Confidential
Final
3 March 2015
Title page
Technical report for the Health Innovation Partnership of the Health
Research Council of New Zealand and National Health Committee
Study title: Epidermal Growth Factor Receptor (EGFR) Mutation Testing
Host Organisation: Auckland UniServices Limited, University of Auckland
Study investigators and affiliations
Professor Mark McKeage, Pharmacology and Clinical Pharmacology and Auckland Cancer Society
Research Centre, University of Auckland
Dr Donald Love, Diagnostic Genetics, LabPLUS, Auckland District Health Board
Mr Phillip Shepherd, Liggins Institute, University of Auckland
Professor Mark Elwood, Epidemiology and Biostatistics, University of Auckland
Dr George Laking, Medical Oncology, Auckland District Health Board
Dr Nichola Kingston, Pathology, LabPLUS, Auckland District Health Board
Dr Christopher Lewis, Respiratory Medicine. Auckland District Health Board
Study date
Start 1st November 2013
Finish 31st October 2014
Signature
Mark McKeage, 7 Nov 2014
1
Confidential
Final
3 March 2015
1 Table of Contents
1
Table of Contents ............................................................................................................................ 2
2
List of Tables ................................................................................................................................... 4
3
Summary ......................................................................................................................................... 5
4
5
3.1
Aims......................................................................................................................................... 5
3.2
Methods .................................................................................................................................. 5
3.3
Results ..................................................................................................................................... 5
3.4
Conclusions ............................................................................................................................. 5
Introduction .................................................................................................................................... 6
4.1
Objective ................................................................................................................................. 6
4.2
Rationale ................................................................................................................................. 6
Aims................................................................................................................................................. 6
5.1
Aim 1 ....................................................................................................................................... 6
5.2
Aim 2 ....................................................................................................................................... 6
5.3
Aim 3 ....................................................................................................................................... 6
6
Research design .............................................................................................................................. 7
7
Clinical laboratory test and retesting study .................................................................................... 8
7.1
Background and Aims ............................................................................................................. 8
7.1.1
7.2
Study design and methods...................................................................................................... 8
7.2.1
Study design .................................................................................................................... 8
7.2.2
Eligibility criteria.............................................................................................................. 8
7.2.3
Study endpoint ................................................................................................................ 8
7.2.4
Study assessments .......................................................................................................... 8
7.3
8
Specific Aim 1 .................................................................................................................. 8
Results ..................................................................................................................................... 8
7.3.1
Tested study population ................................................................................................. 8
7.3.2
Retested study population .............................................................................................. 9
7.3.3
Agreement analysis ....................................................................................................... 10
7.3.4
Summary ....................................................................................................................... 14
Patient cohort study ..................................................................................................................... 15
8.1
Background and Aims ........................................................................................................... 15
8.1.1
Specific Aim 1 ................................................................................................................ 15
8.1.2
Specific Aim 2 ................................................................................................................ 15
8.2
Research design and methods .............................................................................................. 15
2
Confidential
Final
3 March 2015
8.2.1
Study design .................................................................................................................. 15
8.2.2
Eligibility criteria............................................................................................................ 15
8.2.3
Study assessments ........................................................................................................ 15
8.3
Results ................................................................................................................................... 16
8.3.1
Population-based patient cohort .................................................................................. 16
8.3.2
EGFR gene mutation testing in the population-based patient cohort study population
16
8.3.3
Features of eligible cohort patients, and factors related to having had an EGFR gene
mutation test ................................................................................................................................ 17
8.3.4
Features associated with EGFR gene mutations in eligible cohort patients ................. 17
8.3.5
EGFR tyrosine kinase inhibitor (TKI) drug treatment in the population-based patient
cohort study population ............................................................................................................... 18
8.3.6
8.4
9
Mortality outcomes ...................................................................................................... 18
Summary ............................................................................................................................... 18
References .................................................................................................................................... 22
3
Confidential
Final
3 March 2015
2 List of Tables
Table 1 Results of LabPLUS EGFR gene mutation test reports for detecting EGFR gene mutations in
patients samples from the tested study population (n = 826) ............................................................... 9
Table 2 Frequency of specific EGFR gene mutations detected in a New Zealand lung cancer patients
with EGFR gene mutation-positive lung cancer (n=145) from the tested study population. ................. 9
Table 3 Results of the cobas EGFR Mutation Test for detecting EGFR gene mutations in samples from
the retested study population (n=532)................................................................................................. 10
Table 4 Results of sample retesting undertaken by Sequenom MassArray OncoFocus panel
genotyping for the detection of EGFR gene mutations in the retested study population (n=532), using
different Sample ID cut-off sample validity criteria. ............................................................................. 10
Table 5 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping
for the detection of EGFR gene mutations in lung cancer samples from New Zealand patients
(agreement analysis study population). Aggregated results for all EGFR gene mutations using a
Sample ID cut-off for validity of ≥500 amplifiable copies. .................................................................... 11
Table 6 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping
for the detection of EGFR gene exon 20 L858R mutations in lung cancer samples from New Zealand
patients (agreement analysis study population). Sample ID cut-off for validity of ≥500 amplifiable
copies. ................................................................................................................................................... 11
Table 7 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping
for the detection of EGFR gene exon 19 deletion mutations in lung cancer samples from New
Zealand patients (agreement analysis study population). Sample ID cut-off for validity of ≥500
amplifiable copies. ................................................................................................................................ 12
Table 8 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping
for the detection of EGFR gene mutations in lung cancer samples from New Zealand patients.
Aggregate result for Sample ID cut-off for validity of ≥300 amplifiable copies.................................... 13
Table 9 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping
for the detection of EGFR gene mutations in lung cancer samples from New Zealand patients.
Aggregate result for Sample ID cut-off for validity of >0 amplifiable copies........................................ 14
Table 10 Frequency of testing (Cobas EGFR Mutation test) in eligible cohort patients (n=748) ......... 19
Table 11 Results of Cobas EGFR Mutation testing: patients with valid result only (n=414),................ 20
Table 12 EGFR TKI use for patients with Cobas positive test (n= 85) ................................................... 21
4
Confidential
Final
3 March 2015
3 Summary
3.1 Aims
(1) To evaluate the clinical performance of a cobas EGFR Mutation Test being used by LabPLUS
(Auckland City Hospital) for analysing clinical samples from New Zealand lung cancer patients
compared to a reference assay.
(2) To describe the incidence, demographic profiles and patient outcomes for EGFR gene mutationpositive lung cancer in New Zealand.
(3)To ascertain the level and equity of patient access to EGFR gene mutation testing and tyrosine
kinase inhibitor drug treatment for lung cancer patients residing in a large diverse region of NZ.
3.2 Methods
The clinical performance of the cobas EGFR Mutation Test was assessed by agreement analysis by
comparing reports of EGFR gene mutation tests issued by LabPLUS medical laboratory with results of
the retesting of patient tumour DNA extracts by a Sequenom MassArray OncoFocus genotyping
panel system at the University of Auckland facility. Patients eligible for a population-based cohort
were residents of the Auckland, Counties Manakau, Northland and Waitemata District Health
Boards, who had presented with non-small cell lung cancer, of the non-squamous or not-otherwisespecified morphological subtypes, on or after 1 August 2012. Data on their cancer diagnoses, EGFR
gene mutation testing status and access to EGFR tyrosine kinase inhibitor drug treatment were
obtained from the New Zealand Cancer Registry, medical laboratory test listings and patient medical
records. Data analysis used descriptive statistics.
3.3 Results
Agreement analysis demonstrated a positive percentage agreement of 91.2%, negative percentage
agreement of 96.2% and overall percentage agreement of 95.3%, for the detection of EGFR gene
mutations by the cobas EGFR Mutation test, compared to a reference assay, which was the
Sequenom MassArray genotyping panel system. A population-based estimate of the prevalence of
EGFR gene mutations was 20% (95% CI 16.5 to 24.1%) among 414 tested patients eligible for
inclusion in a population-based cohort of New Zealand lung cancer patients (n=748). EGFR gene
mutations were found more commonly among women, SE Asian subjects and non-smokers. EGFR
gene exon 19 deletion and exon 20 L858R mutations, together, accounted for 75% of all EGFR gene
mutations detected in a tested population of New Zealand lung cancer patients. Only 57% of eligible
patients in this population-based New Zealand cohort of lung cancer patients were tested for EGFR
gene mutations, with Maori, males and younger patients, along with those presenting earlier during
the study period, being overrepresented among those not having been tested. About 70% of
patients identified with as having an EGFR gene mutation, were treated with an EGFR tyrosine kinase
inhibitor drug. Older patients and those with localised disease were less likely to receive EGFR
tyrosine kinase inhibitor drug treatment.
3.4 Conclusions
The cobas EGFR Mutation test was an accurate diagnostic assay for detecting EGFR gene mutations
in clinical samples from New Zealand lung cancer patients. EGFR mutation-positive lung cancer is
prevalent among New Zealand lung cancer patients, but uptake of both EGFR gene mutation testing
and EGFR tyrosine kinase inhibitor drug treatment may be influenced by their ethnicity, gender and
age.
5
Confidential
Final
3 March 2015
4 Introduction
4.1 Objective
To assist in the development of recommendations for EGFR gene mutation testing in New Zealand
4.2 Rationale
Lung cancer is a major cause of mortality and morbidity in New Zealand. New Zealand healthcare
leaders previously identified lung cancer as an opportunity for initiatives for improving health.
Mutations in the Epidermal Growth Factor Receptor (EGFR) gene are responsible for initiating and
driving the progression of lung cancer in a subset of patients [1]. International randomised
controlled trials have shown therapeutic beneficial efficacy in using EGFR tyrosine kinase inhibitor
drugs relative to standard chemotherapy, in lung cancer patients whose tumours harbour EGFR
mutations but not in those with lung cancer negative for EGFR mutation [2-6]. International standard
oncology practice guidelines now recommend undertaking EGFR gene mutation testing prior to
systemic treatment of advanced lung cancer patients, and selecting an EGFR tyrosine kinase inhibitor
drugs and systemic chemotherapy for those with or without EGFR mutations, respectively [7].
From August 2012, PHARMAC had made available publicly-funded gefitinib via Special Authority for
patients with EGFR mutation positive lung cancer. However, at the time there was no nationally
standardised process in place for EGFR gene mutation testing in the New Zealand healthcare system.
Previously, very little research had been undertaken about EGFR mutation positive lung cancer in
New Zealand, and its incidence, demographic profiles and clinical outcomes were unknown. This
research was undertaken in the Northern region of New Zealand, whose residents included over one
third of the population of New Zealand, most of its Pacific and Asian people, and a large number of
Maori. Large burdens of lung cancer have been reported in these ethnic groups.With this
background, this study sought to generate new information that would assist in the development of
recommendations for how EGFR gene mutation testing should be done in New Zealand.
5 Aims
5.1 Aim 1
To evaluate the clinical performance of the Roche cobas EGFR Mutation Test being used by LabPLUS
(Auckland City Hospital) for analysing clinical samples from New Zealand lung cancer patients,
compared to a reference assay.
5.2 Aim 2
To describe the incidence, demographic profiles and patient outcomes for EGFR mutation positive
lung cancer in New Zealand.
5.3 Aim 3
To ascertain the level and equity of patient access to EGFR gene mutation testing and tyrosine kinase
inhibitor drug treatment for lung cancer patients residing in a large diverse region of New Zealand.
6
Confidential
Final
3 March 2015
6 Research design
This research consisted of two distinct and related clinical studies, a clinical laboratory test and
retesting study, and a population-based patient-cohort study. These two clinical studies had
separate but overlapping study population groups, including a retested study population and a
population-based patient-cohort study group. The research design and study population groups are
shown in figure 1.
Figure 1 Research design and study populations
Clinical laboratory test and retesting study
Population-based patient-cohort study
Screened study population
n=3175
Tested study population
n=826
Eligible patient-cohort study population
n=748
Retested study population
n=532
Eligible and
tested study
population
n=429
Agreement analysis study population
n=344
7
Eligible but
untested
study
population
n=319
Confidential
Final
3 March 2015
7 Clinical laboratory test and retesting study
7.1 Background and Aims
7.1.1 Specific Aim 1
The specific aim of this study was to evaluate the clinical performance of a cobas EGFR Mutation
Test, when used at LabPLUS (Auckland City Hospital) for analysing clinical samples from New Zealand
lung cancer patients for the detection of EGFR gene mutations, compared to a reference assay.
7.2 Study design and methods
7.2.1 Study design
The clinical laboratory test and retesting study was designed to assess the performance of an EGFR
gene mutation testing strategy applied to New Zealand samples, patients and testing conditions.
7.2.2 Eligibility criteria
Patients were eligible for this laboratory test study if they had been referred to LabPLUS for EGFR
gene mutation testing. Those with remnant tissue DNA extracts available after completion of clinical
diagnostic testing were eligible for the clinical laboratory retesting study.
7.2.3 Study endpoint
The primary endpoint of this clinical laboratory study was the detection of an EGFR gene mutation.
7.2.4 Study assessments
Reports of EGFR gene mutation tests from LabPLUS that were issued since August 2012 were
compiled and collated as anonymised data into a database. Reported test results were recorded as
mutation detected, no mutation detected or invalid result. Any reasons apparent for missing reports
from patients who were identified as having had an EGFR gene mutation test were noted.
Patient tumour DNA extracts were retested using an oncogene mutation detection protocol based
on a Sequenom MassArray OncoFocus genotyping panel system. These analyses were undertaken at
the IANZ accredited Sequenom genotyping facility of the University of Auckland. To ensure samples
met assay requirements, sample quality was assessed using a Sample ID panel, which is a PCR based
MALDI-TOF mass spectrometry method, used to assess viable copy number in the DNA samples prior
to downstream analysis. The OncoFocus genotyping panel is a commercially available set of prevalidated genotyping assays designed to detect 128 different oncogenic somatic mutations in the
EGFR gene, as well as 63 different oncogenic somatic mutations in the BRAF, KRAS and NRAS genes.
To evaluate the clinical performance of the cobas EGFR Mutation Test, results from the cobas EGFR
Mutation Test and Sequenom MassArray OncoFocus genotyping were analysed by agreement
analysis in accordance with best research practice guidelines for the evaluation of diagnostic tests
[8]. Descriptive statistics were used to calculate values for the levels of positive percentage
agreement, negative percentage agreement and overall percentage agreement, and their respective
95% confidence intervals.
7.3 Results
7.3.1 Tested study population
A total of 826 patients were identified who had had EGFR gene mutation tests reported by LabPLUS
using the cobas EGFR Mutation Test (Tested study population).
8
Confidential
Final
3 March 2015
7.3.1.1 EGFR gene mutation test reports
Among the EGFR gene mutation test reports for the 826 patients in the tested study population, 145
tests were reported as an EGFR mutation detected, 584 as no mutation detected, 27 as an invalid
test and the remaining 70 had either no patient record, no report available, a result to follow or the
report was for an EGFR FISH test, as shown in Table 1. The prevalence of EGFR gene mutations in the
tested study population was therefore 145 of 826 or 17.6% (95%CI 14.1 to 21%). The most frequent
type of EGFR gene mutation detected in this tested study population of New Zealand patients with
proven EGFR gene mutation-positive lung cancer were exon 19 deletion mutations (59 of 145
patients or 41%) and exon 21 L858R mutations (50 of 145 patients or 34%). The frequency of these
and other types of EGFR gene mutations detected in this tested study population of New Zealand
lung cancer patients are shown in Table 2.
Table 1 Results of LabPLUS EGFR gene mutation test reports for detecting EGFR gene mutations in patients samples from
the tested study population (n = 826).
cobas EGFR Mutation Test
Mutation
No mutation
detected
detected
145
584
Invalid
27
No patient
No report
Result to
EGFR
record
available
follow
FISH test
47
17
4
2
Total
826
Table 2 Frequency of specific EGFR gene mutations detected using the cobas EGFR Mutation Test in New Zealand lung
cancer patients with EGFR gene mutation-positive lung cancer (n=145) from the tested study population.
Exon involved or compound mutation
18
19
20
21
Unspecified
Compound
Mutation type
G719X
Deletion
Unspecified
Insertion
L858R
Unspecified
Unspecified
G719X plus S768I
Exon 19 deletion plus S768I
Exon 19 deletion plus T790M
Exon 20 insertion plus L858R
Total
Number
5
59
1
17
50
2
2
4
1
2
2
145
Percentage
3.4%
41%
0.7%
12%
34%
1.4%
1.4%
2.8%
0.7%
1.4%
1.4%
100%
7.3.2 Retested study population
Among the 826-patient tested study population, a total of 532 patients had remnant tumour DNA
extract samples available for blinded retesting by the Sequenom MassArray OncoFocus panel
genotyping system. This 532-patient group represented the retested study population.
7.3.2.1 Compilation of LabPLUS test reports in the retested study population
A compilation of the reports of the cobas EGFR Mutation Tests from this 532-patient retested study
population are shown in Table 3. Among the reports for 532 patients in the retested population, 89
9
Confidential
Final
3 March 2015
tests were reported as an EGFR mutation having been detected, 382 as no mutation detected and 10
as an invalid results and the remainder having either no patient record or report available.
Table 3 Results of the cobas EGFR Mutation Test for detecting EGFR gene mutations in samples from the retested study
population (n=532).
cobas EGFR Mutation Test
Mutation
No mutation
detected
detected
89
382
Invalid
10
No patient
No report
record
available
41
10
Total
532
7.3.2.2 Sequenom retesting results
The results of the retesting undertaken by Sequenom MassArray OncoFocus genotyping of samples
from this 532-patient retested study population are shown in Table 4. Retesting of 532 patients
samples for EGFR gene mutations by Sequenom MassArray OncoFocus genotyping showed a
mutation detected in 60 patients, no mutation detected in 329 patients and an invalid result in 143
patients, when the Sample ID sample validity criteria was set to ≥500 amplifiable copies. When the
Sample ID sample validity criteria were reduced to lower numbers of amplifiable copies, the number
of patients with detectable EGFR gene mutations increased.
Table 4 Results of sample retesting undertaken by Sequenom MassArray OncoFocus panel genotyping for the detection
of EGFR gene mutations in the retested study population (n=532), using different Sample ID cut-off sample validity
criteria.
Sample ID Validity Criteria
7.3.3
Sequenom MassArray OncoFocus Genotyping
(amplifiable copies)
Mutation detected
No mutation detected
>0
90
440
2
532
≥300
74
389
69
532
≥500
60
329
143
532
Invalid Total
Agreement analysis
7.3.3.1 Aggregate results
Among the 532-patient retested study population, a total of 344 patients had valid test results from
both the cobas EGFR Mutation Test and Sequenom MassArray OncoFocus panel genotyping system
and were available for inclusion in the agreement analysis. Table 5 shows results aggregated for all
EGFR gene mutations for samples meeting the Sample ID assay manufacturer’s recommended
sample validity criterion (≥500 amplifiable copies). The agreement analysis between the cobas EGFR
Mutation Test and Sequenom MassArray OncoFocus genotyping, for the detection of an EGFR gene
mutation, demonstrated a positive percentage agreement of 91.2%, negative percentage agreement
of 96.2% and overall percentage agreement of 95.3%.
10
Confidential
Final
3 March 2015
Table 5 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping for the detection of
EGFR gene mutations in lung cancer samples from New Zealand patients (agreement analysis study population).
Aggregated results for all EGFR gene mutations using a Sample ID cut-off for validity of ≥500 amplifiable copies.
Sequenom MassArray OncoFocus genotyping
Cobas EGFR Mutation Test
Mutation detected
No mutation detected
Total
Mutation detected
52
11
63
No mutation detected
5
276
281
Total
57
287
344
Positive percentage agreement
52/57 = 91.2% (95%CI; 80 to 100%)
Negative percentage agreement
276/287 = 96.2% (95%CI; 86 to 100%)
Overall percentage agreement
328/344 = 95.3% (95%CI; 90 to 100%)
7.3.3.2 EGFR gene exon 19 deletion and exon 20 L858R mutations
Previous studies had shown that the EGFR exon 19 deletion and exon 20 L858R mutations were
among the most prevalent EGFR gene mutations associated with lung cancer and the most highly
predictive of the therapeutic efficacy of clinical treatment with EGFR tyrosine kinase inhibitor drugs
[1]. In the tested study population of New Zealand lung cancer patients, EGFR exon 19 deletion and
exon 20 L858R mutations accounted for 75% of all of the EGFR gene mutations detected by the
cobas Mutation Test (Table 2).
Results for the agreement analysis for the detection of specific EGFR mutations are shown on Table
6 and 7. The agreement analysis between the cobas EGFR Mutation Test and Sequenom MassArray
OncoFocus genotyping, in detecting EGFR gene exon 20 L858R mutations, demonstrated a positive
percentage agreement of 89.3%, negative percentage agreement of 98.7% and overall percentage
agreement of 98.0% (Table 6). The agreement analysis between the cobas EGFR Mutation Test and
Sequenom MassArray OncoFocus genotyping, in detecting EGFR gene exon 19 deletion mutations,
demonstrated a positive percentage agreement of 90.5%, negative percentage agreement of 99.1%
and overall percentage agreement of 98.5% (Table 7).
Table 6 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping for the detection of
EGFR gene exon 20 L858R mutations in lung cancer samples from New Zealand patients (agreement analysis study
population). Sample ID cut-off for validity of ≥500 amplifiable copies.
Sequenom MassArray OncoFocus genotyping
Cobas EGFR Mutation Test
Mutation detected
No mutation detected
Total
Mutation detected
25
4
29
No mutation detected
3
312
351
Total
28
316
344
11
Confidential
Final
3 March 2015
Positive percentage agreement
25/28 = 89.3% (95%CI; 70.4 to 100%)
Negative percentage agreement
312/316 = 98.7% (95%CI; 93.1 to 100%)
Overall percentage agreement
337/344 = 98.0% (95%CI; 92.6 to 100%)
Table 7 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping for the detection of
EGFR gene exon 19 deletion mutations in lung cancer samples from New Zealand patients (agreement analysis study
population). Sample ID cut-off for validity of ≥500 amplifiable copies.
Sequenom MassArray OncoFocus genotyping
Cobas EGFR Mutation Test
Mutation detected
No mutation detected
Total
Mutation detected
19
3
22
No mutation detected
2
320
322
Total
21
323
344
Positive percentage agreement
19/21 = 90.5% (95%CI; 70.4 to 100%)
Negative percentage agreement
320/323 = 99.1% (95%CI; 93.5 to 100%)
Overall percentage agreement
339/344 = 98.5% (95%CI; 93.1 to 100%)
7.3.3.3 Effect of varying Sequenom sample validity criteria
A total of 143 of 532 patients (27%) from the retested study population were excluded from the
agreement analyses because of invalid results from the Sample ID test of sample validity when the
cut off level for defining sample validity was set to ≥500 amplifiable copies. To investigate whether
biased percentage agreement estimates may have been generated by excluding these subjects, we
explored other sample validity criteria for defining the study population eligible for inclusion in the
agreement analyses, as shown in Table 8 and 9.
The agreement analysis between the cobas EGFR Mutation Test and Sequenom MassArray
OncoFocus genotyping for the detection of an EGFR gene mutation, including samples with ≥ 300
amplifiable copies, demonstrated a positive percentage agreement of 85.5%, negative percentage
agreement of 96.5% and overall percentage agreement of 95.6% (Table 8). The agreement analysis
between the cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping for the
detection of an EGFR gene mutation, including samples with >0 amplifiable copies, demonstrated a
positive percentage agreement of 84.5%, negative percentage agreement of 95.3% and overall
percentage agreement of 93.4% (Table 9). Overall, changing the criteria for sample validity had little
or no effect on levels of negative (96.2% vs 96.5% vs 95.3%) or overall percentage agreement (95.3%
vs 95.6% vs 93.4%) but modestly reduced estimates of positive percentage agreement (91.2% vs
95.6% vs 93.4%), although these values still fell within the 95% confidence interval of the original
estimates.
12
Confidential
Final
3 March 2015
Table 8 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping for the detection of
EGFR gene mutations in lung cancer samples from New Zealand patients. Aggregate result for Sample ID cut-off for
validity of ≥300 amplifiable copies.
Sequenom MassArray OncoFocus genotyping
Cobas EGFR Mutation Test
Mutation detected
No mutation detected
Total
Mutation detected
59
12
71
No mutation detected
16
331
341
Total
69
343
412
Positive percentage agreement
50/69 = 85.5% (95%CI; 73.5 to 97.5%)
Negative percentage agreement
331/343 = 96.5% (95%CI; 91.1 to 100%)
Overall percentage agreement
390/412 = 95.6% (95%CI; 90.7 to 100%)
13
Confidential
Final
3 March 2015
Table 9 Comparison of cobas EGFR Mutation Test and Sequenom MassArray OncoFocus genotyping for the detection of
EGFR gene mutations in lung cancer samples from New Zealand patients. Aggregate result for Sample ID cut-off for
validity of >0 amplifiable copies.
Sequenom MassArray OncoFocus genotyping
Cobas EGFR Mutation Test
Mutation detected
No mutation detected
Total
Mutation detected
71
18
89
No mutation detected
13
367
380
Total
84
385
469
Positive percentage agreement
71/84 = 84.5% (95%CI; 73.6 to 95.3%)
Negative percentage agreement
367/385 = 95.3% (95%CI; 90.2 to 100%)
Overall percentage agreement
438/469 = 93.4% (95%CI; 88.8 to 98.0%)
7.3.4 Summary
This clinical laboratory test and retesting study confirmed that the cobas EGFR Mutation test was an
accurate diagnostic assay for the detection of EGFR gene mutations in clinical samples from New
Zealand lung cancer patients. In previous analytical studies carried out elsewhere, the cobas Gene
Mutation Test had shown high levels of percentage agreement (≥90%) for the detection of EGFR
gene mutations when compared to Sanger sequencing, massively parallel sequencing and to other
allele-specific PCR assays [9-13]. Similar levels of percentage agreements (>90%) were found in the
current study when the cobas EGFR Mutation test was compared to Sequenom MassArray
OncoFocus genotyping. In this population of New Zealand lung cancer patients, EGFR exon 20 L858R
and exon 19 deletion mutations accounted for 75% of all of the EGFR gene mutations detected by
the cobas Mutation Test.
14
Confidential
Final
3 March 2015
8 Patient cohort study
8.1 Background and Aims
8.1.1 Specific Aim 1
To describe the incidence, demographic profiles and patient outcomes from EGFR mutant lung
cancer in New Zealand.
8.1.2 Specific Aim 2
To ascertain the level and equity of access to EGFR gene mutation testing and tyrosine kinase
inhibitor drug treatment for lung cancer patients residing in a large, diverse region of New Zealand.
8.2 Research design and methods
8.2.1 Study design
This study was a population-based cohort study of patients presenting with non-small cell lung
cancer, inclusively of the non-squamous or not-otherwise-specified morphological subtypes, in the
Northern region of New Zealand over an approximately 2-year study period.
8.2.2 Eligibility criteria
Patients eligible for this study were residents of the geographical areas of the Auckland, Counties
Manakau, Northland and Waitemata District Health Boards who presented with non-small cell lung
cancer, of the non-squamous or not-otherwise-specified morphological subtypes, on or after 1
August 2012. Patient with squamous lung cancer, small cell lung cancer or pleural mesothelioma
were excluded. The study population thereby represented the target population recommended for
EGFR gene mutation testing.
8.2.3
Study assessments
8.2.3.1 Subject enrolment and data collection
Potentially eligible subjects were identified by screening case notifications to the New Zealand
Cancer Registry of patients presenting with non-small cell lung cancer of the non-squamous or nototherwise-specified morphological subtypes, over an approximately 2-year period. Lung cancer
registrations were extracted from the New Zealand Cancer Registry, inclusive of all trachea,
bronchus and lung cancers (ICD-10 codes 33-34) with morphology codes indicative of non-squamous
or not-otherwise-specified morphological subtype (morphology codes: 8000, 8010, 8012-14, 8046,
8140, 8246, 8250-5, 8260-3, 8310, 8430, 8480, 8481-2, 8490, 8550, 8560, 8570-3, 8576) from 1
August 2012 to 30 April 2014. No data after April 2014 was available from the New Zealand registry
at the time of the writing of this report. The cancer registry dataset provided, for each case, an NHI
number, year of diagnosis, sex, prioritised ethnic code, domicile code, morphology code, basis for
diagnosis, laboratory code, extent of disease, cancer notes, TNM-T, TNM-N, TNM-M and grade of
tumour code. Patients eligible for inclusion in the cohort study were identified by domicile codes
indicating residence in the geographical areas of the Auckland, Counties-Manakau, Northland and
Waitemata District Health Boards and a date of diagnosis indicating a clinical presentation with lung
cancer between 1 August 2012 and 30 April 2014.
8.2.3.2 EGFR testing and treatment
To identify whether eligible subjects for inclusion in the patient cohort study population had
accessed EGFR gene mutation testing or EGFR tyrosine kinase inhibitor drug therapy, listings of EGFR
15
Confidential
Final
3 March 2015
gene mutation test reports issued by LabPLUS were screened for the NHIs of patients eligible for
inclusion in the population-based study population. Individual patient records were screened for the
reported result of EGFR gene mutation tests and records of drug dispensing and special authority
approvals as evidence of access to EGFR tyrosine kinase inhibitor drug therapy.
8.2.3.3 Data analysis
The proportion of EGFR mutation-positive lung cancer was calculated from the number of new cases
of lung cancer confirmed to harbour EGFR gene mutations over the study period, expressed as a
proportion of either the total population at risk, number of new lung cancers overall, non-small cell
lung cancer, those of the non-squamous or not-otherwise-specified morphological subtypes or that
of other defined subpopulations. Access to publicly-funded EGFR gene mutation testing or tyrosine
kinase inhibitor drug therapy was estimated by calculating the proportion of the total eligible
population who were tested or received treatment. Subgroups of patients were compared with
respect to their demographic profiles, clinical characteristics and survival outcomes. Data were
analysed visually in graphs and tables, and by descriptive statistics such as the calculation of
proportions and their 95% confidence intervals. Comparisons between groups were made by chi
squared test, T-test and multivariate methods such as logistic regression as appropriate for the data.
8.3
Results
8.3.1 Population-based patient cohort
As described above, a population-based cohort of patients was defined from the total of 3175
patients with non-small cell lung cancer of the non-squamous or not-otherwise-specified
morphological subtypes, who were notified to the NZ national Cancer Registry between 1 Jan 2012
and 30 April 2014. Among these 3175 patients, 789 had domicile codes indicating residence in the
geographical areas of the Auckland, Counties-Manakau, Northland or Waitemata District Health
Boards, and a date of diagnosis occurring on or after 1st August 2012. Of these, 41 were notified
based only on a death certificate (32), autopsy report (5), or unknown source (4), with no records
before death, and were excluded, leaving 748 eligible cohort patients.
8.3.2
EGFR gene mutation testing in the population-based patient cohort study
population
Among the 748 patients identified as being eligible for inclusion in the cohort study population, 429
(57%) had Cobas EGFR Mutation testing reports issued by LabPLUS and recorded, and the remaining
319 patients (43%) had no EGFR gene mutation testing reports.
Of these, 86 (20.0%, 95% CI 16.5 to 24.1%), were reported as having a mutation detected. (This is
currently our best estimate of the prevalence of EGFR gene mutations in a population-based group
of patients). This is slightly higher than the prevalence of EGFR gene mutation in all patients tested,
reported earlier in this report as 145 of 826, 17.6% (95%CI 14.1 to 21.0%), but the difference is not
statistically significant.
Among the 429 eligible patients who were tested, 15 (3.5%) had an unsatisfactory or uninterpretable
result. In table 11, these patients have been excluded; the frequency of a mutation in those tested,
excluding invalid results, is 21.0%.
16
Confidential
Final
3 March 2015
8.3.3
Features of eligible cohort patients, and factors related to having had an EGFR
gene mutation test
As shown in Table 10, of the 748 eligible cohort patients, 52% were female, 19% under age 59 and
19% aged 80 and over, and 60% NZ European. Based on registry data, which may not be fully
accurate, 46% had upper lobe cancers, and 50% had distant spread.
The proportions of patients tested for an EGFR gene mutation (overall 57%), was greater in women
(61%, men 53%), increased with age from 29% at ages under 59 to 64% at ages over 80, varied by
ethnicity, being lower in Maori and higher in SE Asians, and increased over time from 50% in AugDec 2012 to 66% in Jan-April 2014. The proportion tested did not vary greatly by site of the tumour,
and was higher in patients with regional or distant disease (based on registry data).
Multivariate analysis was used to assess the effects of inter-related factors. This showed that the
associations with gender, age, and time period were independent. With adjustment for these
factors, the proportion tested in Maori patients was significantly lower (48%) than in NZ Europeans
(57%), but the other ethnic differences, based on smaller numbers of patients, were not significant.
In summary, the group of patients tested contains more women, more older patients, more recently
diagnosed patients, and fewer Maori patients, than the whole eligible cohort population.
8.3.4 Features associated with EGFR gene mutations in eligible cohort patients
Of the 748 eligible cohort patients, 414 had an interpretable Cobas EGFR test. As shown in Table 11,
21% of those tested, with a valid result, had a mutation found.
The proportion with a mutation found was greater in women (27% compared to 12% in men), but
did not vary substantially by age. It varied by ethnicity: compared to the NZ European patients of
whom 18% had mutations, the proportions were significantly higher in SE Asian patients (40%, 95%
CI 27 to 54%). The proportion positive was also higher in the ‘other and unknown’ group. It was
somewhat lower in Maori (10%, 95% CI 5 to 20%). The mutation rate was higher in Pacifica (24%, CI
14 to 39%), but this was not significantly different from the NZ European group.
Of the 414 patients, information on smoking was missing on 134 (32%). For those with information,
mutations were found much more commonly in non-smokers (52%) than in smokers (18%) or exsmokers (17%).
Multivariate analysis was used to assess the effects of inter-related factors. This showed that the
associations with gender and ethnic background were independent, with higher mutation rates in
women, and in SE Asian patients; the lower positivity in Maori was nearly significant (P=0.09). The
positivity rate declined slightly with increasing age, but this association was not significant.
In the multivariate analysis including smoking, restricted to 280 subjects, the higher proportions of
mutations in non-smokers was significant. Adjusting for smoking reduced, but did not remove, the
association seen with gender.
In summary, an EGFR mutation was found in 20% of eligible cohort patients tested using the Cobas
test. This proportion was higher in women, in SE Asian subjects and perhaps in Pacifica, but likely
lower in Maori (each compared to the NZ European group). The proportion with an EGFR gene
mutation was higher in non-smokers. It did not vary much by age.
17
Confidential
Final
3 March 2015
8.3.5
EGFR tyrosine kinase inhibitor (TKI) drug treatment in the population-based
patient cohort study population
In the eligible cohort patients, 86 had an EGFR gene mutation found on the Cobas test. Information
on tyrosine kinase inhibitor (TKI) drug treatment was missing on one patient.
As shown in Table 12, of the 85 with information, 60 (71%) had TKI approval. The proportions with
approval were higher in younger patients, varying from 91% at ages up to 59 to 44% at age 80 and
over, and was lower in the small number of patients with localised disease (2 of 11, 18%). It did not
vary by gender, ethnic background, or tumour site considering the 95% confidence intervals of these
proportions. It varied irregularly by time, with a lower proportion in Jan-June 2013, but there was no
regular trend over time. Multivariate analysis confirmed the associations with age and time period.
8.3.6 Mortality outcomes
Mortality outcomes require further data, and will be assessed later. An analysis of mortality
outcomes is planned for late 2015 when it is expected that over 50% of deaths may have occurred.
8.4 Summary
A population-based estimate of the prevalence of EGFR gene mutations in New Zealand lung cancer
patients was 20% (95% CI 16.5 to 24.1%). EGFR mutations were found more commonly among
women, SE Asian subjects and non-smokers. Only 57% of eligible patients in a population-based New
Zealand cohort of lung cancer patients were tested for EGFR gene mutations, with Maori, males and
younger patients, along with those presenting earlier during the study period, being
underrepresented among those tested. About 70% of patients identified as having an EGFR gene
mutation, were subsequently treated with an EGFR tyrosine kinase inhibitor drug. Older patients and
those with localised disease were less likely to receive EGFR tyrosine kinase inhibitor drug treatment.
18
Confidential
Final
3 March 2015
Table 10 Frequency of testing (Cobas EGFR Mutation test) in eligible cohort patients (n=748)
Factor
Total
Total
Gender
748
391
357
144
227
237
140
444
126
83
68
27
176
194
220
158
41
348
41
166
152
57
27
82
373
209
Age
Ethnic
Time period
Site
Extent
Female
Male
<59
60-69
70-79
80+
NZ European
NZ Maori
Pacific
SE Asian
Other & Unknown
Aug-Dec, 2012
Jan-June, 2013
July-Dec, 2013
Jan-April, 2014
Main bronchus incl. Carina, Hilus
Upper lobe, bronchus or lung
Middle lobe, bronchus or lung
Lower lobe, bronchus or lung
Mixed or unspecified
Localised to organ of origin
Invasion of adjacent tissue or organ
Regional lymph nodes
Distant
Not known
Distribution
Tested
52.3
47.7
19.3
30.3
31.7
18.7
59.4
16.8
11.1
9.1
3.6
23.5
25.9
29.4
21.1
5.5
46.5
5.5
22.2
20.3
7.6
3.6
11.0
49.9
27.9
429
240
189
41
84
104
90
252
61
49
49
18
88
107
130
104
24
192
27
104
82
24
15
56
225
109
Not
tested
319
151
168
103
143
133
50
192
65
34
19
9
88
87
90
54
17
156
14
62
70
33
12
26
148
100
1
univariate analysis for factor; 2multivariate analysis for specific category
19
%
tested
57.4
61.4
52.9
28.5
37.0
43.9
64.3
56.8
48.4
59.0
72.1
66.7
50.0
55.2
59.1
65.8
58.5
55.2
65.9
62.7
53.9
42.1
55.6
68.3
60.3
52.2
95% CI
53.8
56.5
47.8
21.7
31.0
37.7
56.1
52.1
39.9
48.3
60.4
47.8
42.7
48.1
52.5
58.1
43.4
49.9
50.5
55.1
46.0
30.2
37.3
57.6
55.3
45.4
-
60.9
66.1
58.1
36.3
43.5
50.2
71.7
61.3
57.1
69.0
81.3
81.4
57.3
62.0
65.4
72.8
72.2
60.3
78.4
69.6
61.7
55.0
72.4
77.4
65.2
58.8
P1
OR
crude
0.022
1.00
0.71
0.18
0.30
0.43
1.00
1.00
0.72
1.10
1.96
1.52
1.00
1.23
1.44
1.93
1.00
0.87
1.37
1.19
0.83
1.00
1.72
2.96
2.09
1.50
<0.001
0.023
0.027
0.342
0.011
OR
adjusted
P2
0.69
0.17
0.44
0.59
1
1
0.48
0.82
1.60
1.18
1
1.31
1.43
2.27
0.02
<.001
0.001
0.03
0.001
ns
ns
ns
ns
ns
0.001
Confidential
Final
3 March 2015
Table 11 Results of Cobas EGFR Mutation testing: patients with valid result only (n=414).
Factor
Total
Total
Gender
Female
Male
Age
<59
60-69
70-79
80+
Ethnic
NZ European
NZ Maori
Pacific
SE Asian
Other & Unknown
414*
Distribution
Positive
No mutation
% positive
95% limits
P value
86
328
20.8
17.1
-
24.9
235
179
56.8
43.2
64
22
171
157
27.2
12.3
21.9
8.3
-
33.3
17.9
<0.001
95
138
132
49
22.9
33.3
31.9
11.8
23
30
24
9
72
108
108
40
24.2
21.7
18.2
18.4
16.7
15.7
12.5
10.0
-
33.7
29.3
25.6
31.4
0.69
244
59
45
48
18
58.9
14.3
10.9
11.6
4.3
43
6
11
19
7
201
53
34
29
11
17.6
10.2
24.4
39.6
38.9
13.4
4.7
14.2
27.0
20.3
-
22.9
20.5
38.7
53.7
61.4
0.001
Smoking
Note: based on 280 subjects only
Ex-Smoker
149
53.2
26
123
17.4
Non-Smoker
81
28.9
42
39
51.9
Smoker
50
17.9
9
41
18.0
*15 of 429 patients had unsatisfactory or uninterpretable results and were excluded
12.2
41.1
9.8
-
24.3
62.4
30.8
<0.001
20
Confidential
Final
3 March 2015
Table 12 EGFR TKI use for patients with Cobas positive test (n= 85)
Factor
Total
Gender
Age
Ethnic
Time period
Site
Extent
Female
Male
<59
60-69
70-79
80+
NZ European
NZ Maori
Pacific
SE Asian
Other & Unknown
Aug-Dec, 2012
Jan-June, 2013
July-Dec, 2013
Jan-April, 2014
Main bronchus incl. Carina, Hilus
Upper lobe, bronchus or lung
Middle lobe, bronchus or lung
Lower lobe, bronchus or lung
Mixed or unspecified
Localised to organ of origin
Invasion of adjacent tissue or organ
Regional lymph nodes
Distant
Not known
Total
Distribution
TKI yes
no
% yes
95% limits
85
64
21
22
30
24
9
43
7
11
18
6
16
21
29
19
1
36
10
22
16
8
3
15
42
17
75.3
24.7
25.9
35.3
28.2
10.6
50.6
8.2
12.9
21.2
7.1
18.8
24.7
34.1
22.4
1.2
42.4
11.8
25.9
18.8
9.4
3.5
17.6
49.4
20.0
60
44
16
20
21
15
4
30
5
8
12
5
13
10
24
13
1
26
4
15
14
2
0
10
37
11
25
20
5
2
9
9
5
13
2
3
6
1
3
11
5
6
0
10
6
7
2
6
3
5
5
6
70.6
68.8
76.2
90.9
70.0
62.5
44.4
69.8
71.4
72.7
66.7
83.3
81.3
47.6
82.8
68.4
100.0
72.2
40.0
68.2
87.5
25.0
0.0
66.7
88.1
64.7
60.2
56.6
54.9
72.2
52.1
42.7
18.9
54.9
35.9
43.4
43.7
43.6
57.0
28.3
65.5
46.0
20.7
56.0
16.8
47.3
64.0
7.1
0.0
41.7
75.0
41.3
1
univariate analysis for factor; 2multivariate analysis for specific category
21
-
79.2
78.8
89.4
97.5
83.3
78.8
73.3
81.4
91.8
90.3
83.7
97.0
93.4
67.6
92.4
84.6
100.0
84.2
68.7
83.6
96.5
59.1
56.2
84.8
94.8
82.7
P value1
Odds ratio
0.591
1.00
1.45
1.00
0.23
0.17
0.08
1.00
1.08
2.03
1.52
3.81
1.00
0.21
1.11
0.50
0.034
0.986
0.045
0.122
<0.001
1.00
0.00
2.75
10.18
2.52
Multi OR
P2
1
0.23
0.18
0.09
ns
0.05
0.02
1
0.21
0.94
0.54
0.05
ns
ns
Confidential
Final
3 March 2015
9 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Sharma SV, Bell DW, Settleman J, Haber DA: Epidermal growth factor receptor mutations in
lung cancer. Nat Rev Cancer 2007, 7(3):169-181.
Maemondo M: Gefitinib or chemotherapy for non-small cell lung cancer with mutated
EGFR. N Engl J Med 2010.
Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, Seto T, Satouchi M, Tada
H, Hirashima T et al: Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell
lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405):
an open label, randomised phase 3 trial. Lancet Oncol, 11(2):121-128.
Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B,
Margono B, Ichinose Y et al: Gefitinib or carboplatin-paclitaxel in pulmonary
adenocarcinoma. N Engl J Med 2009, 361(10):947-957.
Rosell R, Carcereny E, Gervais R, Vergnenegre A, Massuti B, Felip E, Palmero R, Garcia-Gomez
R, Pallares C, Sanchez JM et al: Erlotinib versus standard chemotherapy as first-line
treatment for European patients with advanced EGFR mutation-positive non-small-cell
lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet
Oncology 2012, 13(3):239-246.
Zhou CC, Wu YL, Chen GY, Feng JF, Liu XQ, Wang CL, Zhang SC, Wang J, Zhou SW, Ren SX et
al: Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR
mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre,
open-label, randomised, phase 3 study. Lancet Oncology 2011, 12(8):735-742.
Keedy VL, Temin S, Somerfield MR, Beasley MB, Johnson DH, McShane LM, Milton DT,
Strawn JR, Wakelee HA, Giaccone G: American Society of Clinical Oncology Provisional
Clinical Opinion: Epidermal Growth Factor Receptor (EGFR) Mutation Testing for Patients
With Advanced Non-Small-Cell Lung Cancer Considering First-Line EGFR Tyrosine Kinase
Inhibitor Therapy. J Clin Oncol 2011, 29(15):2121-2127.
Anonymous: Guidance for Industry and FDA Staff - Statistical Guidance on reporting
Results from Studies Evaluating Diagnostic Tests. In:
http://wwwfdagov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/ucm072101pdf. U.S. Department of Health and Human Services Food and Drug
Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics
Evaluation and Research (CBER); 2006: 36.
O'Donnell P, Ferguson J, Shyu J, Current R, Rehage T, Tsai J, Christensen M, Tran HB, Chien
SS, Shieh F et al: Analytic performance studies and clinical reproducibility of a real-time
PCR assay for the detection of epidermal growth factor receptor gene mutations in
formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer. BMC
Cancer 2013, 13:210.
Benlloch S, Botero ML, Beltran-Alamillo J, Mayo C, Gimenez-Capitan A, de Aguirre I, Queralt
C, Ramirez JL, Cajal SRY, Klughammer B et al: Clinical Validation of a PCR Assay for the
Detection of EGFR Mutations in Non-Small-Cell Lung Cancer: Retrospective Testing of
Specimens from the EURTAC Trial. PLoS One 2014, 9(2).
Kimura H, Ohira T, Uchida O, Matsubayashi J, Shimizu S, Nagao T, Ikeda N, Nishio K:
Analytical performance of the cobas EGFR mutation assay for Japanese non-small-cell lung
cancer. Lung Cancer 2014, 83(3):329-333.
Lopez-Rios F, Angulo B, Gomez B, Mair D, Martinez R, Conde E, Shieh F, Tsai J, Vaks J, Current
R et al: Comparison of molecular testing methods for the detection of EGFR mutations in
formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer. J Clin
Pathol 2013, 66(5):381-385.
22
Confidential
13.
Final
3 March 2015
Wong ATC, To RMY, Wong CLP, Chan WK, Ma ESK: Evaluation of 2 Real-Time PCR Assays for
In Vitro Diagnostic Use in the Rapid and Multiplex Detection of EGFR Gene Mutations in
NSCLC. Diagnostic Molecular Pathology 2013, 22(3):138-143.
23