Outcomes of First-Generation EGFR-TKIs Against Non-Small-Cell Lung Cancer Harboring Uncommon EGFR Mutations: A Post Hoc Analysis of the BE-POSITIVE Study

Accepted Manuscript Outcomes of first-generation EGFR-TKIs against non-small-cell lung cancer harboring uncommon EGFR mutations: a post-hoc analysis of the BE-POSITIVE study. Sara Pilotto, Antonio Rossi, Tiziana Vavalà, Alessandro Follador, Marcello Tiseo, Domenico Galetta, Alessandro Morabito, Massimo Di Maio, Olga Martelli, Orazio Caffo, Pier Luigi Piovano, Diego Cortinovis, Nicoletta Zilembo, Clelia Casartelli, Giuseppe Luigi Banna, Antonio Ardizzoia, Maria Luisa Barzelloni, Alessandra Bearz, Giovenzio Genestreti, Claudia Mucciarini, Virginio Filipazzi, Jessica Menis, Elisa Rizzo, Fausto Barbieri, Erika Rijavec, Fabiana Cecere, Gianluca Spitaleri, Emilio Bria, Silvia Novello PII:

S1525-7304(17)30165-1

DOI:

10.1016/j.cllc.2017.05.016

Reference:

CLLC 657

To appear in:

Clinical Lung Cancer

Received Date: 24 January 2017 Revised Date:

19 May 2017

Accepted Date: 23 May 2017

Please cite this article as: Pilotto S, Rossi A, Vavalà T, Follador A, Tiseo M, Galetta D, Morabito A, Di Maio M, Martelli O, Caffo O, Piovano PL, Cortinovis D, Zilembo N, Casartelli C, Banna GL, Ardizzoia A, Barzelloni ML, Bearz A, Genestreti G, Mucciarini C, Filipazzi V, Menis J, Rizzo E, Barbieri F, Rijavec E, Cecere F, Spitaleri G, Bria E, Novello S, Outcomes of first-generation EGFR-TKIs against non-smallcell lung cancer harboring uncommon EGFR mutations: a post-hoc analysis of the BE-POSITIVE study., Clinical Lung Cancer (2017), doi: 10.1016/j.cllc.2017.05.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Article Type: Original study. Title: Outcomes of first-generation EGFR-TKIs against non-small-cell lung cancer harboring

RI PT

uncommon EGFR mutations: a post-hoc analysis of the BE-POSITIVE study. Authors:

Sara Pilotto1*, Antonio Rossi2*, Tiziana Vavalà3, Alessandro Follador4, Marcello Tiseo5, Domenico

SC

Galetta6, Alessandro Morabito7, Massimo Di Maio8, Olga Martelli9, Orazio Caffo10, Pier Luigi Piovano11, Diego Cortinovis12, Nicoletta Zilembo13, Clelia Casartelli14, Giuseppe Luigi Banna15,

M AN U

Antonio Ardizzoia16, Maria Luisa Barzelloni2, Alessandra Bearz17, Giovenzio Genestreti18, Claudia Mucciarini19, Virginio Filipazzi20, Jessica Menis4, Elisa Rizzo21, Fausto Barbieri22, Erika Rijavec23, Fabiana Cecere24, Gianluca Spitaleri25, Emilio Bria1§, Silvia Novello3§.

§

Equally contributors (Emilio Bria and Silvia Novello share the last co-authorship).

Affiliations:

Medical Oncology, University of Verona, Azienda Ospedaliera Universitaria Integrata di Verona,

EP

1

TE D

* Equally contributors (Sara Pilotto and Antonio Rossi share the first co-authorship).

P.zza L. A. Scuro 10, 37134 Verona, Italy; 2Division of Medical Oncology, S.G. Moscati Hospital,

AC C

Contrada Amoretta, 83100 Avellino, Italy; 3Department of Oncology, University of Torino AOU San Luigi, Regione Gonzole 10, 10043 Orbassano (TO), Italy; 4Department of Oncology, University Hospital of Udine, Piazzale S. Maria della Misericordia 15, 33100 Udine, Italy; 5Medical Oncology Unit, University Hospital of Parma, via Gramsci, 14, 43126 Parma, Italy; 6Medical Oncology Unit, Clinical Cancer Center Giovanni Paolo II, viale Orazio Flacco, 65, 70124 Bari, Italy; 7Thoracic Medical Oncology, National Cancer Institute, Fondazione “G. Pascale”, via Mariano Semmola 80131, Napoli, Italy; 8Medical Oncology, Mauriziano Hospital, Department of Oncology, University of Turin, Italy; 9Medical Oncology, S. Giovanni-Addolorata Hospital, via di 1

ACCEPTED MANUSCRIPT S. Stefano Rotondo 5a, 00184 Roma, Italy; 10Medical Oncology Unit, Santa Chiara Hospital, Largo Medaglie D’oro 9, 38122 Trento, Italy; 11Medical Oncology Unit, AO SS. Antonio Biagio e Cesare Arrigo, via Venezia 16, Alessandria, Italy; 12Medical Oncology Unit, AOU San Gerardo, via Giambattista Pergolesi 33, 20900 Monza, Italy; 13Department of Medical Oncology, Fondazione

RI PT

IRCCS Istituto Nazionale dei Tumori, via Giacomo Venezian 1, Milano, Italy; 14Medical Oncology Unit, Valduce Hospital, via Dante Alighieri 11, 22100 Como, Italy; 15Division of Medical

Oncology, AO Cannizzaro Hospital, via Messina 829, 95126 Catania, Italy; 16Medical Oncology

SC

Unit, A. Manzoni Hospital via dell’Eremo 9/11, 23900 Lecco, Italy; 17Department of Medical Oncology, National Institute for Cancer Research, via Franco Gallini 2, Aviano (PN), Italy; Department of Medical Oncology, IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura

M AN U

18

dei Tumori (IRST), via Piero Maroncelli 40, 47014 Meldola, Italy; 19Department of Oncological Medicine, Ramazzini Hospital, via Guido Molinari 2, 41012 Carpi (MO), Italy; 20UOC Medical Oncology, AO Luigi Sacco, via Giovanni Battista Grassi 74, 20157 Milano, Italy; 21EORTC

TE D

Headquarters, Avenue E. Mounier 83, 1200 Bruxelles, Belgium; 22Department of Oncology and Hemathology, AOU of Modena, viale del pozzo 71, Modena, Italy; 23Lung Cancer Unit, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, 16132

EP

Genova, Italy; 24Medical Oncology Unit, University Hospital Careggi, Largo Brambilla 3, 50134 Firenze, Italy; 25Division of Thoracic Oncology, European Institute of Oncology, via Ripamonti

AC C

435, 20141 Milano, Italy. Corresponding author:

Dr. Sara Pilotto, M.D., University of Verona, Medical Oncology, Azienda Ospedaliera Universitaria Integrata, P.le L.A. Scuro 10, 37124, Verona, Italy, ph. +390458128502, +390458128140; e-mail: [email protected]

2

ACCEPTED MANUSCRIPT MicroAbstract: NSCLC harboring uncommon EGFR mutations (any other mutation than deletion 19 or L858R) present a variable sensitivity to EGFR-TKIs. With the final aim to enrich knowledge about

RI PT

uncommon EGFR mutations, we performed a post-hoc analysis of the BE-POSITIVE trial. Thirtyfive patients were included out of the original 312 EGFR-mutated cases. The results of our analysis support the existence of a strong heterogeneity within patients harboring uncommon EGFR

mutations, which implies the necessity to stratify the subgroups of rare mutations in individual

SC

entities with different clinical perspectives.

M AN U

Structured abstract:

Purpose: BE-POSITIVE was the first Italian multicenter observational study reporting the outcomes of first-generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFRTKIs) in a “real-life” Caucasian EGFR-mutated non-small-cell lung cancer (NSCLC) population.

TE D

The sharing of multi-institutional experiences represents a crucial strategy to enrich knowledge about uncommon EGFR mutations. Therefore, we performed a post-hoc analysis of BE-POSITIVE study.

EP

Patients and methods: Data of advanced NSCLC patients with uncommon EGFR mutations

AC C

receiving first-line first-generation EGFR-TKIs in 24 Italian Hospitals was collected. This analysis aimed to evaluate overall survival (OS), progression-free survival (PFS), overall response rate (ORR) of EGFR-TKIs in NSCLC patients harboring uncommon EGFR mutations. Results: Thirty-five patients harboring uncommon EGFR mutations (any mutation other than deletion 19 or L858R) were included out of the original 312 EGFR-mutated cases. Most of them were females (57.1%), former smokers (65.7%), with adenocarcinoma (88.6%). The most frequent EGFR mutations were G719X (17.2%), L861Q (14.2%). The population presented an ORR of 25.7%, a median PFS of 5.19, a median OS of 14.49 months. When stratified according to type of EGFR mutation, median OS ranged from 3.65 months for unspecified mutations to 21.29 for double 3

ACCEPTED MANUSCRIPT EGFR mutations. Median PFS ranged from 1.77 months for unspecified mutations to 20.83 for concomitant EGFR-ALK alteration. ORR varied from 0% in exon 18, 20 and double gene alteration to 66.6% in exon 19. Conclusion: Our study supports the existence of a strong outcome heterogeneity within patients

RI PT

harboring uncommon EGFR mutations, which needs to be clarified to achieve a real personalized treatment strategy.

AC C

EP

TE D

M AN U

SC

Keywords: lung cancer; uncommon EGFR mutations; gefitinib; erlotinib; BE-POSITIVE study.

4

ACCEPTED MANUSCRIPT Introduction Epidermal growth factor receptor (EGFR) activating mutations are detected in approximately 1117% of Caucasian patients affected by advanced non-small-cell lung cancer (NSCLC) with

RI PT

adenocarcinoma histotype 1-3. To date, some specific alterations in the exons 18, 19 and 21 of the tyrosine kinase (TK) domain of the EGFR gene have been identified as predictor of EGFR TK inhibitors (TKIs) efficacy 4. Although to date more than 250 EGFR alterations have been described, the two most frequently reported are: 1) short in-frame deletions of the exon 19 (Del19), which

SC

usually involve aminoacids among codons 746 to 750 (E746-A750) and, among them, the deletion

M AN U

of five aminoacids (2235_2249delELREA) has been reported as the most frequent (40-45%); 2) a point mutation (CTG to CGG) in exon 21 at nucleotide 2573, which results in substitution of leucine by arginine at codon 858 (L858R), representing about 80-85% of all point mutations 5. EGFR Del19 and L858R mutations are detected up to 90% of all the EGFR-mutated lung adenocarcinoma and are strongly associated with response to TKIs targeting EGFR 6-8.

TE D

Nevertheless, a series of other alterations involving the EGFR gene and globally recognized as ‘uncommon mutations’ have been further identified and their predictive impact in determining

EP

sensitivity or resistance to EGFR-TKIs is still uncertain 9. This heterogeneous group includes also the T790M mutation, deriving from the substitution of threonine 790 with methionine, which

AC C

accounts for approximately 50% of the acquired resistance mechanisms to EGFR-TKIs 10, 11. Their low frequency and wide variety strongly complicate the understanding of the biological role and the clinical perspectives of uncommon EGFR mutations, which were frequently excluded from pivotal clinical trials to guarantee a homogeneous population. Nevertheless, their incidence (for instance, approximately 2-3% for G719X mutations in exon 18 and 1-2% for L861Q mutations in exon 21) 12 is comparable to other rare druggable driver alterations currently under investigation, such as ROS1 and RET translocations 13.

5

ACCEPTED MANUSCRIPT Recently, a multicenter Italian observational study, called BE-POSITIVE, was performed to evaluate physicians’ choices and outcomes of second-line treatment in EGFR-mutated patients, who progressed after first-line EGFR-TKIs 14. In order to investigate the efficacy of gefitinib and erlotinib in patients with uncommon EGFR mutations in the context of a “real-life” Caucasian

AC C

EP

TE D

M AN U

SC

RI PT

EGFR-mutated population, we performed a post-hoc analysis of the BE-POSITIVE study.

6

ACCEPTED MANUSCRIPT Patients and Methods Patient population We analysed the data of advanced NSCLC patients with uncommon EGFR mutations who received

RI PT

first-line EGFR-TKI in 24 Italian Hospitals from June 2009 to May 2013. Patients’ records were collected by each institution, centrally stored and analysed by the Coordinating Center.

Patients with histological or cytological confirmed NSCLC diagnosis, stage IIIB-IV by the AJCC Staging Manual 7th edition, presence of uncommon EGFR activating mutation (defined as any

SC

mutation other than Del19 or L858R), detected on tumor specimens obtained by either surgical or

M AN U

needle biopsy/aspiration procedures and locally sequenced for mutational analysis, were included. Those EGFR mutations, other than Del19 or L858R, but not better-characterized are defined as “unspecified” or “unknown” mutations.

The study protocol was approved by the Institutional Review Board of the Coordinating Center: Thoracic Oncology Unit, Department of Oncology, San Luigi Gonzaga University Hospital

TE D

(Orbassano-Turin, Italy) and by all participating centres. Treatment

EP

Eligible patients received gefitinib, 250 mg/day, or erlotinib, 150 mg/day. Treatment was administered until disease progression or unacceptable toxicity.

AC C

Timing and sequence of subsequent treatments, at progression of disease to EGFR-TKI, were at physicians’ discretion. Clinical assessments

Baseline and subsequent disease assessments were at physicians’ discretion and based on personal clinical and routine experience. Usually, a computed tomography (CT) scans of chest, upper abdomen and brain, were performed at baseline and, following routine clinical practice, to monitor

7

ACCEPTED MANUSCRIPT the response, its duration and disease progression. Best response was recorded for first and subsequent line of treatment. The primary objective of the BE-POSITIVE study was to describe outcomes in terms of activity, efficacy and safety of second-line therapy in EGFR-mutated patients who progressed to first-line

RI PT

TKI. Herein, we performed a retrospective post-hoc analysis of the BE-POSITIVE study, aimed to evaluate the efficacy, in terms of overall survival (OS) and progression-free survival (PFS), the activity, in term of overall response rate (ORR), and tolerability of first-line EGFR-TKIs in patients

SC

affected by NSCLC harboring uncommon EGFR mutations, within a national context and in a “real life” Caucasian population.

M AN U

The disease response was defined according to Response Evaluation Criteria in Solid Tumors (RECIST criteria version 1.1). Toxicity was assessed by using Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Statistical analysis

TE D

OS was measured from the first day of the EGFR-TKI treatment until the day of death or until last follow-up visit. PFS was measured from the first day of treatment until the first objective or clinical

EP

sign of disease progression, death without progression or until last follow-up visit without progression. According to the Italian Medicines Agency (AIFA), the disease assessment was

AC C

performed for gefitinib every 3 months and for erlotinib after the first 2 months of treatment and then every 3 months. Survival data were estimated by the Kaplan-Meier method. According to the descriptive statistics, categorical variables were summarized by frequencies and percentages, and continuous covariates in term of median, range and numbers of observations.

8

ACCEPTED MANUSCRIPT Results Patient population From June 2009 to May 2013, 312 patients with advanced NSCLC harboring activating EGFR

RI PT

mutations were included in the BE-POSITIVE study. We identified 35 patients with uncommon EGFR mutations and all of them were included in the present analysis.

The demographic and disease characteristics of the patients are reported in Table 1. Median age at

SC

diagnosis was 70 years (range 42-86), the majority of patients were females (20, 57.1%) and former smoker (23, 65.7%), affected by NSCLC with adenocarcinoma histotype (31, 88.6%), usually in

M AN U

stage IIIB (4, 11.4%) or IV (24, 68.6%) at diagnosis.

The specific types of uncommon EGFR mutations detected in this analysis are described in Table 2. The most frequent EGFR mutations were G719X and L861Q, detected in 6 (17.2%) and 5 (14.2%) patients, respectively. The individual characteristics of each patient with uncommon EGFR

TE D

mutations are reported in Supplementary Table 1. Uncommon mutations and outcomes

The Kaplan–Meier curves of OS and PFS for patients harboring common versus uncommon EGFR

EP

mutations in the BE-POSITIVE trial population are reported in Supplementary Figure 1.

AC C

The Kaplan–Meier curves of OS for uncommon EGFR mutations are reported in Figure 1. The median OS was 14.49 months for all uncommon mutations, with a 1-year OS of 60.0% (Figure 1 Panel A). When stratified according to the type of EGFR mutations (Figure 1 - Panel B), we observed a median OS of 17.00 months in uncommon mutations of exon 18, 12.21 months in exon 19, 15.44 months in exon 20, 14.49 months in exon 21, 21.29 months in the subgroup carrying two concomitant EGFR mutations and 3.65 months in the subgroup with an unspecified mutation. For the patient harboring both an uncommon EGFR mutation and ALK translocation, the median OS was not reached. 9

ACCEPTED MANUSCRIPT The Kaplan–Meier curves of PFS for uncommon EGFR mutations are reported in Figure 2. The median PFS was 5.19 months for all uncommon mutations, with a 1-year PFS of 31.4% (Figure 2 Panel A). When stratified according to the type of EGFR mutations (Figure 2 - Panel B), we observed a median PFS of 8.38 months in uncommon mutations of exon 18, 5.95 months in exon

RI PT

19, 7.77 months in exon 20, 5.16 months in exon 21, 6.93 months for the subgroup carrying two concomitant EGFR mutations, 20.83 for the patient harboring both an uncommon EGFR mutation and ALK translocation and 1.77 months for the subgroup with an unspecified mutation.

SC

Concerning response to EGFR-TKIs in patients with uncommon EGFR mutations (Table 3), the ORR for all patients, regardless of the type of EGFR-TKI administered, was 25.7%. In particular,

M AN U

patients with NSCLC harboring uncommon EGFR mutations of exon 18, 20 and the concomitant EGFR mutation and ALK translocation did not obtain any objective response; for the other mutations, the ORR was 66.6% for exon 19, 40.0% for exon 21, 33.3% for the subgroup of patients carrying two concomitant EGFR mutations and 14.3% for the subgroup with an unspecified

TE D

mutation (Table 3).

The disease control rate (DCR) was 60.0% in the overall population of uncommon EGFR-mutated (Table 3). When analyzed according to the type of EGFR mutation, the DCR ranges from 28.6% for

EP

the subgroup with an unspecified mutation to 100% for the patient with concomitant EGFR mutation and ALK translocation (Table 3).

AC C

Median duration of treatment (DOT) was 9.3 months in the overall population of uncommon EGFR-mutated (Table 3). The DOT was higher in the exon 18 subgroup (14.9 months) and in the patient harboring both EGFR mutation and ALK translocation (21.2 months), and lower in the subgroup with an unspecified EGFR mutation (1.8 months) (Table 3). No unexpected adverse events have been observed with first-line EGFR-TKIs, regardless of the type of uncommon EGFR mutation (Supplementary Table 2). Second-line treatment characteristics 10

ACCEPTED MANUSCRIPT Second-line treatments and efficacy data are reported in Table 4. At data cutoff (February 1st 2015), 2 patients were still on treatment with first-line EGFR-TKI, 33 progressed and 25 received a second-line systemic therapy, but only for 23 full information were available. Among chemotherapy regimens, platinum-doublets were the preferred option, with pemetrexed in combination with

RI PT

cisplatin or carboplatin as the favorite choice. Single-agent chemotherapy was administered to 7 patients, while 3 patients received only best supportive care and in 1 case a re-challenge with EGFR-TKI was done with afatinib (Table 4).

SC

To examine whether the sequence of platinum doublet after EGFR-TKIs treatment affected OS, we performed a further subgroup analysis. The survival time tended to be longer among patients

M AN U

receiving first-line EGFR-TKI followed by platinum-doublet compared with the survival time among those patients treated with EGFR-TKI followed by second-line single-agent therapy (HR =

AC C

EP

TE D

0.24; p = 0.067).

11

ACCEPTED MANUSCRIPT Discussion The establishment of reliable evidence for uncommon EGFR mutations is strongly limited by their low frequency (10-12% of all EGFR mutations), their heterogeneity (approximately 594 EGFR

RI PT

mutation types are reported in the COSMIC database) and by the fact that many pivotal clinical trials excluded patients with these rare EGFR genotypes 9. Considering these intrinsic limitations, the sharing of single or multi-institutional experiences is likely to represent the crucial strategy to

SC

enrich the global knowledge and improve the personalized treatment strategy. With this aim, we performed a post-hoc analysis of the BE-POSITIVE study in order to investigate in the context of a

patients with uncommon EGFR mutations.

M AN U

“real-life” Caucasian EGFR-mutated NSCLC population the efficacy of gefitinib and erlotinib in

The frequency of uncommon EGFR mutations in our series is 11.2% (35/312), consistent with other available data 15-18. Nevertheless, the frequency estimations of rare EGFR mutations appears

TE D

extremely wide, ranging from 2% 19, 20 to 25% 21. Several factors could affect the estimation of the real frequency of EGFR mutations, such as the variability in the detection methods 22, the publication bias caused by the exclusion of uncommon genotypes from clinical trials and

EP

metanalysis 23, and the existence of compound EGFR mutations of difficult classification 24, 25. In our analysis, the most frequent single uncommon EGFR mutations are G719X (2%, 6/312) and

26, 27

AC C

L861Q (1.6%, 5/312), which we observed with similar percentages compared with the literature 12, . Moreover, we reported uncommon variants of exon 19 deletions (1.6%, 5/312) and single

cases of exon 20 alterations including one insertion in the exon 20, one T790M and one S768I mutations. Although different types of alteration may involve the exon 20 28, exon 20 insertions are described to be among the most frequent rare EGFR alterations, ranging from 1% to 17% according to the type of population 26, 29. In our series, we also observed the 2% of compound EGFR mutations, including G719X, L858R and S768I complex mutations, which represent the most frequently detected double EGFR variants 25, 30. Regarding patients’ characteristics, comparing to 12

ACCEPTED MANUSCRIPT the original BE-POSITIVE study where never smokers are predominant (64.1%) 14, most of the patients harboring uncommon EGFR mutations in our analysis were former smokers (65.7%). This finding is consistent with other published evidence supporting a potential correlation between smoking habit and rare EGFR genotypes 17, 31.

RI PT

Recently, several subgroup analyses suggest that NSCLC patients harboring uncommon EGFR mutations may obtain a variable clinical benefit (greater or less) from treatment with EGFR-TKIs compared with classical activating mutations 5, 19, 21, 30-36. Although the general scientific consensus

SC

supports the inferior activity and efficacy of EGFR-TKIs in patients with uncommon mutations, the recent increase in the available data revealed that some rare EGFR genotypes could maintain a

M AN U

certain degree of sensibility to targeted therapy 9, 15, 37. At this regard, also in our post-hoc analysis of the BE-POSITIVE study, the results in the overall uncommon EGFR-mutated population appear inferior to what is usually expected in classical EGFR mutations (ORR 25.7%, median PFS 5.19 months and median OS 14.49 months). Nevertheless, we observed a consistent heterogeneity of

TE D

treatment outcome for patients harboring different uncommon EGFR mutations. Among patients harboring G719X mutations, no objective responses occurred, with a DCR of 66.7%, a median PFS of 8.38 months and a median OS of 17 months. In vitro studies demonstrated

EP

that NSCLC cell lines expressing the G719S mutation are less sensitive to gefitinib compared to L858R mutant cell lines 38, whereas exon 18 alterations seems to be highly sensitive to second-

AC C

generation TKIs afatinib and neratinib 27. For what concerns the clinical experience, the data reported by Wu et al. of 15 G719 mutant patients treated with erlotinib or gefitinib, are similar to our results (ORR 53.3%, median PFS 8.1 months and median OS 16.4 months) 30, whereas a shorter OS compared to classical EGFR mutations is reported with gefitinib in the post-hoc analysis of the NEJ002 study 34. In patients with alternative exon 19 deletions, we observed an ORR of 66.6%, a median PFS of 5.95 months and a median OS of 12.21 months. Considering the heterogeneity of exon 19 deletions

13

ACCEPTED MANUSCRIPT (including at least 30 variants), to date clinical data regarding their predictive impact are still limited and controversial 39. In the subgroup of exon 20 alterations, no objective responses occurred with a DCR of 75.0%, a median PFS of 7.77 months and a median OS of 15.44 months. Several studies suggested that first-

RI PT

generation EGFR-TKIs might be less effective in NSCLC patients with exon 20 uncommon

alterations than in those with common mutations 16, 40. Although preliminary preclinical evidence supported the sensitivity of EGFR S768I mutant lung adenocarcinoma to EGFR-TKIs 41, 42,

SC

conflicting findings are available from clinical series 21, 30. Moreover, the S768I mutation is

frequently detected in association with other EGFR mutations, complicating the understanding of its

M AN U

reliable predictive impact 16.

Regarding the L861Q mutations, we observed a DCR of 60.0%, a median PFS of 5.16 months and a median OS of 14.49 months. Our results are similar to those published by Yasuda et al. who reported the outcome of 15 patients with this mutation treated with erlotinib or gefitinib 43. In the

TE D

NEJ002 study, OS with gefitinib was significantly shorter in patients with rare EGFR mutations (G719X or L861Q) compared with patients with common mutations (11.9 versus 29.3 months; p<0.001), without significant difference in the chemotherapy arm 34. By contrary, L861Q

EP

demonstrated an increased sensitivity to afatinib supported by the combined analysis of LUX-Lung 2,3 and 6 trials describing an ORR of 56% (9/16) 16 and by recent in vitro findings 42.

AC C

The evolution in the analysis of the molecular background of EGFR-mutated NSCLC, led to the discovery of compound EGFR mutations (double or multiple) with a plausible enhanced sensitivity to EGFR-TKIs. For example, complex mutations involving L858R and G719X seem to affect the response to EGFR-TKIs in different ways according to the other associated mutations 25, 44. Our population of double EGFR-mutated NSCLC exhibit the best outcome among rare EGFR mutations, with a median OS of 21.29 months. Although a wide heterogeneity limits the generalization of the results and probably each couple of EGFR alterations owns a peculiar biological behavior, this finding is similar to other published series 26, 40, 45. By contrary, a recent 14

ACCEPTED MANUSCRIPT retrospective analysis exploring the sensitivity to EGFR-TKIs of co-mutation compared with single mutation, reported a significantly shorter median PFS for co-mutation compared with those with single mutation (5.7 versus 12.3 months; p=0.02) with a worse response rate (38% versus 89%; p<0.001) 46. Similarly, in other series of EGFR-mutated advanced lung cancers the presence of

RI PT

additional coexisting mutations decreases the expected benefit of EGFR-TKIs 47, 48.

The patient affected by NSCLC harboring both EGFR mutation and ALK translocation in our analysis, obtained a prolonged benefit with EGFR-TKIs. At this regard, several case reports and

SC

retrospective analyses are available exploring the potential predictive impact of this double gene alteration, with conflicting results in term of sensitivity to EGFR-TKIs and/or crizotinib 49, 50.

M AN U

Finally, in our analysis we identified four NSCLC patients harboring unspecified mutations who did not demonstrate any relevant benefit from first-generation EGFR-TKIs. Nevertheless, there are no relevant predictive information available about these unspecified subtypes of uncommon EGFR mutations, useful to compare our findings.

TE D

For what concerns the data about second-line treatment in uncommon EGFR-mutated NSCLC, the results in term of response rate (25.0%) and PFS (5.06 months) of patients treated with chemotherapy are similar to those reported in the context of the BE-POSITIVE trial, supporting the

EP

lack of a greater benefit deriving from chemotherapy in this subgroup of rare EGFR genotypes. Moreover, although only a little number of events were available, the median OS reported for these

AC C

patients was 12.16 months versus 24.5 months of the BE-POSITIVE study 14. To conclude, although specific clinical recommendations could not be provided with the available data, the general scientific consensus supports that the EGFR mutations involving exons 18 to 21 are usually sensitive to EGFR-TKIs, with the exception of some exon 20 mutations, including T790M, and exon 20 insertions. Nevertheless, globally considered, patients with uncommon EGFR mutations seem to be less responsive to EGFR-TKIs therapy than NSCLC patients harboring common mutations. Regarding the type of TKI, although to date the main insight into the clinical responsiveness of uncommon EGFR mutations has been obtained with afatinib, no validated 15

ACCEPTED MANUSCRIPT difference in term of activity between first and second generation anti-EGFR TKIs are available in order to support the use of one TKI instead of another. Finally, the existence of a strong molecular cancer heterogeneity (i.e. the presence of additional coexisting mutations) suggests that the overall

AC C

EP

TE D

M AN U

SC

RI PT

genomic profile of tumors harboring uncommon EGFR mutations could be even more complex.

16

ACCEPTED MANUSCRIPT Conclusions This patient series reports outcome data in the context of a NSCLC population receiving first-line first-generation TKIs (gefitinib and erlotinib), while the few available data to date are mainly

RI PT

obtained in patients treated with afatinib. In conclusion, our analysis, although limited by its retrospective nature and the small number of included patients, supports the existence of a strong outcome heterogeneity in patients harboring different uncommon EGFR mutations, which needs to

SC

be clarified to achieve a real personalized treatment strategy. Short Clinical Practice Points

M AN U

NSCLC harboring uncommon EGFR mutations (any mutation other than deletion 19 or L858R) represent a heterogeneous group of diseases whose sensitivity or resistance to EGFR-TKIs is still uncertain. Considering the low incidence of each specific uncommon EGFR mutation, the sharing of single or multi-institutional experiences may enrich the global knowledge about this peculiar

TE D

clinical situation. With this aim, our analysis, although limited by its retrospective nature and the small number of included patients, is consistent with other data supporting the existence of a strong heterogeneity of outcome for patients harboring different uncommon EGFR mutations, which

EP

implies the need to stratify the subgroup of rare mutations in individual entities with different

AC C

clinical perspectives. In addition, this patient cohort reports outcome data in the context of a NSCLC population receiving first-line first-generation TKIs (gefitinib and erlotinib), while the few available data to date are mainly obtained in patients treated with afatinib. The incessant collecting and reporting of clinical data, together with the improvement in genotyping techniques to detect EGFR alterations and the in vitro/in vivo investigations of novel TKIs, may contribute to further increase the clinical benefit deriving from targeted therapy in EGFR-mutated NSCLC patients.

17

ACCEPTED MANUSCRIPT Acknowledgments S.P. and E.B. are supported by a specific grant of the Italian Association for Cancer Research (AIRC, My First AIRC Grant n° 14282); S.P. was supported by a Young Investigational Award of

RI PT

the International Association for the Study of Lung Cancer (IASLC). Conflict of interest

Antonio Rossi declared a role as Speaker Bureau for Roche, Boehringer Ingelheim, AstraZeneca,

SC

advisory board's participation for AstraZeneca, Eli-Lilly. Alessandro Morabito declared speaker’s fee from AstraZeneca, Roche, Boehringer Ingelheim. Massimo Di Maio declared honoraria from

M AN U

AstraZeneca and Boehringer Ingelheim. Diego Cortinovis declared a consultancy role for Boehringer Ingelheim, boards’ participation for Novartis, Roche, Eli Lilly and speaker’s fee from Boehringer Ingelheim and Pfizer. Emilio Bria declared a consultancy role for Celgene, boards’ participation for Novartis, AstraZeneca, Pierre-Fabre, and speaker’s fee from Pfizer and MSD.

TE D

Silvia Novello declared a role as Speaker Bureau for Eli Lilly, MSD, BMS, Roche, Boehringer

AC C

EP

Ingelheim, AstraZeneca. All other authors have no conflict of interest to declare.

18

ACCEPTED MANUSCRIPT References

6.

7. 8. 9.

10.

11.

12.

13. 14.

15.

16.

RI PT

SC

5.

M AN U

4.

TE D

3.

EP

2.

Douillard JY, Ostoros G, Cobo M, et al. First-line gefitinib in Caucasian EGFR mutationpositive NSCLC patients: a phase-IV, open-label, single-arm study. British journal of cancer. 2014;110:55-62. Kris MG, Johnson BE, Berry LD, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. Jama. 2014;311:1998-2006. Barlesi F, Mazieres J, Merlio JP, et al. Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT). Lancet. 2016;387:1415-1426. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. The New England journal of medicine. 2004;350:2129-2139. Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer science. 2007;98:1817-1824. Linardou H, Dahabreh IJ, Kanaloupiti D, et al. Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. The Lancet. Oncology. 2008;9:962-972. Rosell R, Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. The New England journal of medicine. 2009;361:958-967. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nature reviews. Cancer. 2007;7:169-181. Kobayashi Y, Mitsudomi T. Not all epidermal growth factor receptor mutations in lung cancer are created equal: Perspectives for individualized treatment strategy. Cancer science. 2016;107:1179-1186. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS medicine. 2005;2:e73. Yu HA, Arcila ME, Hellmann MD, Kris MG, Ladanyi M, Riely GJ. Poor response to erlotinib in patients with tumors containing baseline EGFR T790M mutations found by routine clinical molecular testing. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2014;25:423-428. Massarelli E, Johnson FM, Erickson HS, Wistuba, II, Papadimitrakopoulou V. Uncommon epidermal growth factor receptor mutations in non-small cell lung cancer and their mechanisms of EGFR tyrosine kinase inhibitors sensitivity and resistance. Lung cancer. 2013;80:235-241. Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nature medicine. 2012;18:378-381. Vavala T, Follador A, Tiseo M, et al. BE-POSITIVE: Beyond progression after tyrosine kinase inhibitor in EGFR- positive non small cell lung cancer patients: Results from a multicenter Italian observational study. Lung cancer. 2016;95:73-81. Xu J, Jin B, Chu T, et al. EGFR tyrosine kinase inhibitor (TKI) in patients with advanced non-small cell lung cancer (NSCLC) harboring uncommon EGFR mutations: A real-world study in China. Lung cancer. 2016;96:87-92. Yang JC, Sequist LV, Geater SL, et al. Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6. The Lancet. Oncology. 2015;16:830-838.

AC C

1.

19

ACCEPTED MANUSCRIPT

22.

23.

24.

25.

26.

27.

28.

29.

30.

RI PT

SC

21.

M AN U

20.

TE D

19.

EP

18.

Lohinai Z, Hoda MA, Fabian K, et al. Distinct Epidemiology and Clinical Consequence of Classic Versus Rare EGFR Mutations in Lung Adenocarcinoma. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2015;10:738-746. Roengvoraphoj M, Tsongalis GJ, Dragnev KH, Rigas JR. Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients. Cancer treatment reviews. 2013;39:839-850. De Pas T, Toffalorio F, Manzotti M, et al. Activity of epidermal growth factor receptortyrosine kinase inhibitors in patients with non-small cell lung cancer harboring rare epidermal growth factor receptor mutations. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2011;6:18951901. Wu YL, Kim JH, Park K, Zaatar A, Klingelschmitt G, Ng C. Efficacy and safety of maintenance erlotinib in Asian patients with advanced non-small-cell lung cancer: a subanalysis of the phase III, randomized SATURN study. Lung cancer. 2012;77:339-345. Chiu CH, Yang CT, Shih JY, et al. Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Treatment Response in Advanced Lung Adenocarcinomas with G719X/L861Q/S768I Mutations. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2015;10:793-799. Khoo C, Rogers TM, Fellowes A, Bell A, Fox S. Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond. Translational lung cancer research. 2015;4:126-141. Oxnard GR, Janne PA. Power in numbers: meta-analysis to identify inhibitor-sensitive tumor genotypes. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013;19:1634-1636. Chen Z, Feng J, Saldivar JS, Gu D, Bockholt A, Sommer SS. EGFR somatic doublets in lung cancer are frequent and generally arise from a pair of driver mutations uncommonly seen as singlet mutations: one-third of doublets occur at five pairs of amino acids. Oncogene. 2008;27:4336-4343. Kobayashi S, Canepa HM, Bailey AS, et al. Compound EGFR mutations and response to EGFR tyrosine kinase inhibitors. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2013;8:45-51. Beau-Faller M, Prim N, Ruppert AM, et al. Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2014;25:126-131. Kobayashi Y, Togashi Y, Yatabe Y, et al. EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs. Clinical cancer research : an official journal of the American Association for Cancer Research. 2015;21:5305-5313. Wu JY, Wu SG, Yang CH, et al. Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14:4877-4882. Arcila ME, Nafa K, Chaft JE, et al. EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics. Molecular cancer therapeutics. 2013;12:220-229. Wu JY, Yu CJ, Chang YC, Yang CH, Shih JY, Yang PC. Effectiveness of tyrosine kinase inhibitors on "uncommon" epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2011;17:3812-3821.

AC C

17.

20

ACCEPTED MANUSCRIPT

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

RI PT

SC

35.

M AN U

34.

TE D

33.

EP

32.

Pallis AG, Voutsina A, Kalikaki A, et al. 'Classical' but not 'other' mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with nonsmall cell lung cancer. British journal of cancer. 2007;97:1560-1566. Costa DB, Nguyen KS, Cho BC, et al. Effects of erlotinib in EGFR mutated non-small cell lung cancers with resistance to gefitinib. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14:7060-7067. Sequist LV, Martins RG, Spigel D, et al. First-line gefitinib in patients with advanced nonsmall-cell lung cancer harboring somatic EGFR mutations. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2008;26:2442-2449. Watanabe S, Minegishi Y, Yoshizawa H, et al. Effectiveness of gefitinib against non-smallcell lung cancer with the uncommon EGFR mutations G719X and L861Q. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2014;9:189-194. Arrieta O, Cardona AF, Corrales L, et al. The impact of common and rare EGFR mutations in response to EGFR tyrosine kinase inhibitors and platinum-based chemotherapy in patients with non-small cell lung cancer. Lung cancer. 2015;87:169-175. Baek JH, Sun JM, Min YJ, et al. Efficacy of EGFR tyrosine kinase inhibitors in patients with EGFR-mutated non-small cell lung cancer except both exon 19 deletion and exon 21 L858R: a retrospective analysis in Korea. Lung cancer. 2015;87:148-154. Klughammer B, Brugger W, Cappuzzo F, et al. Examining Treatment Outcomes with Erlotinib in Patients with Advanced Non-Small Cell Lung Cancer Whose Tumors Harbor Uncommon EGFR Mutations. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2016;11:545-555. Jiang J, Greulich H, Janne PA, Sellers WR, Meyerson M, Griffin JD. Epidermal growth factor-independent transformation of Ba/F3 cells with cancer-derived epidermal growth factor receptor mutants induces gefitinib-sensitive cell cycle progression. Cancer research. 2005;65:8968-8974. Chung KP, Wu SG, Wu JY, et al. Clinical outcomes in non-small cell lung cancers harboring different exon 19 deletions in EGFR. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012;18:3470-3477. Chen D, Song Z, Cheng G. Clinical efficacy of first-generation EGFR-TKIs in patients with advanced non-small-cell lung cancer harboring EGFR exon 20 mutations. OncoTargets and therapy. 2016;9:4181-4186. Hellmann MD, Reva B, Yu H, et al. Clinical and in vivo evidence that EGFR S768I mutant lung adenocarcinomas are sensitive to erlotinib. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2014;9:e73-74. Banno E, Togashi Y, Nakamura Y, et al. Sensitivities to various epidermal growth factor receptor-tyrosine kinase inhibitors of uncommon epidermal growth factor receptor mutations L861Q and S768I: What is the optimal epidermal growth factor receptor-tyrosine kinase inhibitor? Cancer science. 2016;107:1134-1140. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. The Lancet. Oncology. 2012;13:e2331. Doss GP, Rajith B, Chakraborty C, NagaSundaram N, Ali SK, Zhu H. Structural signature of the G719S-T790M double mutation in the EGFR kinase domain and its response to inhibitors. Scientific reports. 2014;4:5868. Oikawa T, Ohira T, Otani K, Hagiwara M, Konaka C, Ikeda N. Clinical usefulness of gefitinib for non-small-cell lung cancer with a double epidermal growth factor receptor mutation. Molecular and clinical oncology. 2015;3:329-333. Barnet M, O'Toole S, Horvath L, et al. Epidermal Growth Factor Receptor (EGFR) comutated advanced non-small cell lung cancer (NSCLC) and response to EGFR tyrosine

AC C

31.

21

ACCEPTED MANUSCRIPT

RI PT

SC

M AN U

50.

TE D

49.

EP

48.

AC C

47.

kinase inhibitors (TKIs): A Brief Report. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2016. Bria E, Pilotto S, Amato E, et al. Molecular heterogeneity assessment by next-generation sequencing and response to gefitinib of EGFR mutant advanced lung adenocarcinoma. Oncotarget. 2015;6:12783-12795. Eng J, Woo KM, Sima CS, et al. Impact of Concurrent PIK3CA Mutations on Response to EGFR Tyrosine Kinase Inhibition in EGFR-Mutant Lung Cancers and on Prognosis in Oncogene-Driven Lung Adenocarcinomas. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2015;10:17131719. Pilotto S, Bria E, Peretti U, et al. Lung adenocarcinoma patient refractory to gefitinib and responsive to crizotinib, with concurrent rare mutation of the epidermal growth factor receptor (L861Q) and increased ALK/MET/ROS1 gene copy number. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2013;8:e105-106. Ulivi P, Chiadini E, Dazzi C, et al. Nonsquamous, Non-Small-Cell Lung Cancer Patients Who Carry a Double Mutation of EGFR, EML4-ALK or KRAS: Frequency, ClinicalPathological Characteristics, and Response to Therapy. Clinical lung cancer. 2015.

22

ACCEPTED MANUSCRIPT Figures Figure 1. Kaplan–Meier curves of OS for uncommon EGFR mutations. Panel A: OS for all uncommon EGFR mutations; panel B: OS according to the different type of EGFR mutation.

RI PT

Legend - Figure 1: OS, overall survival; NR, not reached; CI, confidence interval. Figure 2. Kaplan–Meier curves of PFS for uncommon EGFR mutations. Panel A: PFS for all uncommon EGFR mutations; panel B: PFS according to the different type of EGFR mutation.

AC C

EP

TE D

M AN U

SC

Legend - Figure 2: PFS, progression free survival; NR, not reached; CI, confidence interval.

23

ACCEPTED MANUSCRIPT Supplementary material Supplementary Table 1. Characteristics of each patient with uncommon EGFR mutations. Supplementary Table 2. Main safety results of first-line EGFR-TKIs, according to the type of

Legend - Supplementary Table 2: GI, gastrointestinal.

RI PT

EGFR mutation.

Supplementary Figure 1. Kaplan–Meier curves for patients harboring common versus uncommon

SC

EGFR mutations in the BE-POSITIVE trial population. Panel A: OS; panel B: PFS.

AC C

EP

TE D

reached; CI, confidence interval.

M AN U

Legend – Supplementary Figure 1: OS, overall survival; PFS, progression free survival; NR, not

24

ACCEPTED MANUSCRIPT

Table 1. Demographic and disease characteristics of patients (n = 35). Patients Number (%) Median age (range)

Performance Status sec. ECOG at diagnosis 0 1 2 Smoking habit

8 (22.9) 21 (60.0) 6 (17.1)

M AN U

Never Former <= 10 pack/yrs >10 pack/yrs <=20 >20 pack/yrs <=30 >30 pack/yrs <=40 >40 pack/yrs Unknown

20 (57.1) 15 (42.9)

SC

Female Male

RI PT

70 (42-86) Gender

Stage at diagnosis

12 (34.3) 23 (65.7) 6 (26.1) 1 (4.3) 3 (13.0) 4 (17.4) 7 (30.4) 2 (8.8)

2 (5.7) 2 (5.7) 3 (8.6) 4 (11.4) 24 (68.6)

Lung Bone Lymph nodes Pleura Brain Liver Adrenal cortex

22 (62.9) 8 (22.9) 13 (37.1) 10 (31.3) 9 (25.7) 5 (14.3) 5 (14.3)

Adenocarcinoma Large Cell Carcinoma Squamous Cell Carcinoma Sarcomatoid carcinoma

31 (88.6) 2 (5.7) 1 (2.9) 1 (2.9)

TE D

IB IIA IIB IIIB IV

AC C

EP

Site of metastasis *

Histotype

* Frequencies of site metastasis are not cumulative frequencies.

ACCEPTED MANUSCRIPT

Table 2. Types of uncommon EGFR mutations detected in this analysis. Patients Number (%) 6 (17.2) p.G719X p.G719A (c.2156T>C) p.G719C (c.2154_2155GG>TT) Exon 19

4 (11.4) 1 (2.9) 1 (2.9)

RI PT

Exon 18

6 (17.2)

SC

mut and delE746_A750 delL747_S752 delE746_A750 in homozygosis complex mutation in the untranslated region CDS 2234-2258 del747_751 Exon 20

4 (11.4)

Exon 21

M AN U

ins20 p.T790M Polymorphism 787 p.S768I

1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9)

5 (14.2)

p.L861Q

Double EGFR mutation

TE D

Double gene alteration

EP

1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9)

1 (2.9)

del19; ALK rearrangement

Others unspecified

AC C

5 (14.2)

6 (17.2)

del19; p.T790M p.L858R; p.T790M p.L858R; p.S768I p.L858R; p.L838V p.L858R; p.E709A del19; p.G719X

Unknown

1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9) 2 (5.7)

1 (2.9) 4 (11.4)

p.G824A p.G857T mutation exon 21 NOS p.L768G (c.CTG>CAG)

1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9) 3 (8.5)

Legend - Table 2. CDS, coding DNA sequence; NOS, not otherwise specified.

3 (8.5)

ACCEPTED MANUSCRIPT

Table 3. Response to EGFR-TKIs in patients with uncommon EGFR mutations.

n

All

Best Response to EGFR-TKIs CR (%)

PR (%)

SD (%)

PD (%)

35

1 (2.8)

8 (22.8)

12 (34.3)

14 (40.0)

Exon 18

6

0

0

4 (66.7)

2 (33.3)

Exon 19

6

1 (16.7)

3 (50.0)

1 (16.7)

Exon 20

4

0

0

Exon 21

5

0

Double EGFR mutation

6

Double gene alteration Not specified Unknown

ORR (%)

DCR (%)

DOT (months)

9 (25.7)

21 (60.0)

9.3

RI PT

Type of mutation

4 (66.7)

14.9

1 (16.7)

4 (66.6)

5 (83.3)

9.0

3 (75.0)

1 (25.0)

0

3 (75.0)

9.1

2 (40.0)

1 (20.0)

2 (40.0)

2 (40.0)

3 (60.0)

4.8

0

2 (33.3)

1 (16.7)

3 (50.0)

2 (33.3)

3 (50.0)

6.8

1

0

0

1 (100.0)

0

0

1 (100.0)

21.2

7

0

1 (14.3)

1 (14.3)

5 (71.4)

1 (14.3)

2 (28.6)

1.8

M AN U

SC

0

Legend - Table 3. TKIs, tyrosine kinase inhibitors; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; ORR, overall response rate; DCR, disease control rate; DOT,

AC C

EP

TE D

duration of treatment.

ACCEPTED MANUSCRIPT

Table 4. Second-line treatments and efficacy data. Patients

ORR

N

%

Events

Median (95% CI) (Months)

All

23

17.4

17

4.14 (2.14, 7.85)

12

12.09 (3.98, NR)

Chemotherapy (N=16)

16

25.0

12

5.06 (2.04, 10.22)

7

12.16 (2.89, NR)

cisplatin + pemetrexed

3

33.3

2

7.0

1

12.3

PFS

4

50

1

1

0

1

carboplatin + paclitaxel

1

100

3

pemetrexed

3

0

3

gemcitabine

3

0

1

navelbine

1

0

3

0

afatinib

1

0

crizotinib

2

0

4

0

radiotherapy

1

0

BSC

3

0

1

2.8

10.4

0

NR

4.2

0

NR

2.2

2

7.6

5.1

3

5.5

1.9

0

NR

M AN U

Other

Median (95% CI) (Months)

0

1

3.98

1

3.98

0

NR

0

NR

1

4.0

1

4.0

4

2.64

4

3.10

1

5.1

1

8.6

0.3

3

0.3

TE D

Targeted Agent (N=3)

2.7

Events

RI PT

carboplatin + pemetrexed carboplatin + gemcitabine

OS

SC

All EGFR-TKI with second line treatment

3

Legend - Table 4: ORR, overall response rate; PFS, progression free survival; OS, overall survival;

AC C

EP

CI, confidence interval; NR, not reached; BSC, best supportive care.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT