION TEST · Roche Molecular Systems, Inc. · May 7, 2015
- P110020 — COBAS 4800 BRAF V600 MUTATION TEST · Roche Molecular Systems, Inc. · Aug 17, 2011
Submission Summary (Full Text)
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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)
I. GENERAL INFORMATION
Device Generic Name: Real-time PCR test
Device Trade Name: cobas® EGFR Mutation Test
Device Procode: OWD
Applicant’s Name and Address: Roche Molecular Systems, Inc. (RMS)
4300 Hacienda Drive
Pleasanton, CA 94588-2722
Date(s) of Panel Recommendation: None
Premarket Approval Application (PMA) Number: P120019
Date of FDA Notice of Approval: May 14, 2013
Expedited: Granted priority review status on December 14, 2012 because the device addresses an unmet medical need, as demonstrated by significant clinically meaningful advantage.
II. INDICATIONS FOR USE
The cobas® EGFR Mutation Test is a real-time PCR test for the qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the epidermal growth factor receptor (EGFR) gene in DNA derived from formalin-fixed paraffin-embedded (FFPET) human non-small cell lung cancer (NSCLC) tumor tissue. The test is intended to be used as an aid in selecting patients with NSCLC for whom Tarceva® (erlotinib), an EGFR tyrosine kinase inhibitor (TKI), is indicated.
Specimens are processed using the cobas® DNA Sample Preparation Kit for manual sample preparation and the cobas z 480 analyzer for automated amplification and detection.
III. CONTRAINDICATIONS
None.
IV. WARNINGS AND PRECAUTIONS
The warnings and precautions can be found in the cobas® EGFR Mutation Test labeling.
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V. DEVICE DESCRIPTION
The cobas® EGFR Mutation Test is based on two processes:
1. The cobas® DNA Sample Preparation Kit provides reagents for manual specimen preparation to obtain genomic DNA from formalin-fixed, paraffin-embedded tissue (FFPET).
2. The cobas® EGFR Mutation Test kit provides reagents for automated real-time PCR amplification and detection of the EGFR mutations.
Two external run controls are provided and the EGFR exon 28 wild-type allele serves as an internal, full process control.
Specimen Preparation
FFPET specimens are processed and genomic DNA is isolated using the cobas® DNA Sample Preparation Kit. A deparaffinized 5-μm section of an FFPET specimen is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is spectrophotometrically determined and adjusted to a fixed concentration of 5 ng/μL with 25 μL used in the amplification and detection mixture.
PCR Amplification and Detection
Target Selection and Amplification
The cobas® EGFR Mutation Test kit uses primers that define specific base-pair sequences for each of the targeted mutations. For the exon 19 deletion mutations, base pair sequences that range from 125 to 141 are targeted and; for the exon 21 L858R mutation, a 138 base pair sequence is targeted; for the internal control in exon 28, a 87 base pair sequence is targeted. Amplification occurs only in the regions of the EGFR gene between the primers; the entire EGFR gene is not amplified.
The cobas® EGFR Mutation Test uses allele-specific PCR (AS-PCR) chemistry for amplification and detection. The selected AS-PCR primers specifically amplify the targeted mutant sequences over the wild-type sequences and/or other human genomic DNA. The cobas® EGFR Mutation Test is designed to detect exon 19 deletion mutations and exon 21 L858R substitution mutation of the EGFR gene. The cobas® EGFR Mutation Test is designed to use two master mix reagents which are run in two separate wells. For detecting exon 19 deletion mutations, fourteen AS-PCR primers, one common primer, and one common probe are included in Master Mix 1 (MMx1). For detecting exon 21 L858R substitution mutation, one AS-PCR primer, one common primer, and one common probe are included in Master Mix 2 (MMx2).
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A derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. Briefly, the PCR reaction mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05-AS1 DNA polymerase, in the presence of divalent metal ion and excess dNTPs, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy which includes the targeted base-pair regions of the EGFR gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.
Selective amplification of target nucleic acid from the specimen is achieved in the cobas® EGFR Mutation Test by the use of AmpErase® (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP) which are included in the Master Mix reagents. The AmpErase® enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine, but not DNA containing thymidine. Deoxyuridine is always present in amplicon due to the use of dUTP as one of the nucleotide triphosphates in the Reaction Mix reagent; therefore, only amplicon contains deoxyuridine. The AmpErase® enzyme is inactive at temperatures above 55°C, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon.
## Automated Real-time Detection
The cobas® EGFR Mutation Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5' to 3' nuclease activity of the Z05-AS1 DNA polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Two different reporter dyes are used to label the mutations targeted by the test. Amplification of the targeted EGFR sequences are detected independently across three reactions by measuring fluorescence at the two characteristic wavelengths in dedicated optical channels.
## Instrument and Software
The cobas® 4800 system is controlled by the cobas® 4800 system software which provides the core software engines and user interfaces. This core system software was designed to allow multiple assays to be performed on the system using analyte specific analysis package software (ASAP). The cobas z 480 analyzer component of the test system also has its own internal instrument control software which is driven by the core software.
A dedicated Control Unit computer runs the cobas® 4800 system software and provides an interface to the cobas z 480 and Laboratory Information System (LIS). The computer also processes the fluorescent signals with the analyte specific analysis package and
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stores the test results in a controlled database. The complete system allows a user to create a test work order for each specimen either manually or automatically when connected to a LIS. A software wizard guides the user through the necessary steps to perform a run which includes z 480 maintenance handling, test selection, specimen ID entry, reagent and microwell plate bar code entry, microwell plate loading and run start.
The cobas® 4800 System tracks each specimen during processing and analysis on the cobas z 480 analyzer. Once the thermal run is complete the ASAP software processes the fluorescence data using data analysis algorithms, assesses the validity of the controls and determines the results using the assay specific result interpretation logic. The software then provides the results to the user in three formats; a printable PDF results report, a GUI based result viewer and a result export file that can be exported to the LIS.
The cobas® 4800 System software includes the cobas® 4800 EGFR Analysis Package (AP) software, which contains an algorithm to determine sample results and run validity. The overall cobas® 4800 System components are shown in the diagram below:
## Interpretation of Results
If the run is valid, then the cycle-to-threshold (Ct) and CtR values for each sample will be evaluated against acceptable ranges for each channel. The CtR value is determined by calculating the difference between the observed Ct for the exon 19 deletion or exon 21 L858R mutation and the corresponding Internal Control (IC) Ct value from the same Master Mix. Ct values are not available to the user. The table below summarizes how the results from each amplification Master Mix are combined to provide an overall result.
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| Master Mix 1 Result | Master Mix 2 Result | Reported Result |
| --- | --- | --- |
| Valid Mutation Not Detected | Valid Mutation Not Detected | Valid Mutation Not Detected |
| Valid Mutation Not Detected | Valid Exon 21 L858R Mutation Detected | Valid Exon 21 L858R Mutation Detected |
| Valid Mutation Not Detected | Invalid | Invalid |
| Valid Exon 19 Deletion Mutation Detected | Valid Mutation Not Detected | Valid Exon 19 Deletion Mutation Detected |
| Valid Exon 19 Deletion Mutation Detected | Valid Exon 21 Deletion Mutation Detected | Valid Exon 19 Deletion and Exon 21 L858R Mutation Detected |
| Valid Exon 19 Deletion Mutation Detected | Invalid | Invalid |
| Invalid | Valid Mutation Not Detected | Invalid |
| Invalid | Valid Exon 21 L858R Mutation Detected | Invalid |
## Result Interpretation of the cobas® EGFR Mutation Test
| Test Result | Mutation Result | Interpretation |
| --- | --- | --- |
| Mutation Detected (MD) | Exon 19 Deletion
Exon 21 L858R
(More than one mutation may be present) | Mutation detected in specified targeted EGFR region. |
| Mutation Not Detected* (MND) | N/A | Mutation not detected in targeted EGFR regions |
| Invalid | N/A | Specimen result is invalid. Repeat the testing of specimens with invalid results following the instructions outlined in the “Retesting of Specimens with Invalid Results” section. |
| Failed | N/A | Failed run due to hardware or software failure. Contact your local Roche office for technical assistance |
*A Mutation Not Detected result does not preclude the presence of a mutation in the targeted EGFR regions because results depend on percent mutant sequences, adequate specimen integrity, absence of inhibitors, and sufficient DNA to be detected.
## Test Controls
One EGFR mutant control and one EGFR negative control are provided. The EGFR wild-type allele on exon 28 serves as an internal, full process control.
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1. EGFR Mutant Control (MUT): The Mutant Control is a blend of six DNA plasmids containing specified EGFR mutation sequences and cell line DNA that is wild-type for EGFR. The Mutant Control is composed of plasmids representing the most frequently observed mutation for each mutation class detected by the test. The Mutant Control will be included in every run and will serve as a process control for amplification and detection. The Mutant Control must yield Cycle Threshold (Ct) values for the Internal Control (IC), exon 19 deletion mutation, and L858R mutation within the respective acceptable ranges for the run to be considered valid.
2. EGFR Negative Control: The Negative Control is a full process contamination control for a given test batch of specimens. No reagent is provided for the Negative Control; instead a blank vial containing no specimen (specimen diluent only) is processed through specimen preparation and the resulting eluate is subsequently diluted and amplified and detected. The Negative Control Ct values must be either not detected or greater than the pre-established Ct maximum value for the exon 19 deletion and L858R mutation groups and the IC for the run to be considered valid.
3. EGFR WT Internal Control (IC): The Internal Control in EGFR exon 28 from test specimens serves as a full process control. This control ensures that every step of the process from specimen preparation to amplification and detection has been completed successfully.
Acceptable Ct Values for Control Reactions
| Sample Types | Master Mix | Ct for Channel 1 (FAM, probe) | Ct for Channel 4 (Cy5.5, IC probe) |
| --- | --- | --- | --- |
| MUT Positive Control (Exon 19 Deletion) | 1 | 30.3 ≤ Ct ≤ 38.1 | 22.0 ≤ Ct ≤ 28.0 |
| Negative Control (Exon 19 Deletion) | 1 | Ct > 40 | Ct > 35 |
| MUT Positive Control (L858R) | 2 | 32.3 ≤ Ct ≤ 40.8 | 22.0 ≤ Ct ≤ 28.0 |
| Negative Control (L858R) | 2 | Ct > 40 | Ct > 35 |
VI. ALTERNATIVE PRACTICES AND PROCEDURES
There are no other FDA-cleared or approved alternatives for EGFR mutation testing of formalin-fixed, paraffin-embedded NSCLC tissue for the selection of patients who are eligible for first-line treatment with Tarceva® (erlotinib).
VII. MARKETING HISTORY
The cobas® EGFR Mutation Test has not been marketed in the United States or any foreign country.
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# VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH
Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect EGFR test results and subsequently improper patient management decisions in non-small cell lung cancer (NSCLC) treatment.
For the specific adverse events that occurred in the clinical studies, please see Section X below.
# IX. SUMMARY OF PRECLINICAL STUDIES
## A. Laboratory Studies
For the non-clinical studies described below, percentage of tumor was assessed by pathology review. Bi-directional Sanger sequencing and massively parallel sequencing (MPS) were used to select the specimens for testing. Percentage of mutation of NSCLC FFPET specimen was determined using a MPS method.
### 1. Correlation with Reference Method for Phase 3 Samples
The analytical performance of the cobas® EGFR Mutation Test was assessed by comparing it to two reference methods – bidirectional Sanger sequencing and MPS – using 487 formalin-fixed paraffin-embedded lung tumor specimens from patients with advanced NSCLC who screened for the Phase 3 EURTAC trial of erlotinib vs. cisplatin-based chemotherapy. The clinical and demographic characteristics of the patients whose specimens were available for this retrospective testing were comparable to those of otherwise eligible patients (n=557) whose specimens were not available for retesting.
A total of 1,276 patients were screened for the EURTAC trial using a combination of laboratory developed tests, collectively referred to as the clinical trial assay (CTA). After excluding ineligible patients and those without CTA results, 1,044 patients were potentially eligible for the current study. Among the 1,044 eligible patients, 225 patients had samples that were mutation positive by CTA, 792 had samples that were wild-type by CTA, and 27 had samples with inconclusive results by CTA. Of the 1,044 potentially eligible patients, 487 specimens were available for retesting with the cobas® EGFR Mutation Test. More details are included in “Accountability of PMA Cohort” section below.
All 487 specimens were tested in a blinded fashion with both the cobas® EGFR Mutation Test and Sanger sequencing. Of those, 406 had valid results by both the cobas test and Sanger sequencing, 38 invalid results were observed by the cobas test and Sanger sequencing, 38 invalid results by Sanger sequencing only, and 5 invalid results by cobas testing only. Among the 487 specimens available for retesting with the cobas® EGFR Mutation Test, 444 specimens gave valid cobas test results were also tested with MPS. Of those, there were 36 invalid results by MPS; thus, 408 had valid results by both cobas testing and MPS sequencing. The analytical accuracy of the
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cobas® EGFR Mutation Test compared with each reference method was evaluated by estimating the positive percentage agreement (PPA), negative percentage agreement (NPA), and overall percentage agreement (OPA) and their corresponding 95% confidence intervals (CIs) for exon 19 deletions and L858R mutations in aggregate, and separately.
A total of 406 samples with valid cobas test and Sanger sequencing results were included in the agreement analysis. The agreement analysis between the cobas® EGFR Mutation Test results and Sanger sequencing results for the detection of the EGFR mutation demonstrated a PPA of 96.6%, a NPA of 88.3%, and an OPA of 90.6%. The PPA, NPA, and OPA in the detection of exon 19 deletion mutations were all greater than 92%. The PPA, NPA, and OPA in the detection of L858R mutations compared were all greater than 95%.
cobas® EGFR Mutation Test vs. Sanger Sequencing Using Phase 3 Specimens
| cobas® EGFR Mutation Result | Sanger Sequencing Result | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Result in aggregate | | | Exon 19 deletion | | | Exon 21 Mutation | | |
| | MD | MND | Total | MD | MND | Total | MD | MND | Total |
| MD | 112 | 34 | 146 | 71 | 25 | 96 | 41 | 9 | 50 |
| MND | 4 | 256 | 260 | 2 | 308 | 310 | 2 | 354 | 356 |
| Total | 116 | 290 | 406 | 73 | 333 | 406 | 43 | 363 | 406 |
| PPA (95% CI) | 112/116 = 96.6%
(91.5%, 98.7%) | | | 71/73 = 97.3%
(90.5%, 99.2%) | | | 41/43 = 95.3%
(84.5%, 98.7%) | | |
| NPA (95% CI) | 256/290 = 88.3%
(84.1%, 91.5%) | | | 308/333 = 92.5%
(89.2%, 94.9%) | | | 354/363 = 97.5%
(95.4%, 98.7%) | | |
| OPA (95% CI) | 368/406 = 90.6%
(87.4%, 93.1%) | | | 379/406 = 93.3%
(90.5%, 95.4%) | | | 395/406 = 97.3%
(95.2%, 98.5%) | | |
MD denotes Mutation Detected
MND denotes Mutation Not Detected
A total of 408 samples with valid cobas test and MPS results were included in the agreement analysis. The agreement analysis between the cobas® EGFR Mutation Test results and MPS sequencing results for the detection of the EGFR mutation demonstrated a PPA of 94.0%, a NPA of 97.7%, and an OPA of 96.3%. The PPA, NPA, and OPA in the detection of exon 19 deletion mutations were all greater than 95%. The PPA, NPA, and OPA in the detection of L858R mutations were all greater than 90%.
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cobas® EGFR Mutation Test vs. MPS Sequencing Using Phase 3 Specimens
| cobas® EGFR Mutation Result | MPS Result | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Result in aggregate | | | Exon 19 deletion | | | Exon 21 Mutation | | |
| | MD | MND | Total | MD | MND | Total | MD | MND | Total |
| MD | 142 | 6 | 148 | 94 | 1 | 95 | 48 | 5 | 53 |
| MND | 9 | 251 | 260 | 4 | 309 | 313 | 5 | 350 | 355 |
| Total | 151 | 257 | 408 | 98 | 310 | 408 | 53 | 355 | 408 |
| PPA (95% CI) | 142/151 = 94.0%
(89.1%, 96.8%) | | | 94/98 = 95.9%
(90.0%, 98.4%) | | | 48/53 = 90.6%
(79.7%, 95.9%) | | |
| NPA (95% CI) | 251/257 = 97.7%
(95.0%, 98.9%) | | | 309/310 = 99.7%
(98.2%, 99.9%) | | | 350/355 = 98.6%
(96.7%, 99.4%) | | |
| OPA (95% CI) | 393/408 = 96.3%
(94.0%, 97.8%) | | | 403/408 = 98.8%
(97.2%, 99.5%) | | | 398/408 = 97.5%
(95.5%, 98.7%) | | |
MD denotes Mutation Detected
MND denotes Mutation Not Detected
## 2. Analytical Sensitivity
### a. Analytical Sensitivity - Limit of Blank (LoB)
To assess performance of the cobas® EGFR Mutation Test in the absence of template and to ensure that a blank sample or a sample with wild-type DNA does not generate an analytical signal that might indicate a low concentration of mutation, samples with no template and NSCLC FFPET EGFR wild-type specimens were evaluated.
i. Limit of Blank (LoB) no template – DNA Specimen Diluent reagent was run as the sample with no template. None of the replicates tested across each sample panel and reagent lot gave a “Mutation Detected” result in either channel (FAM or Cy5.5). Cts can be measured out to 55 cycles and results reported as “NaN” for “Not a Number”, indicating no growth curve was observed and no Ct value was determined.
ii. Limit of Blank (LoB) FFPET Specimens – NSCLC FFPET EGFR wild-type specimens were tested. Specifically, 32 specimens wild-type for exon 19 deletion, 36 specimens wild-type for exon 21 L858R mutation, and 18 specimens wild-type for both exon 19 and exon 21 mutations were tested using 50 ng DNA per amplification. There were no detectable Ct values in the FAM channel (EGFR mutation) in the presence of EGFR wild-type DNA isolated from NSCLC FFPET specimens.
### b. Analytical Sensitivity - Limit of Detection (LoD)
Replicate cobas® EGFR Mutation Test measurements were performed on dilution panel members that contained various amounts of genomic DNA and various percentages of the EGFR mutation, which bracketed the expected analytical sensitivity of the cobas® EGFR Mutation Test. The study was performed by testing dilution panels prepared from FFPET specimen blends.
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Specimen FFPET blends – Multiple FFPET specimen DNA extracts for specific exon 19 deletion mutations and the exon 21 L858R substitution mutation were blended with EGFR wild-type FFPET specimen extracts to achieve blends with samples targeting 10, 5.0, 2.5 and 1.25% mutation level as determined by a MPS method, that was validated for detecting the EGFR exon 19 deletions and exon 21 L858R mutation only. Serial dilutions of each specimen blend were prepared and eight (8) replicates of each panel member were run using each of 3 cobas® EGFR Mutation Test kit lots (n=24/panel member). The sensitivity of each sample was determined by the lowest amount of DNA that gave an EGFR “Mutation Detected” rate of at least 95% for the targeted mutation. The study results are summarized in the table below.
Sensitivity of the cobas® EGFR Mutation Test using FFPET Specimen Blends
| EGFR Mutation | EGFR Nucleic Acid Sequence | Percent Mutation in the Panel Member to achieve ≥95% “Mutation Detected” Rate with 50 ng DNA input per reaction well (N=24 replicates) |
| --- | --- | --- |
| Exon 19 Deletion | 2235_2249del15 | 1.4 |
| | 2236_2250del15 | 2.5 |
| | 2238_2252del15 | 2.4# |
| | 2239_2248>C | 2.2 |
| | 2240_2254del15 | 7.2 |
| | 2240_2257del18 | 13.4** |
| | 2237_2253>TTGCT* | 6.32 |
| | 2237_2255>T* | 4.08 |
| | 2239_2256del18* | 4.74 |
| | 2238_2252del15* | 5.45# |
| | 2239_2257>GT* | 6.02 |
| Exon 21 L858R ## | 2573 T>G | 4.0 |
| | 2573 T>G | 4.2 |
| | 2573 T>G | 4.3 |
| | 2573 T>G | 5.3 |
* Only targeting level of approximately 5% mutation was tested for these non-predominant exon 19 deletion mutations present in the EURTAC cohort. Specimens DNA blends were tested across 3 study sites.
** Analytical sensitivity of the cobas® EGFR Mutation Test for detecting this mutation is greater than 10% mutation level using the standard input of 50 ng per reaction well.
# Two independent specimens for the exon 19 deletion (2238_2252del15) were tested.
## Four independent specimens for the exon 21 L858R mutation were tested.
The studies support the claim that the cobas® EGFR Mutation Test can detect 5% EGFR mutant alleles in a background of 95% wild-type alleles in formalin-fixed, paraffin-embedded tumor samples when using 50 ng DNA per amplification reaction (50 μL), with the exception of the 2240_2257del18 exon 19 deletion mutation, which is detected at a sensitivity of >10% (see table above). Although the analytical sensitivity studies have been conducted, the cobas® EGFR Mutation Test is for the
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qualitative detection of the EGFR mutation and is not intended to for quantitative measurements.
c. Analytical Sensitivity – Ct Range and RFI Validation
The Relative Fluorescence Increase (RFI) value intrinsically linked to the Ct value was used as part of an initial assessment for potentially positive mutation results. The final mutation result for a specimen was determined by the observed Ct and CtR values. The preliminary Ct and CtR specifications for the exon 19 deletion and exon 21 mutation calls were established based on testing results for 178 FFPET specimens using one lot of reagents for the cobas® EGFR Mutation Test. Each specimen was tested with one replicate, yielding a total of 178 replicates for analysis. The reference method was massively parallel sequencing (MPS).
The preliminary specifications for exon 19 deletion mutations were confirmed using a data set that included a total of 770 replicates drawn from 204 FFPET specimens. The preliminary specifications for exon 21 L858R mutation were confirmed using a data set that included a total of 640 replicates drawn from 222 FFPET specimens. Multiple lots of reagents were used to generate the confirmatory datasets, which did not include replicates used to establish the preliminary specifications.
Ct and CtR cut-offs for the cobas® EGFR Mutation Test was evaluated by comparing sensitivity and specificity observed for the results of the confirming dataset via Receiver Operating Characteristic (ROC) analysis. The confirmatory test results yielded similar results to the preliminary testing results and indicated that acceptable combination of specificity and sensitivity are optimized for FFPET clinical specimens at the cut-off value listed in the table below. Ct and CtR values are not reported as part of the cobas® test results.
CtR and Ct Threshold Ranges for the cobas® EGFR Mutation Test
| Master Mix and Channel | Mutation or Internal Control | CtR Min | CtR Max | Ct Min | Ct Max |
| --- | --- | --- | --- | --- | --- |
| MMx1 FAM | Exon 19 Deletion | -5.00 | 9.00 | 15.00 | 33.00 |
| MMx2 FAM | Exon 21 L858R | -5.00 | 11.00 | 15.00 | 34.00 |
3. Analytical Sensitivity – Genomic DNA Input Range
Various genomic DNA input amounts may result from DNA quantitation errors and/or variation in the amount of degraded DNA. To evaluate the effects of various genomic DNA input amounts, genomic DNA of five DNA input concentrations of 50, 12.5, 3.1, 0.8 and 0.2 ng per amplification reaction were evaluated as part of the LoD - Specimen FFPET blends study. The study results supported the recommended DNA input of 50 ng per PCR reaction for the cobas® EGFR Mutation Test.
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# 4. Analytical Sensitivity - Minimum Tumor Content
A total of 66 independent EGFR mutant specimens (i.e., 35 of exon 19 deletion mutants and 31 exon 21 L858R mutants) with tumor content ranging from 25% to 99% were tested to determine the minimum tumor content required for detecting the EGFR mutation in NSCLC specimens. None of the specimens evaluated had both the exon 19 deletion mutation and the exon 21 L858R mutation. Each specimen was tested without macrodissection (neat), and after macrodissection. The observed CtR values for the neat and macrodissected slides were analyzed using Deming regression and the Bland-Altman plot (differences vs. mean). The results support the use of samples whose tumor content is greater than 25% without macrodissection.
In the Phase 3 EURTAC trial of erlotinib vs. cisplatin-based chemotherapy, NSCLC FFPET specimens with less than 10% tumor content were macrodissected prior to EGFR mutation analysis. A subset of the screened EURTAC samples was evaluated for EGFR mutation status by both the cobas® EGFR Mutation Test and a MPS method. Tables below included NSCLC specimens with valid paired results of EGFR Exon 19 or L858R mutations combined from both the cobas® EGFR Mutation Test and the MPS sequencing. Using the MPS as the reference method, results showed that macro-dissection of NSCLC FFPET sections with less than 10% tumor content demonstrated comparable analytical accuracy to NSCLC FFPET section without macro-dissection. Together, these studies support that macro-dissection is required for NSCLC FFPET sections with less than 10% tumor content prior to testing with the cobas® EGFR Mutation Test.
Performance of the cobas® EGFR Mutation Test for NSCLC FFPET Specimens with Tumor Contents ≤ 10% (Macro-dissected)
| cobas® EGFR Mutation Test (Test Method) | MPS (Reference Method) | | |
| --- | --- | --- | --- |
| | Mutation Detected^a | Mutation Not Detected^b | Total |
| Mutation Detected | 35 | 3 | 38 |
| Mutation Not Detected | 1 | 52 | 53 |
| Total | 36 (39.6%) | 55 (60.4%) | 91 |
| PPA (95% CI) | 35/36 = 97.2% (85.8%, 99.5%) | | |
| NPA (95% CI) | 52/55 = 94.5% (85.1%, 98.1%) | | |
| OPA (95% CI) | 87/91 = 95.6% (89.2%, 98.3%) | | |
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Performance of the cobas® EGFR Mutation Test for NSCLC FFPET Specimens with Tumor Contents > 10% (Not Macro-dissected)
| cobas® EGFR Mutation Test (Test Method) | MPS (Reference Method) | | |
| --- | --- | --- | --- |
| | Mutation Detected^{a} | Mutation Not Detected^{b} | Total |
| Mutation Detected^{a} | 107 | 3 | 110 |
| Mutation Not Detected^{b} | 8 | 199 | 207 |
| Total | 115 (36.3%) | 202 (63.7%) | 317 |
| PPA (95% CI) | 107/115 = 93.0% (86.9%, 96.4%) | | |
| NPA (95% CI) | 199/202 = 98.5% (95.7%, 99.5%) | | |
| OPA (95% CI) | 306/317 = 96.5% (93.9%, 98.1%) | | |
a Mutation Detected indicates the presence of EGFR Exon 19 or L858R Mutations Combined.
b Mutation Not Detected indicates the absence of EGFR Exon 19 or L858R Mutations Combined.
Note: CI = (score) confidence interval.
## 5. Analytical Specificity
### a. Primer and Probe Specificity
Sequence information and alignment of the primers and probes with the EGFR gene was provided. A traditional Basic Local Alignment Search Tool (BLAST) search was performed for all of the oligonucleotides (primers and probes) as well as the target EGFR exons and amplicons using the human reference genome GRCh37. The traditional BLAST search was conducted using BLASTN 2.2.10 to assess short matches between the query (i.e., oligonucleotides and target sequences) and the database sequences. Based on two threshold parameters, T and S, sequences are reported as potential matches. A ThermoBLAST analysis (BLASTN 2.2.17, version 1.2.2.4.0) was also conducted to assess potential mismatches in hybridization, including stabilizing G-T mismatches. Based on the combined results of the BLAST searches, no potentially cross reacting sequences other than the targeted sequences were identified.
### b. Cross Reactivity
Cross-reactivity of the cobas® EGFR Mutation Test to other EGFR exon 19 and exon 21 mutations were evaluated using the Phase 3 EURTAC clinical trial specimens and EGFR plasmids. While the cobas® EGFR Mutation Test demonstrated cross-reactivity to the mutations listed in the tables below, analytical performance of the cobas® EGFR Mutation Test in detecting these mutations has not been evaluated.
#### i. EURTAC clinical trial specimens
The cobas® EGFR Mutation Test gave “Mutation Detected” results for the following EGFR mutations observed in the EURTAC clinical trial specimens. Analytical performance of the cobas® EGFR Mutation Test in detecting these mutations has not been evaluated.
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Mutations Observed in the Phase 3 EURTAC Cohort Determined to Cross-React with the cobas® EGFR Mutation Test
| Exon 19 – Mutation Call Exon 19 Deletion | | |
| --- | --- | --- |
| Mutation | AA Change | COSMIC ID13 |
| 2234_2251>AAT | K745_T751>K | Not Found |
| 2236_2244del9 | E746_R748>E | Not Found |
| 2236_2252AT | E746_T751>I | 26680 |
| 2236_2263>GAAGCAT | E746_A755>E | Not Found |
| 2237_2251>AAC | E746_751T>E | Not Found |
| 2239_2253>CAA | L747_T751>Q | 51527 |
| 2237_2257>TCT | E746_P753>VS | 18427 |
| 2239_2251>C | L747_T751>P | 12383 |
ii. EGFR mutation plasmids – Plasmid constructs containing the non-predominant mutation for the exon 19 deletion (n=20) and exon 21 L858R mutation (n=1) were blended with wild-type genomic DNA to create 5% mutant sample with 50 ng DNA input per PCR. Results demonstrated that the cobas® EGFR Mutation Test cross-reacts to the following mutations at a ≥ 86% hit rate. Independently, the EGFR exon 19 L747S substitution mutation was also tested at a genomic copy number equivalent to 50 ng/PCR input and confirmed for cross reactivity.
Mutations Determined to Cross-React with the cobas® EGFR Mutation Test
| Exon 19 – Mutation Call Exon 19 Deletion | | |
| --- | --- | --- |
| Mutation | AA Change | COSMIC ID* |
| 2237_2251del15 | E746_T751>A | 12678 |
| 2239_2253del15 | L747_T751del | 6254 |
| 2239_2247del9 | L747_E749del | 6218 |
| 2235_2252>AAT | E746_T751>I | 13551 |
| 2236_2253del18 | E746_T751del | 12728 |
| 2237_2254del18 | E746_S752>A | 12367 |
| 2238_2255del18 | E746_S752>D | 6220 |
| 2238_2248>GC | L747_A750>P | 12422 |
| 2238_2252>GCA | L747_T751>Q | 12419 |
| 2239_2258>CA | L747_P753>Q | 12387 |
| 2240_2251del12 | L747_T751>S | 6210 |
| 2233_2247del15 | K745_E749del | 26038 |
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| 2253_2276del24 | S752_I759del | 13556 |
| --- | --- | --- |
| 2235_2248>AATTC | E746_A750>IP | 13550 |
| 2237_2252>T | E746_T751>V | 12386 |
| 2235_2251>AATTC | E746_T751>IP | 13552 |
| 2235_2255>AAT | E746_S752>I | 12385 |
| 2239_2256>CAA | L747_S752>Q | 12403 |
| 2240T>C | L747S | 26704 |
| Exon 21 –Mutation Call L858R | | |
| --- | --- | --- |
| Mutation | AA Change | COSMIC ID* |
| 2573_2574TG>GT | L858R | 12429 |
Catalogue of Somatic Mutations in Cancer (COSMIC), 2011, v.51, http://www.sanger.ac.uk/genetics/CGP/cosmic
## c. Microorganisms and EGFR Homologs
Specificity of the cobas® EGFR Mutation Test was evaluated by testing lung-related microorganisms, and plasmids of EGFR homologs, i.e., plasmids containing the sequences from each of the HER2, HER3, and HER4 genetic regions analogous to the sequences in EGFR exons 19 and 21 amplified by the cobas® EGFR Mutation Test.
i. **EGFR homolog panels** – EGFR is a member of four structurally related epidermal receptor tyrosine kinase proteins: EGFR/HER1, HER2, HER3 and HER4. The target regions in EGFR exons 19 and 21 were evaluated against the analogous HER2, HER3, and HER4 sequences. To test specific sequences for cross-reactivity, plasmids containing the sequences from each of the HER2, HER3, and HER4 genetic regions analogous to the sequences in EGFR exons 19 and 21 amplified by the cobas® EGFR Mutation Test were created. Each plasmid DNA was isolated and assigned a copy number using a spectrofluorometric method. A control condition without plasmid DNA was included. Each plasmid was added at 15,850 copies/PCR, the genomic copy number equivalent to 50 ng/PCR input. Results indicated that the observed mutations for all tested FFPET specimens matched the expected mutation as determined by sequencing, in the presence and absence of the added HER gene plasmid DNA. Thus, the HER-2, HER-3 and HER-4 analogs of EGFR exons 19 and 21 had no effect on the results obtained with the cobas® EGFR Mutation Test, indicating that the EGFR homolog plasmids do not cross-react with the test.
ii. **Testing of lung-related microorganisms** – *Streptococcus pneumoniae* and *Haemophilus influenzae* at 1 × 10⁶ colony forming units (CFU) were found not to cross react or interfere with the cobas® EGFR Mutation Test when added to specimens containing
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wild-type and mutant EGFR sequences during the tissue lysis step. Presence of *Pseudomonas aeruginosa* and *Aspergillus Niger* at approximately 100 CFU/mL in EGFR MMx1, EGFR MMx2, and EGFR MMx3 were found not to cross react or interfere with the performance of the cobas® EGFR Mutation Test.
## 6. Interference – Effects of Necrotic Tissue
To evaluate the potential interference of high necrotic tissue content in NSCLC FFPET specimens using the cobas® EGFR Mutation Test, 20 NSCLC FFPET specimens, including 8 mutant specimens (4 with exon 19 deletion mutations, 4 with the L858R mutation) and 12 wild-type specimens were evaluated. Percent necrosis, as identified by a pathologist, varied from 0-60% for mutant FFPET specimens and 0-85% for wild-type FFPET specimens.
DNA isolated from two 5-μm sections for each of the 20 NSCLC FFPET specimens was run in duplicates using one lot of reagent. Eight mutant specimens with tumor content of 35-80% and 14-82% mutation were used in this study to assess the impact of the necrotic tissues. All observed results matched the expected results for all the specimens tested. Data supported that necrotic tissue content up to 60% for EGFR mutant and 85% in wild-type NSCLC FFPET specimens do not interfere with the call results for the cobas® EGFR Mutation Test.
## 7. Interference – Triglycerides or Hemoglobin
To evaluate the potential interference of triglycerides and hemoglobin on the performance of the cobas® EGFR Mutation Test, five conditions were tested for each of 10 NSCLC FFPET specimens:
- Hemoglobin (2 mg/mL)
- Buffer Control for Hemoglobin
- Triglycerides (37 mM)
- Buffer Control for Triglycerides
- Neat (No Substance)
Five 5-μm sections were obtained from each of the 10 NSCLC EGFR FFPET specimens. Each of the sections was deparaffinized and spiked with one of the five potential interfering materials in tissue pellet suspension prior to DNA extraction. The levels of triglycerides (37 mM) and hemoglobin (2 mg/mL) were equal to the levels recommended to be tested by the Clinical and Laboratory Standards Institute (CLSI) EP7-A2, Appendix D 2005. Following deparaffinization and the spiking of potential interfering substances, genomic DNA was isolated from each of the spiked tissue specimens using the cobas® EGFR Mutation Test.
Two mutant specimens with tumor content of 55-65% and 15-20% mutation were used in this study to assess the impact of the interference at approximately 3-fold to 4-fold analytical sensitivity. All observed results matched the expected results at the levels of triglycerides and hemoglobin
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tested, indicating that triglycerides and hemoglobin do not interfere with the performance of the cobas® EGFR Mutation Test.
## 8. Interference – Drugs
To evaluate the potential interference of therapeutic drugs which may be present in NSCLC FFPET specimens used to test with the cobas® EGFR Mutation Test, 10 NSCLC FFPET specimens were tested with drugs (i.e., albuterol, ipratropium, fluticasone, ceftazidime, imipenem, cilastin, piperacillin, tazobactam, betadine, lidocaine) and the solvents used to dissolve each drug. Eighteen 5-μm sections were obtained from each of the 10 NSCLC EGFR FFPET specimens. Each of the sections was deparaffinized and spiked with tested drugs or solvent in tissue pellet suspension prior to DNA extraction. The levels of potential interfering substance were equal to the levels recommended to be tested by the Clinical and Laboratory Standards Institute (CLSI) EP7-A2, Appendix D 2005 or at 3x Cmax value as recommended by the drug’s package insert with the exception of betadine, which is a topical solution that was tested as 10μL of a 10% (w/v) solution. Following deparaffinization and the spiking of potential interfering substances, genomic DNA was isolated from each of the spiked tissue specimens using the cobas® EGFR Mutation Test. Four mutant specimens with tumor content of 25-65% and ~20% mutation were used in this study to assess the impact of the interference at 4-fold of analytical sensitivity (LoD = 5%). All observed results matched the expected results for all conditions tested, indicating the tested drugs do not interfere with the performance of the cobas® EGFR Mutation Test.
## 9. Reproducibility
An external study was performed to assess the reproducibility of the cobas® EGFR Mutation Test across 3 external testing sites (2 operators per site), 3 reagent lots, and 5 non-consecutive testing days, with a 13-member panel of DNA samples extracted from FFPET sections of NSCLC wild-type (WT) and mutant tumor specimens. This panel included the L858R mutation in exon 21 and five different exon 19 deletion mutations, which covers majority of the EGFR mutations detected in NSCLC patients. Of 92 runs, 90 (97.8%) were valid. A total of 2,340 tests were performed on the 13 panel members in 90 valid runs; all test results were valid. There were no “Mutation Detected” results in 180 valid tests of WT panel members, producing 100% agreement. Agreements were 100% for 10 of the 12 mutant panel members. For panel member EX19_2240_2257del18 at 5% Mutation, agreement was 62.8% (67 of 180 test results were Mutation not Detected). For panel member EX19_2240_2257del18 at 10% Mutation, agreement was 99.4% (1 of 180 test result was “Mutation not Detected”). Results by overall agreement are presented below. The coefficient of variation (CV) was <6% in all mutation panel members. Within each panel member, the CV was <3.5%. For external control the overall CV was <1.3%. The CV% was <0.5% for between-lots and <1.2% for within-lot.
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Overall Agreement Estimates by Panel Member in the cobas® EGFR Mutation Test Reproducibility Study
| Panel Member | Number of Valid Tests | Agreement (N) | % Agreement (95% CI)^{a} |
| --- | --- | --- | --- |
| Wild-type | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2235_2249del15 - 5% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2235_2249del15 - ≤10% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2236_2250del15 - 5% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2236_2250del15 - ≤10% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2239_2248>C - 5% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2239_2248>C - ≤10% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2240_2254del15 - 5% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2240_2254del15 - ≤10% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX19_2240_2257del18 - 5% Mutation | 180 | 113 | 62.8 (55.3, 69.9)* |
| EX19_2240_2257del18 - ≤10% Mutation | 180 | 179 | 99.4 (96.9, 100.0)* |
| EX21_2573T>G=L858R - 5% Mutation | 180 | 180 | 100 (98.0, 100.0) |
| EX21_2573T>G=L858R - ≤10% Mutation | 180 | 180 | 100 (98.0, 100.0) |
Note: Results were in agreement when a Mutant Type panel member had a valid result of Mutation Detected or when Wild-type panel member had a valid result of Mutation Not Detected.
$95\%$ CI = 95% exact binomial confidence interval.
* Analytical sensitivity of the cobas® EGFR Mutation Test for detecting this mutation is greater than 10% mutation level using the standard input of 50 ng per reaction well.
The total precision mean, standard deviation, and CV (%) of CtR attributed to lot, site/instrument, operator, day, and within run by panel member are summarized below. Across all variance components, the total CV (%) was <6% in all mutant panel members. Within each component, the CV (%) was ≤3.5% across all mutant panel members.
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Reproducibility for Mutant Tumor Specimens
| | | | Standard Deviation and Percent Coefficient of Variation | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Lot | | Site/Inst. | | Operator | | Day | | Within-Run | | Total |
| Panel Member | N | Mean Ctr | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV | CV |
| EX19_2235_2249del15 - 5% Mutation | 180 | 5.6 | 0.135 | 2.4% | 0.090 | 1.6% | 0.011 | 0.2% | 0.075 | 1.3% | 0.129 | 2.3% | 0.220 |
| EX19_2236_2250del15 - 5% Mutation | 180 | 7.0 | 0.017 | 0.2% | 0.094 | 1.3% | 0.000 | 0.0% | 0.088 | 1.3% | 0.168 | 2.4% | 0.213 |
| EX19_2239_2248>C - 5% Mutation | 180 | 6.5 | 0.071 | 1.1% | 0.150 | 2.3% | 0.000 | 0.0% | 0.066 | 1.0% | 0.228 | 3.5% | 0.289 |
| EX19_2240_2254del15 - 5% Mutation | 180 | 7.5 | 0.071 | 0.9% | 0.187 | 2.5% | 0.013 | 0.2% | 0.093 | 1.2% | 0.199 | 2.6% | 0.298 |
| EX19_2240_2257del18 - 5% Mutation | 113 | 8.5 | 0.113 | 1.3% | 0.141 | 1.6% | 0.000 | 0.0% | 0.000 | 0.0% | 0.292 | 3.4% | 0.344 |
| EX21_2573T>G=L858R - 5% Mutation | 180 | 9.0 | 0.154 | 1.7% | 0.138 | 1.5% | 0.000 | 0.0% | 0.122 | 1.4% | 0.145 | 1.6% | 0.280 |
| EX19_2235_2249del15 - ≤10% Mutation | 180 | 4.9 | 0.154 | 3.1% | 0.085 | 1.7% | 0.000 | 0.0% | 0.063 | 1.3% | 0.123 | 2.5% | 0.224 |
| EX19_2236_2250del15 - ≤10% Mutation | 180 | 4.9 | 0.025 | 0.5% | 0.124 | 2.5% | 0.045 | 0.9% | 0.051 | 1.0% | 0.121 | 2.5% | 0.188 |
| EX19_2239_2248>C - ≤10% Mutation | 180 | 5.7 | 0.082 | 1.4% | 0.161 | 2.8% | 0.000 | 0.0% | 0.055 | 1.0% | 0.190 | 3.4% | 0.268 |
| EX19_2240_2254del15 - ≤10% Mutation | 180 | 6.5 | 0.054 | 0.8% | 0.182 | 2.8% | 0.000 | 0.0% | 0.072 | 1.1% | 0.170 | 2.6% | 0.265 |
| EX19_2240_2257del18 - ≤10% Mutation | 179 | 7.4 | 0.213 | 2.9% | 0.235 | 3.2% | 0.052 | 0.7% | 0.160 | 2.2% | 0.250 | 3.4% | 0.438 |
| EX21_2573T>G=L858R - ≤10% Mutation | 180 | 8.5 | 0.177 | 2.1% | 0.148 | 1.7% | 0.026 | 0.3% | 0.101 | 1.2% | 0.124 | 1.4% | 0.281 |
Note: SD = Standard Deviation, CV = Coefficient of Variation.
## 10. Repeatability
To evaluate the repeatability of the cobas® EGFR Mutation test, duplicate results were obtained on 6 NSCLC FFPET specimens with known sequencing results, including 2 EGFR mutant specimens (one exon 19 deletion and one exon 21 L858R) and 4 wild-type specimens. For EGFR mutants, the tumor content were 65% and 70%, and the percentage of mutation were 22% and 40%. Each of the 6 NSCLC FFPET specimens were tested in duplicate by 2 operators using 2 different cobas® DNA Sample Preparation Kit / cobas® EGFR Mutation Test
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kit reagent lot combinations across 4 days; the testing was split between two cobas z 480 analyzers. A total of 32 replicates were evaluated per sample. Overall, 190 of the 192 replicates yielded the expected call, giving an overall call accuracy of 99% across the study.
## 11. Lot-to-Lot Reproducibility
The cobas® EGFR Mutation Test utilizes two separate kits: (1) The cobas® DNA Sample Preparation kit for isolation of DNA from NSCLC FFPET specimens, and (2) the cobas® EGFR Mutation Test for the amplification and detection of the isolated DNA for its EGFR mutation status. Eight NSCLC FFPET specimens were tested with nine combinations of 3 lots of the cobas® DNA Sample Preparation Kit and 3 lots of the cobas® EGFR Mutation Test kit. The 8 NSCLC FFPET specimens included 4 mutant specimens with either exon 19 deletion or exon 21 L858R mutation, and 4 EGFR wild-type specimens. The tumor content in EGFR mutant specimens ranged from 50% to 80% and mutation percentage ranged from 22% to 91%. The test results were analyzed for each specimen, and all observed results matched expected results (100% correct calls). The mean Ct and % CV values for each channel were summarized across lots and no trend in Ct values was observed.
## 12. Specimen Handling – Reproducibility
The reproducibility of the cobas® DNA Sample Preparation Kit was examined using sections taken from three FFPET specimen blocks, one containing an exon 19 deletion mutation (2235-2249del15), one containing an L858R mutation (2573T>G), and one that is wild-type. From each of the three specimens, thirty-six 5-μm curl sections were obtained. Each site tested twelve curl sections for each specimen. The specimen sections for a given specimen were randomized and tested over six days across three sites using three lots of the cobas® DNA Sample Preparation Kit, yielding a total of 36 replicates per specimen.
On each test day, each operator isolated and tested the DNA from two NSCLC FFPET curl sections for each specimen. One operator conducted the testing using one cobas z 480 analyzer per site. One lot of the cobas® EGFR Mutation Test kit reagents was used in combination with all three lots of the cobas® DNA Sample Preparation Kit. All mutant and wild-type specimen results were valid and yielded the expected call result (correct call =100%, 36/36 for each specimen), supporting the reproducibility and repeatability for the cobas® EGFR Mutation Test at the pre-analytical step of DNA isolation.
## 13. Specimen Handling – Curl Versus Slide Equivalency
To evaluate the equivalence of using DNA extracted from 5-μm unmounted NSCLC FFPET sections (FFPET "curls") and DNA extracted from NSCLC FFPET sections mounted on slides (FFPET "slides"), 50 NSCLC FFPET specimens (26 wild-type, 22 mutant specimens; and 2 invalid specimens for exon 21 by Sanger sequencing) were tested. The mutant specimens included 12
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exon 19 specimens (covering 5 different mutations) and 10 exon 21 L858R mutation specimens. Two (2) sections were sliced from each NSCLC FFPET specimen; one section was mounted on a slide and the other "curl" section placed into a microfuge tube, and prepared according to directions. Both the slide and curl sections for each specimen were tested using one lot combination of the cobas® DNA Sample Preparation Kit and cobas® EGFR Mutation Test kit.
Tumor content ranged from 25% to 80% for mutant specimens, and from 1% to 85% for wild-type specimens. The results demonstrated 93.8% (90/96) agreement between unmounted NSCLC FFPET sections and FFPET sections mounted on slides. To investigate the inconsistent result for the discordant specimens, MPS sequencing of the original specimens was performed and new FFPET sections were analyzed. The investigation results suggested possible operator-related contamination event prior to or during the sample processing procedure.
## 14. Specimen Handling – Macrodissection
The accuracy of samples following macrodissection was evaluated with EURTAC clinical trial specimens that had less than 10% tumor content. Refer to Section 4 on "Analytical Sensitivity - Minimum Tumor Content" above for more details.
## 15. Guard banding
The objective of the guard banding studies was to establish the robustness of the PCR conditions for the cobas® EGFR Mutation Test. Guard banding studies were performed on the cobas® EGFR Mutation Test Thermal Cycling Profile, components of the Working Master Mix, and Proteinase K concentration (for DNA isolation procedure).
Three FFPET NSCLC specimen blocks, one containing an exon 19 deletion mutation (2239-2245del15), one containing an L858R mutation (2573T>G), and one that is wild-type, were used. At least ten 5-μm sections were obtained from each of the three specimens and processed to isolate DNA using a single lot of the cobas® DNA Specimen Preparation Kit according to the cobas® EGFR Mutation Test Instructions For Use. After processing, all replicates of each specimen were combined to make a pool of extracted DNA. Three replicates of each specimen pool were tested for each condition using a single reagent lot of the cobas® EGFR Mutation Test.
### a. Thermal Cycling Profile
The thermal cycling profile was guard banded by varying both the denaturation and annealing temperatures by ±1°C. All replicates of each specimen pool produced their expected results. For each specimen tested, the average Ct for each guard band condition was within 1 Ct of the average Ct of the control condition. The Ct difference from control
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condition for all specimens combined ranged from -0.19 to 0.52. The results showed that the cobas® EGFR Mutation Test is able to tolerate variations of ±1°C of the thermal cycling profile in denaturation and annealing temperatures.
b. **Working Master Mix (MMx)**
The components of the PCR amplification mixture (MMx, MGAC, and Sample) were tested by varying each individual component volumetrically while keeping the other two components at the control volumes. Each EGFR MMx reagent volume was tested using the control condition as well as varying the volume by ±10%. The MGAC reagent volume was evaluated using the control condition as well as varying the volume by ±10%. The sample input volume was assessed using the control condition as well as varying the volume by ±20%. All replicates of each specimen pool produced their expected results. For each specimen tested, the average Ct for each guard band condition was within 1 Ct of the average Ct of the control. The Ct difference from control condition for all specimens combined ranged from -0.27 to 0.26. The results showed that the cobas® EGFR Mutation Test is able to tolerate volume differences of ±10% for each MMx reagent, ±10% of the MGAC reagent, and ±20% of DNA sample.
c. **Proteinase K (PK)**
During sample preparation, the NSCLC FFPET specimen is lysed by incubation at an elevated temperature with a proteinase and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. For each specimen processed by the cobas® DNA Sample Preparation kit, 70 μL of PK and 180 μL of DNA Tissue Lysis Buffer are added. The mixture is first incubated at 56°C for 60 minutes and then at 90°C for 60 minutes.
Proteinase K was guard banded by varying the PK volume (±20%), the first incubation temperature (±2°C), and the first incubation time (±25%) using the cobas® DNA Sample Preparation kit. Following the cobas® EGFR Mutation Test Instructions For Use, three operators each processed one replicate of three NSCLC FFPET specimens for nine PK conditions. A single reagent lot was used in this study and a total of three runs were performed. All replicates of each specimen produced their expected results. For each PK condition tested, the average Ct for each guard band condition was within 1 Ct of the average Ct of the control condition. The Ct difference from control condition for all specimens combined ranged from -0.26 to 0.32. The results showed that the cobas® EGFR Mutation Test is able to tolerate differences of ±20% for PK volume, ±2°C for the first incubation temperature, and ±25% for the first incubation time.
16. **Cross-Contamination**
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AmpErase® (Uracil- N-glycosylase) is used in the test to avoid carryover contamination. Studies were performed to evaluate the incidence of false-positive results when replicates of NSCLC FFPET specimens with high and low EGFR mutation percentages were run adjacent to replicates of an EGFR wild-type FFPET specimen. Cross contamination with the cobas® EGFR Mutation Test was evaluated by co-processing two exon 19 deletion (25.0% and 92.1% mutation) and two exon 21 L858R mutations (16.5% and 89.0% mutation) specimens with four wild-type specimens in an alternating pattern. One lot of the cobas® EGFR Mutation Test and a total of eight specimens were tested. Each of the mutant specimens was paired with a wild-type specimen for testing.
For each run, 12 replicates of each mutant NSCLC FFPET specimen were paired with 12 replicates for the corresponding wild-type specimen and they were co-processed in an alternating pattern. A total of five runs were performed for each mutant/ wild-type specimen pair in one day, resulting in a total of 60 replicates per specimen tested per day. Testing for all four specimen pairings was completed across four days. A total of 8 cobas z 480 analyzers were used by a total of 5 operators where the same 5 operators were used for each day of testing. The observed calls for all 479 valid tests matched the expected call. The average CtR, average Ct, associated SD, and %CV were analyzed for each specimen, combining results over different runs per specimens. No trend in CtR and Ct values was observed over the five consecutive runs across different days.
## 17. Stability Studies
### a. Clinical Specimens, FFPET Blocks
The stability of NSCLC FFPET specimen blocks stored at room-temperature (15°C to 32°C) was evaluated using 6 NSCLC FFPET specimens, including 4 EGFR wild-type and 2 EGFR mutant specimens. The 2 EGFR mutant specimens were 1 exon 19 deletion (39.4% mutation) and 1 exon 21 L858R mutation (14.4% mutation) specimens. Two sections were obtained from each of the specimens with tumor content ranged from 35% to 75%. The specimen blocks were stored at 32°C with testing conducted at 0, 3, 6, 9, and 13 months using one lot of the reagent. For all specimens except one replicate tested, the observed results matched the expected results, and no trend in Ct values was detected. One replicate of a wild-type specimen tested at 9 month did not yield the expected result. This result is likely due to decrease of tumor tissue when the block is cut through. The results support that NSCLC FFPET clinical specimens are stable for up to 13 months when stored at 15-30°C
### b. Clinical Specimen, Slide-Mounted and Slide Curl
To demonstrate the stability of sections from NSCLC FFPET clinical specimens when mounted on a slide ("slide") or when not mounted on a slide ("curl"), 6 NSCLC FFPET specimens, including 3 EGFR mutant and 3 EGFR wild-type specimens were evaluated. The 3 EGFR mutant
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specimens were 2 exon 19 deletion (23.7% and 71.0% mutation) and 1 exon 21 L858R mutation (14.4% mutation) specimens. Multiple 5-micron sections were obtained from each of the 6 NSCLC FFPET specimens, mounted or not mounted on slides (one section per slide). The slides and curls were stored at 2-8°C and 32°C and tested after 0, 15, 51 days and 6 months. At each time point, two slides and two curls for each specimen and storage temperature were processed using one lot of the reagent. Results from the 6 specimens matched the expected results (based on sequencing results) at all time points. No trend in Ct values was detected over these testing conditions. These results indicated that sections stored as slides or curls are stable for 6 months at 2-8°C and 32°C storage for testing with the cobas® EGFR Mutation Test.
c. Extracted DNA From FFPET Specimens
Stability of DNA extracted from NSCLC FFPET specimens was evaluated using 4 EGFR wild-type specimens, 2 exon 19 deletion mutant specimens (25.0% and 78.4% mutation), and 2 exon 21 L858R mutant specimens (16.5% and 88.6% mutation). DNA extracts obtained from each NSCLC FFPET specimens were tested as follows:
i. after storage at -20°C for 15, 31 or 61 days,
ii. after storage at 2 to 8°C for 0, 8, 15, 31, or 61 days,
iii. after storage at 32°C for 2, 4, or 8 days,
iv. after one, two, or three freeze-thaw cycles consisting of storage at -20°C, thawing, re-freezing and sampling at the specified -20°C time points.
After storage at -20°C, 2 to 8°C, or 32°C, results from the 8 specimens matched the expected results (based on sequencing results) at all time points, indicating that DNA extracted from NSCLC FFPET specimens using the cobas® DNA Specimen Preparation Kit is stable for at least 60 days when stored at 2-8°C or -20°C. The results also indicated that the extracted DNA is stable for at least 8 days when stored at 32°C, and up to three freeze/thaw cycles when stored at -20°C. No trend in Ct values was detected over these testing conditions.
d. Working (Activated) Master Mix
To evaluate the stability of cobas® EGFR Mutation Test Working (activated) Master Mixes stored at 2-8°C and 32°C for up to 125 minutes, 8 NSCLC FFPET specimens, including 4 EGFR mutant and 4 EGFR wild-type specimens, were tested using 1 lot of reagent. The 4 EGFR mutant specimens were 2 exon 19 deletion (78.4% and 25.0% mutation) and 2 exon 21 L858R mutation (16.5% and 88.6% mutation) specimens. Working (activated) Master Mix was prepared by adding EGFR Master Mix to Magnesium Acetate, and then stored at 2-8°C for 0, 35, 65, and 125 minutes, and at 32°C for 0, 35, 65, and 125 minutes. Multiple 5-micron sections were obtained from each of the 8 NSCLC FFPET specimens. Each of the sections was processed to isolate DNA using the cobas® DNA
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Sample Preparation Kit. DNA obtained from a single specimen was combined, resulting in a pool of extracted DNA for each specimen. After the indicated storage time, duplicate samples of DNA extracts from each of 8 NSCLC specimens were combined with the Working Master Mixes and amplified and detected using the cobas® EGFR Mutation Test. All valid specimens' results matched the expected results at all time points, indicating that cobas® EGFR Mutation Test working (activated) Master Mix is stable for at least two hours when stored at at 2-8°C or 32°C.
e. **Extracted DNA Plus Working (Activated) Master Mix**
To evaluate the stability of the combination of extracted DNA from NSCLC FFPET specimens and cobas® EGFR Mutation Test Working (activated) Master Mixes, 8 NSCLC FFPET specimens, including 4 EGFR mutant and 4 EGFR wild-type specimens, were tested using 1 lot of reagent. The 4 EGFR mutant specimens were 2 exon 19 deletion (78.4% and 25.0% mutation) and 2 exon 21 L858R mutation (16.5% and 88.6% mutation) specimens. DNA was extracted from each of 8 NSCLC FFPET specimens using the cobas® DNA Specimen Preparation Kit, combined with Working (activated) Master Mixes, and stored at 2-8°C for 0, 35, 65, and 125 minutes, and at 32°C for 0, 35, 65, and 125 minutes. After the indicated storage time, the combined DNA extract/Working Master Mixes were amplified and detected using the cobas® EGFR Mutation Test. Results from the 8 specimens matched the expected result (based on sequencing results) at all time points after storage at 2-8°C or 32°C for 0, 35, 65, and 125 minutes. No trend in Ct values was detected over these testing conditions. These results indicated that DNA extracted from NSCLC FFPET specimens using the cobas® DNA Specimen Preparation Kit and the cobas® EGFR Mutation Test Working Master Mix is stable for up to 125 minutes when stored at 2-8°C or 32°C prior to the start of amplification.
f. **Open Vial, cobas® EGFR Mutation Test Kit Reagents**
To determine the open vial stability of the reagents in the cobas® EGFR Mutation Test kit, 2 kits were used to test 6 NSCLC FFPET specimens, including 2 EGFR mutant and 4 EGFR wild-type specimens. The 2 EGFR mutant specimens were 1 exon 19 deletion (78.4% mutation) and 1 exon 21 L858R mutation (38.2% mutation) specimens. One kit was tested on Days 0, 15, 21, and 31 and the second kit was tested on Days 0, 45, 61, and 91. The two kits and testing time points were done to demonstrate the open vial stability of the cobas® EGFR Mutation Test kit reagents for up to 30 days and 90 days with up to 4 uses per kit. Multiple 5-μm sections were obtained from each NSCLC FFPET specimens. All results from the 6 specimens matched the expected result when the cobas® EGFR Mutation Test kit reagents were used 4 times over 91 days when stored at 2-8°C between uses. No trend in Ct values was detected over these open-
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vial storage periods. The results indicate that the open vial stability of the cobas® EGFR Mutation Test kit reagents is at least 90 days.
g. **cobas® EGFR Mutation Test**
Stability of the cobas® EGFR Mutation Test Kit and its components were assessed at various time points after storage at 2-8°C (real time) in upright and inverted orientations using 3 lots of the kit reagents. The test samples were the EGFR Mutant Control (EGFR MC) and the DNA Specimen Diluent (DNA SD), which serve as a positive control and negative control in the cobas® EGFR Mutation Test. Eight replicates of the EGFR MC and DNA SD were tested at each storage condition. Stability was evaluated by performing functional testing at 4 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 13 months, 18 months, 19 months, 24 months, and 25 months. For a given storage condition at any time point to pass, the eight replicates of the EGFR MC and DNA SD must be within the pre-specified ranges of Ct, which are identical to the Ct values for assessing validity of a run and assigning mutation status. To date, testing has been completed and met the acceptance criteria through 18 months storage at 2-8°C for all three lots of the kit reagents. Current real-time stability data support stability of the cobas® EGFR Mutation Test at 2-8°C for 18 month. Real-time stability studies are ongoing to support the 24 months expiry.
h. **cobas® DNA Sample Preparation Kit**
Stability of the cobas® DNA Sample Preparation Kit, including open-vial stability, was demonstrated in P110020 approval of the cobas® 4800 BRAF V600 Mutation Test.
i. **Shipping**
To evaluate the tolerance of the cobas® EGFR Mutation Test Kit to temperature extremes that can occur during shipping of the product, four different temperature profiles were tested according to the tables below. For each temperature profile, five kits were each subjected to the two Category A profiles, Category B, and the 2-8°C condition. The kits were all from one production lot of reagents, which was stored at 2-8°C for 15 months prior to the start of the shipping stability study.
At the beginning of the study, all of the kits were stored at 2-8°C. Ten kits were transferred to begin the two Category A storage protocols, five kits were transferred to Category B storage protocol, and five kits remained stored at 2-8°C for the control condition. The test samples were the EGFR Mutant Control (EGFR MC) and the DNA Specimen Diluent (DNA SD), which serve as a positive control and a negative control in the cobas® EGFR Mutation Test, respectively. Eight (8) replicates of the EGFR MC and DNA SD are assessed for each temperature profile. The shipping stability study was repeated on kits that have been stored at the recommended temperature (2-8°C) for a minimum of 15 months. Results
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indicated that the reagents and controls in the cobas® EGFR Mutation Test are not affected by the temperature extremes that can occur during shipping of the product under any of the four shipping categories studied.
## Temperature Profiles Followed During Stressing of the Reagent Kits
| Steps in the Profile | | Temperature Profile for Testing Each Shipping Category | | | Steps in the Profile | Temperature Profile for Testing Each Shipping Category | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Step | Duration | Category A37 (5 Kits) | Category A-20 (5 Kits) | 2-8°C Control (5 Kits) | | | |
| 1 | 2 Days | 4°C | 4°C | 4°C | Step | Duration | Category B (5 Kits) |
| 2 | 20 Hours | 32°C | 32°C | | 1 | 2 Days | 4°C |
| 3 | 5 Days | 15°C | 15°C | | 2 | 20 Hours | 25°C |
| 4 | 20 Hours | 32°C | 32°C | | 3 | 5 Days | 15°C |
| 5 | 10 Days | 15°C | 15°C | | 4 | 20 Hours | 25°C |
| 6 | 5 Days | 37°C | -20°C | | 5 | 10 Days | 15°C |
| | | | | | 6 | 5 Days | 25°C |
## 18. Antimicrobial Effectiveness Testing (AET)
To assess the effectiveness of the preservatives in the cobas® EGFR Mutation Test kit components, AET was performed on kit components (i.e., EGFR Master Mixes, EGFR Mutant Control, Magnesium Acetate Reagent and DNA Specimen Diluent). Each kit component was stored at 2-8°C and tested at Month 0 and Month 13. At each time point interval, a total of five microorganisms (Staphylococcus aureus, Candida albicans, Aspergillus niger, Pseudomonas aeruginosa and Escherichia coli) were individually spiked in each of the kit components on Day 0, and the colony forming units (CFU) count in log10 was determined on Day 14 and Day 28. To date, all components have passed 13 months of AET. Testing is ongoing.
Functional AET of the Master Mix reagents in the presence of two microorganisms (Pseudomonas aeruginosa and Aspergillus niger) was also conducted. The Master Mix reagents, with and without added microorganisms (100 CFU/mL) were stored at the recommended storage temperature (2-8°C), and tested at Day 0, Months 3, 6, 9, 12, 15, 18, 21 and 25 using the cobas® EGFR Mutation Test. The EGFR Mutant Control and DNA Specimen Diluent are being used as samples at each time point. For a given time point to pass, the EGFR Mutant Control and Sample Diluent must be within the pre-specified ranges for the EGFR Mutant Control and EGFR Negative Control. All test results for all conditions tested to date were valid and passed up to 18 month. There is no impact of microbial contamination on the functional performance of the cobas® EGFR Mutation Test at 2-8°C for up to 18 months.
## B. Animal Studies
None.
## C. Additional Studies
None.
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# X. SUMMARY OF PRIMARY CLINICAL STUDY
The EURTAC¹ study and the EURTAC bridging study was the basis of for the PMA approval decision. The Phase 3 EURTAC clinical study was conducted in Europe. Justification to support Outside-the-US (OUS) clinical data was provided in sNDA 21743, supplement 18. The international, multi-center, randomized Phase 3 EURTAC study (ML20650) tested the administration of Tarceva® (erlotinib) versus standard platinum-based chemotherapy to support a supplemental New Drug Application (sNDA 21743) for first-line treatment of patients with advanced NSCLC whose tumors harbor EGFR activating mutations. The EURTAC study was sponsored by F. Hoffmann-La Roche, Ltd., and was conducted by the Spanish Lung Cancer Group (SLCG) at approximately 45 centers in 3 countries (Spain, France and Italy). Justifications for using foreign data to support device approval in U.S. was provided in sNDA 21743, supplement 18. The study dates were from February 1, 2007 (first patient screened) to April 11, 2012 (clinical data cut-off). The EGFR mutation status of tumor tissue was assessed for patient eligibility using a clinical trial assay (CTA) at one single site, which is at the Molecular Biology Laboratory of the Medical Oncology Service-ICO at Germans Trias I Pujol University Hospital, Badalona, collaborator of the SLCG.
In the EURTAC study, the EGFR mutation status of the patients was determined at a central laboratory using Sanger sequencing first for determination of EGFR mutation status, followed by confirmatory testing for exon 19 deletions (GeneScan) and exon 21 L858R mutations (TaqMan PCR). The cobas® EGFR Mutation Test was developed after patients had been screened and enrolled in EURTAC study using the CTA. Retrospective testing of tissue specimens from patients screened from the EURTAC study was performed using the cobas® EGFR Mutation Test. A bridging study was conducted to assess the concordance of the cobas® EGFR Mutation Test results with the CTA used to select patients for the EURTAC trial. To establish the clinical utility of the cobas® EGFR Mutation Test, clinical outcomes (progression-free survival or PFS and response rates) for all patients enrolled in the EURTAC trial (i.e., CTA-positive) were compared to the outcomes of patients whose specimens were mutation-positive upon retrospective testing with the cobas® EGFR Mutation Test (i.e., cobas-positive). A summary of the clinical study is presented below.
## A. Study Design
**EURTAC Study:** The EURTAC study was a Phase 3, multicenter, open-label, randomized study of erlotinib treatment versus chemotherapy in patients with advanced non-small-cell carcinoma of the lung who present activating mutations in the tyrosine kinase (TK) domain of EGFR. Randomization of 174 patients with CTA mutation detected results was stratified according to ECOG performance status and EGFR mutation (i.e., exon 19 deletion vs. exon 21 L858R substitution mutation). Randomized patients were assigned 1:1 to receive chemotherapy or erlotinib treatment as summarized in the figure below. Among the 174 patients enrolled in EURTAC; 86 were allocated to receive erlotinib, 87 to receive chemotherapy, and 1 was excluded because the patient started treatment prior to randomization. The
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database for this PMA reflected data collected through February 01, 2007 and April 11, 2012.
# EURTAC Study Design
ECOG – Eastern Cooperative Oncology Group
The treatment arms were well-balanced with respect to general demographic characteristics, with the exception of gender (female, 78% in the chemotherapy arm vs. 67% in the erlotinib arm) and smoking status (current smoker, 14% in the chemotherapy arm vs. 8% in the erlotinib arm; past smoker, 14% in the chemotherapy arm vs. 26% in the erlotinib arm; and never smoked, 72% in the chemotherapy arm vs. 66% in the erlotinib arm). The overall study population comprised more females than males (72.8% and 27.2%, respectively) and the vast majority of patients were of the same race (White). The median age was 65 years for both the chemotherapy and erlotinib arms
The primary objective of the ERUTAC study was to compare investigator assessed progression-free survival (PFS) in the two treatment arms of the study (conventional chemotherapy vs. erlotinib) in patients with NSCLC in advanced stages (stages IIIB and IV) who have not received previous chemotherapy or any other systemic antitumor therapy for their disease and who present mutations in the TK domain of the EGFR. A two-sided log-rank test was used for testing the difference in PFS and overall survival between the erlotinib and chemotherapy arms. The primary efficacy analysis was performed at a two-sided significance level of 0.025. All other tests were two-sided at a 5% significance level. The primary analysis population for
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efficacy was the intent-to-treat (ITT) population, also defined as the fully analysis set (FAS) in the study protocol. All patients who received at least one dose of trial medication and had at least one safety follow-up, whether withdrawn prematurely or not, were included in the safety analysis. The safety parameters were analyzed and presented according to the therapy the patient received.
Data from this study were monitored by an Independent Data Monitoring Committee (IDMC), which was responsible for reviewing the efficacy and safety data from the pre-planned interim analysis based on the pre-defined statistical analysis plan. The IDMC consisted of two oncologists and a biostatistician with expertise in oncology trials.
## 1. Clinical Inclusion and Exclusion Criteria For Patient Enrollment
### Inclusion Criteria
1. Before beginning the specific procedures in the protocol, informed consent was obtained in writing from the patient and documented before witnesses.
2. Histologic diagnosis of NSCLC, stage IV or stage IIIB with malignant pleural effusion or N3 tumors not candidates for thoracic irradiation who present with exon 19 deletions or exon 21 mutation in the tyrosine kinase domain of EGFR.
3. Measurable or evaluable disease.
4. Patients over 18 years.
5. Performance status ≤ 2 on the Eastern Cooperative Oncology Group (ECOG) scale.
6. Adequate bone marrow reserve:
a) Hemglobin ≥ 9 g/dL
b) Absolute neutrophil count > 1500/L
c) Platelet count > 100,000/L
7. Adequate kidney function.
8. Adequate liver function.
9. Patients must be accessible for treatment and follow-up.
10. Patients capable of proper therapeutic compliance and accessible for correct follow-up.
11. Women of childbearing age must have a negative serum or urine pregnancy test within 7 days before beginning treatment.
12. Patients of both sexes of childbearing age, including women who had their last menstrual period in the last 2 years, must use an effective contraceptive method.
13. Oral swallowing capability.
14. Patients with asymptomatic and stable cerebral metastases receiving medical treatment were eligible for the study. Patients who received radiation therapy for their cerebral metastases before the initiation of systemic treatment for NSCLC were also eligible.
15. Absence of intestinal transit problems, such as malabsorption syndrome, chronic intestinal inflammatory disease, or other pathologies that, in the judgment of the investigator, can alter absorption of the medication.
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Exclusion Criteria
1. Women who were pregnant or in the period of lactation.
2. Women of childbearing age who presented a positive pregnancy test result in the baseline evaluation or who did not undergo this test.
3. Sexually active men and women (of childbearing age) who were not willing to use contraceptive methods during the study.
4. Previous treatment with chemotherapy for metastatic disease. The administration of neoadjuvant or adjuvant chemotherapy was allowed as long as it was completed ≥ 6 months before entering the study.
5. Previous treatment with therapeutic agents targeting EGFR.
6. Patients could have received radiotherapy as long as the irradiated lesion was not the only target lesion for evaluating response and as long as radiotherapy had been completed before initiating the study treatment (a 2-week period was recommended).
7. Treatment with an investigational drug agent during the 3 weeks before enrollment in the study.
8. Any known significant ophthalmologic anomaly of the ocular surface. The use of contact lenses was not recommended.
9. Pre-existent motor or sensorial neurotoxicity grade ≥ 2 according to the National Cancer Institute – Common Terminology Criteria (NCI-CTC) AE scale.
10. Evidence of spinal cord compression.
11. Incapacity to take oral medication or previous surgical procedures that affect absorption and imply the need for intravenous or parenteral feeding.
12. Other serious diseases or clinical conditions.
13. Absolute contraindication for steroid use.
14. Dementia or significantly disturbed mental state that could interfere with the patient’s understanding and granting of informed consent.
15. History of another neoplasm other than carcinoma in situ of the uterine cervix, basal cell skin carcinoma treated adequately, or prostate carcinoma with a good prognosis (Gleason ≤ 6) treated radically. History of another neoplasm treated curatively and without evidence of disease in the last 5 years.
2. Follow-up Schedule
Patients were followed for efficacy:
Patients in each treatment arm underwent scheduled clinical and tumor assessments via scans every 6 weeks until confirmation of disease progression and thereafter, patients were followed every 3 months until death to document follow-up of adverse events (AEs) (if present), date of death and further-line treatments.
Patients were followed for safety:
Adverse events (AE) were recorded as they were encountered during the study and up until 28 days after final administration of study medication and classified according to the NC…
