The epidermal growth factor receptor (EGFR) pathway

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1 ORIGINAL ARTICLE Changes in Plasma Mass-Sectral Profile in Course of Treatment of Non-small Cell Lung Cancer Patients with Eidermal Growth Factor Recetor Tyrosine Kinase Inhibitors Chiara Lazzari, MD,* Anna Sreafico, MD,* Angela Bachi, PhD, Heinrich Roder, PhD, Irene Floriani, PhD, Daniela Garavaglia, PhD, Angela Cattaneo, PhD, Julia Grigorieva, PhD, Maria Grazia Viganò, MD,* Cristina Sorlini, MD,* Domenico Ghio, MD, Maxim Tsyin, PhD, Alessandra Bulotta, MD,* Luca Bergamaschi, MD,* and Vanesa Gregorc, MD* Introduction: Our revious study showed that retreatment serum or lasma Matrix-Assisted Laser Desortion/Ionization Time-of- Flight Mass Sectrometry may redict clinical outcome of nonsmall cell lung cancer (NSCLC) atients treated with eidermal growth factor recetor (EGFR) tyrosine kinase inhibitors (TKIs). In this study, lasma roteomic rofiles of NSCLC atients were evaluated in the course of EGFR TKIs theray. Materials and Methods: Plasma samles were collected at baseline, in the course of gefitinib theray and at treatment withdrawal. Samles were analyzed by Matrix-Assisted Laser Desortion/Ionization Time-of-Flight Mass Sectrometry. Acquired sectra were classified by the VeriStrat test into good and oor rofiles. The association between VeriStrat classification and rogression-free survival (PFS) and overall survival (OS), and tyes of clinical rogression, was analyzed. Results: Plasma samles from 111 NSCLC atients treated with gefitinib were rocessed. VeriStrat good classification at baseline correlated with longer PFS (hazard ratio [HR], 0.54; 95% confidence interval, ; 0.005) and OS (HR, 0.40; 95% confidence interval, ; ), when comared with VeriStrat oor. Multivariate analysis confirmed longer PFS (HR, 0.52; 0.025) and OS (HR, 0.44; 0.001) in atients classified as VeriStrat good, when VeriStrat was considered as a time-deendent variable. About one-third of baseline good classifications had changed to oor at the time of treatment withdrawal; rogression in these atients was associated with the develoment of new lesions. *Deartment of Oncology, San Raffaele Scientific Institute; Biomolecular Mass Sectrometry Unit, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy; Biodesix, Steamboat Srings, Colorado; Istituto di Ricerche Farmacologiche Mario Negri ; and Deartment of Radiology, San Raffaele Scientific Institute, Milan, Italy. Address for corresondence: Vanesa Gregorc, San Raffaele Scientific Institute, Via Olgettina, Milan, Italy. gregorc.vanesa@hsr.it The first two authors equally contributed to the work. Coyright 2011 by the International Association for the Study of Lung Cancer ISSN: /12/ Conclusions: Our findings suort the role of VeriStrat in the assistance in treatment selection of NSCLC atients for EGFR TKI theray and its otential utility in treatment monitoring. Key Words: Non-small cell lung cancer, Eidermal growth factor recetor tyrosine kinase inhibitors, Proteomics, Resistance to eidermal growth factor tyrosine kinase inhibitor. (J Thorac Oncol. 2012;7: 40 48) The eidermal growth factor recetor (EGFR) athway lays a key role in the develoment and rogression of non-small cell lung cancer (NSCLC) and many other malignant eithelial tumors. 1 Major achievements in the treatment of lung, colorectal, and head and neck cancer were associated with the develoment of targeted drugs inhibiting the EGFR athway. 2 Gefitinib and erlotinib are oral EGFR tyrosine kinase inhibitors (EGFR TKIs) shown to be active in NSCLC atients. 3 7 Nevertheless, desite a high exression level of EGFR recetor in most lung cancers, only a certain fraction of NSCLC atients benefits from EGFR TKIs. 8 Identification of biomarkers redictive of resonse to treatment became a focus of the clinical research in the recent decade. Multile studies have shown a ositive correlation between activating EGFR gene mutations (exon 19 deletion, exon 18 G719X, and exon 21 L858R mutations), resonse rate, and rogression-free survival (PFS) for EGFR-TKI theraies Results from recent hase III clinical trials, comaring gefitinib and chemotheray arms in front line (IPASS, First SIGNAL, NEJ002, and WJTOG 3405) and in second line (INTEREST), 19,20 have led to the regulatory aroval of gefitinib in Euroe in all lines of theray for atients carrying EGFR mutations. At the same time, the INTEREST study showed that in unselected and EGFR wild-tye atients, gefitinib and docetaxel rovide similar PFS and overall survival (OS). 19,20 The BR.21 trial 7 (hase III trial of erlotinib versus lacebo in reviously treated advanced NSCLC atients) showed that erlotinib significantly rolonged OS, comared with lacebo, leading 40 Journal of Thoracic Oncology Volume 7, Number 1, January 2012

2 Journal of Thoracic Oncology Volume 7, Number 1, January 2012 Plasma Mass-Sectral Profile in Treatment NSCLC to accetance of erlotinib as a treatment in advanced NSCLC atients after the failure of first line chemotheray. Nevertheless, correlative studies erformed in a subset of atients with available tissue enrolled in the BR.21 trial showed that EGFR mutations were rognostic for OS but were not redictive, whereas increased EGFR coy number was both rognostic and redictive for OS benefit. 21 None of measured biomarkers was redictive of OS in the INTEREST trial. 20 Considering that rolonged OS and stabilization of the disease are imortant criteria of benefit from treatment, esecially in advanced lines of treatment, and taking into account the low frequency of EGFR mutations in the Caucasian oulation (aroximately 10%), 22 and ossible discordance in the mutation status between rimary NSCLC and corresonding metastatic tumors, 23,24 and absence of consensus on the role of molecular markers in second-line treatment selection in EGFR wild-tye atients, it is of clinical imortance to find additional indeendent biomarkers redictive of benefit from treatment. The insufficient availability of tumor tissue for molecular analysis, which ranges between 20 and 30% even in large well-designed clinical trials, makes the discovery and validation of a serum or lasma-based redictive test esecially desirable. In addition, such a noninvasive test, if it were correlated with a switch from drug sensitivity to drug resistance, would be extremely valuable in monitoring the onset of the acquired resistance that eventually develos in the majority of atients, even those with good initial clinical resonse. 25 In our revious multiinstitutional study, Matrix-Assisted Laser Desortion/Ionization Time-of-Flight Mass Sectrometry (MALDI ToF MS) was used to build, develo, and indeendently validate a serum/lasma test (VeriStrat), able to identify, before treatment, a subset of NSCLC atients with better outcome from EGFR TKI theray in terms of time to rogression and OS. 26 The algorithm in the core of the test uses the integrated intensities of eight mass sectral eaks and assigns a classification label good or oor or indeterminate. The identity of the eaks constituting the test and the underlying biological mechanism related to the VeriStrat signature are subjects of ongoing investigations. The test was develoed using a training set of samles from three different cohorts of atients treated with gefitinib and validated in blinded fashion in two other indeendent cohorts treated with gefitinib and erlotinib and in three control cohorts of atients treated with chemotheray and surgery. The blinded validation showed statistically significant searation in terms of both OS and in terms of time to rogression in NSCLC atients treated with EGFR TKIs, whereas no statistically significant searation was observed in cohorts treated with chemotheray or surgery. The original study was followed by multile clinical validation studies in NSCLC and in other eithelial tumors. Alication of VeriStrat to a subset of samles from the BR.21 trial showed that the test has a significant rognostic comonent, i.e., demonstrates a searation by VeriStrat for OS and PFS both in lacebo and in treatment arms. However, VeriStrat good atients received statistically significant benefit from erlotinib theray over lacebo, whereas in VeriStrat oor atients, the searation was not statistically significant. In addition, VeriStrat classification was redictive of objective resonse to erlotinib and significantly correlated with disease control rate in the treatment arm. 27 VeriStrat redicted OS of atients with head and neck squamous cell carcinoma (HNSCC) treated with gefitinib, erlotinib/bevacizumab, and cetuximab and PFS in colorectal cancer atients treated with cetuximab; a chemotheray cohort again showed no statistically significant survival difference. 28 In the case of combination theray targeting EGFR and vascular endothelial growth factor (VEGF), the magnitude of searation, in comarison with monotheraies, increased dramatically. 29 The body of results obtained demonstrated the alicability of VeriStrat in various tyes of eithelial cancers and in a variety of targeted theraies, including EGFR-TKIs, anti-egfr, and anti-vegf antibodies and their combinations. The deendence of the magnitude of the searation of survival curves between VeriStrat good and oor atients on the articular treatment suggests the relation of the test not just to the natural history of the disease of a atient, but to the theray itself, and rovides additional evidence of VeriStrat redictive roerties. The results obtained in the analyses of retreatment samles insired the current study of the stability of VeriStrat classification in the course of gefitinib treatment of NSCLC atients, as well of the ossible correlation of its changes with disease rogression, and of the otential utility of the test for treatment monitoring. MATERIALS AND METHODS Patient and Samle Characteristics This is a retrosective study of samles from NSCLC atients treated with gefitinib in advanced lines of treatment at the Scientific Institute San Raffaele University Hosital of Milan, Italy. Patients rovided written informed consent for the study; analyses were erformed under the rotocol aroved by the local institutional review boards. Eligibility criteria included atients aged 18 years and older with cytological or histological diagnosis of advanced or inoerable NSCLC; Eastern Cooerative Oncology Grou erformance status (ECOG PS) 2; adequate heatic (total bilirubin 2.5 the uer limit of normal and AST 2.5 normal) function, treatment with gefitinib at the recommended dose (250 mg/daily). Plasma samles were collected at baseline, after 1 month and concomitantly with CT scan evaluation erformed every other month until withdrawal from treatment with EGFR TKIs for either toxicity or rogression. Progression was defined according to RECIST criteria, version 1.0. CT scan evaluation was erformed by a designated radiologist. The collected blood was centrifuged at 2000g for 10 minutes at 4 C, and lasma was searated, aliquoted, and roerly stored at 80 C at Scientific Institute San Raffaele University Hosital of Milan, Italy, until analysis. MALDI ToF Mass Sectrometry Proteomic sectra were collected in the Biological Sectra Unit at the Scientific Institute San Raffaele University Hosital, Milan, Italy. Mass sectra were generated on a Voyager DE-STR MALDI ToF mass sectrometer (Alied Coyright 2011 by the International Association for the Study of Lung Cancer 41

3 Lazzari et al. Journal of Thoracic Oncology Volume 7, Number 1, January 2012 Biosystems, Framingham, MA). Plasma was thawed on ice and diluted 1:10 in deionized water. One microliter of each diluted samle was sotted on the MALDI target and 1 l of matrix solution (35 mg/ml sinainic acid [Sigma, St. Louis, MO], 50% acetonitrile [Burdick & Jackson, Muskegon, MI], and 0.1% trifluoroacetic acid [Sigma]) was added. The solution was mixed by drawing the mixture u and down into the iette ti 10 times and then exelling it. The MALDI lates were then allowed to dry at room temerature in a dark lace. Samles were sotted in trilicate on the MALDI target and at least three sectra were collected for each samle. Positive ion mass sectra were acquired in linear mode in an automated manner. One hundred fifty shots were collected from four unique locations within the erimeter of each MALDI sot to generate an average sectrum from 600 indeendent sectra for each lasma secimen. Raw sectra were sent electronically to Biodesix (Steamboat Srings, CO) and analyzed using the reviously validated VeriStrat algorithm (Biodesix, Inc.) that assigns either good or oor or indeterminate classification to a samle. EGFR Mutational Status and EGFR Gene Amlification All secimens were obtained from the original biosy, before any treatment. GenomicDNA was derived from tumor tissue after laser cature microdissection. Deletions in exon 19 (del 19) were determined by length analysis after olymerase chain reaction amlification with the use of a FAMlabeled rimer in an ABI Prism 3130 DNA Analyzer (Alied Biosystems). Exon 21 oint mutations in codon 858 were detected with a 5 nuclease PCR assay (TaqMan assay) using FAM and VIC MGB-labeled robes for the wild tye and the mutant sequence, resectively. All mutants were confirmed by DNA sequencing. Gene coy number er cell was investigated by fluorescence in situ hybridization (FISH) using the LSI EGFR SectrumOrange/CEP 7 SectrumGreen robe (Vysis; Abbott Laboratories, IL), according to a ublished rotocol. 30 High EGFR gene coy number was defined as high olysomy ( 4 coies in 40% of cells) or gene amlification (resence of tight gene clusters; a gene: chromosome ratio er cell of 2; or 15 coies of EGFR er cell in 10% of cells analyzed). Statistical Analysis PFS was defined as the time from the beginning of gefitinib treatment to first aearance of rogressive disease or death from any cause. Patients known to be alive, and who had not rogressed at the time of analysis were censored at their last available follow-u assessment. OS was defined as the time from the beginning of gefitinib treatment to the date of death from any cause. Patients not reorted as having died at the time of the analysis were censored at the date they were last known to be alive. Survival curves were estimated using the Kalan-Meier method. Cox roortional hazards models were used for univariate and multivariate analyses to test demograhic characteristics, clinical features, and VeriStrat rofile (included as time-deendent variable) for their associations with PFS and OS. Variables found to be associated ( 0.10) with PFS and OS in the univariate model were included in the multivariate analysis. A logistic regression model was used to assess the association of demograhic characteristics and clinical features with VeriStrat status at baseline. Correlation between EGFR amlification by FISH and VeriStrat classification was assessed with 2 test. Results were exressed as hazard ratios (HRs) or odds ratios (ORs) and relative 95% confidence intervals (CIs). The statistical significance was set at 0.05 for a bilateral test. Analyses were carried out with SAS Software, version 9.1 (SAS Institute, Cary, NC). RESULTS Patients Characteristics Consecutive lasma samles from NSCLC atients, admitted to Scientific Institute San Raffaele Hosital from October 2001 until December 2004 and included in the revious study, 26 were collected in the course of treatment and evaluated in this study. One hundred eleven atients having received gefitinib for a median duration of 3.5 (range, ) months were analyzed. Patients characteristics are shown in Table 1. Patients were redominantly males (77%), ever smokers (84%), with ECOG-PS of 0 1 (82%). The majority of them (96%) had advanced disease and 50% had adenocarcinoma histology. Median age was 68 years, ranging from 36 to 91 years. Most of the atients (72%) were treated with gefitinib in a second/third-line setting; 21% of atients received gefitinib as a first-line theray because of their clinical conditions and comorbidities. In the course of treatment and follow-u, 110 atients rogressed and 109 deceased. Median PFS and OS were 3.4 (interquartile range, ) and 8.3 (interquartile range, ) months, resectively, as exected in locally advanced and metastatic NSCLC atients (Sulementary digital content 1, htt://links.lww.com/jto/a166). VeriStrat Classification at Baseline VeriStrat classification was erformed at baseline, after 1 month of gefitinib theray, and every 2 months concomitantly to CT scan evaluation until withdrawal in a total of 476 lasma samles. At baseline, 69% of atients were classified as VeriStrat good and 28% as VeriStrat oor. Concordantly with reviously ublished results, 26 only 3% of atients had an indeterminate classification; they were excluded from the statistical analyses. Patients classified as VeriStrat good had longer PFS (HR, 0.54; 95% CI, ; 0.005) and OS (HR, 0.40; 95% CI, ; ) than VeriStrat oor atients (Figures 1 and 2). VeriStrat good classification was associated with adenocarcinoma histology and ECOG PS both in univariate and multivariate analyses. For all other variables, no statistically significant correlation was detected (Table 2). VeriStrat Classification in the Course of Treatment In the course of treatment (before rogression or withdrawal for other reasons), 98 of 111 (88%) atients main- 42 Coyright 2011 by the International Association for the Study of Lung Cancer

4 Journal of Thoracic Oncology Volume 7, Number 1, January 2012 Plasma Mass-Sectral Profile in Treatment NSCLC TABLE 1. Main Patient Characteristics Characteristic N % No. of atients 111 Age, yr Median (range) 68 (36 91) Sex Male Female Smoking history Ever smoke Never smoke Unknown 6 ECOG-PS Unknown 2 Histology Adenocarcinoma Squamous carcinoma NSCLC BAL 7 6 Stage Ib/IIb/IIIa 5 4 IIIb IV Line of treatment I II III IV 7 6 V 1 1 Treatment duration, mo Median (range) 3.5 (0.7 47) NSCLC, non-small cell lung cancer. tained their baseline VeriStrat classification and only 13 (11%) resented one or more intraindividual changes of label (from good to oor or vice versa). VeriStrat classification of individual atients in the course of gefitinib treatment, along with PFS and OS, is resented in Figure 3. At treatment withdrawal, the number of VeriStrat good rofile atients decreased from 69 to 51%, whereas the number of VeriStrat oor rofile atients increased from 28 to 43%; 6 atients (6%) were indeterminate. The data are summarized in Table 3. Twenty of 71 (28%) of good classified atients shifted to a oor rofile at withdrawal, and in 90% of these cases they either stoed treatment because of the evidence of new lesions or died early. Within this subgrou, 61% of atients resented new lesions. Patients who shifted from good to oor classification had a higher risk of develoing new lesions in comarison with other atients (OR, 2.9; 95% CI, ; 0.049). Nevertheless, new lesions were also observed in 22% of atients who remained good at rogression. Interestingly, in 12% of the cases that remained good at rogression with new lesions, the brain was the only site of new metastases. Of 32 baseline VeriStrat oor classified atients, 27 (84%) maintained a oor rofile at treatment withdrawal, 96% of whom remained steadily oor : 7 of them died within 1 month from the beginning of treatment and 20 atients rogressed early during gefitinib theray. Of the five atients who changed rofile, one (3%) became indeterminate and four (12%) shifted to good. Two of these four atients had a ronounced clinical benefit and remained on treatment for 3 years. VeriStrat classification, considered as a time-deendent variable, had a statistically significant effect both on PFS and OS. Good classification was associated with longer PFS both in univariate (HR, 0.54; 95% CI, ; 0.004) and multivariate (HR, 0.52; 95% CI, ; 0.025) analysis. Good VeriStrat classification was associated with longer OS both in univariate and multivariate analysis (HR, 0.35; 95% CI, ; and HR, 0.44; 95% CI, FIGURE 1. Progression-free survival (PFS) according to baseline VeriStrat classification (HR, 0.54; 95% CI, ; 0.005). Coyright 2011 by the International Association for the Study of Lung Cancer 43

5 Lazzari et al. Journal of Thoracic Oncology Volume 7, Number 1, January 2012 FIGURE 2. Overall survival (OS) according to baseline VeriStrat classification (HR, 0.40; 95% CI, ; ). TABLE 2. Univariate and Multivariate Logistic Regression Models Assessing the Association Between Demograhic and Clinical Characteristics and Veristrat Good Profile Characteristics Univariate OR (95% CI) Multivariate OR (95% CI) Sex Male Reference Not included Female 2.17 ( ) Smoking history Never smoke Reference Not included Ever smoke 0.32 ( ) ECOG-PS 0 Reference Reference ( ) ( ) ( ) ( ) Histology Other Reference Reference Adenocarcinoma 2.49 ( ) ( ) Platinum-based I line CHT No Reference Not included Yes 1.47 ( ) ECOG-PS, ECOG erformance status; CHT, chemotheray; CI, confidence interval; OR, odds ratio ; 0.001, resectively). Smoking history, ECOG-PS, latinum-based first line chemotheray (only in univariate analysis), and resence of metastases seemed to significantly affect PFS and OS in both models. Results are reorted in Table 4. EGFR Gene Amlification and Mutational Analysis Of 111 atients, 17 (15%) had an adequate tissue samle to erform both EGFR gene amlification and EGFR mutation analysis; for 34 atients (30%), only EGFR gene amlification was assessable. Mutation analysis identified one EGFR mutation (exon 21 L858R). No correlation between known EGFR status and baseline VeriStrat classification was found (Sulementary digital content 2, htt://links.lww.com/jto/a167). DISCUSSION In our revious work, VeriStrat was develoed as a test for atient selection for EGFRTKIs treatment in NSCLC. Further studies showed that it was also alicable to other eithelial cancers, such as colorectal and HNSCC, and to other targeted theraies, including anti-egfr and anti- VEGF, actually demonstrating larger effects in cases of combination treatments. In the studied chemotheray-treated cohorts of NSCLC and HNSCC atients, the test did not show significant searation of survival curves between 44 Coyright 2011 by the International Association for the Study of Lung Cancer

6 Journal of Thoracic Oncology Volume 7, Number 1, January 2012 Plasma Mass-Sectral Profile in Treatment NSCLC FIGURE 3. VeriStrat classification of individual atients in course of gefitinib theray along with rogression and death. TABLE 3. Veristrat Profile at Baseline and at Treatment Withdrawal Veristrat Profile Treatment Withdrawal Good Poor Indeterminate Total Baseline Good 51 (89) 20 (42) 5 (83) 76 (69) Poor 4 (7) 27 (56) 1 (17) 32 (28) Indeterminate 2 (4) 1 (2) 0 (0) 3 (3) Total 57 (51) 48 (43) 6 (6) 111 Values resented as N (%). good and oor subgrous. 26,28 Retrosective analysis of the available samles from the BR.21 trial showed that the VeriStrat test has a significant rognostic comonent, i.e., OS and PFS were significantly different between VeriStrat good and oor grous not only in the erlotinib arm but also in the lacebo arm. Nevertheless, VeriStrat good atients received statistically significant benefit from the targeted theray when comared with lacebo, whereas in the VeriStrat oor grou, this difference was not statistically significant. Resonse to treatment was also significantly correlated with the VeriStrat good classification. 27 Aarently, differences in PFS and OS of VeriStrat good and oor atients are caused by the combination of their rognostic characteristics and differences in their reaction to secific treatments. The relative imact of both comonents deends on the articular treatment and requires further investigation. The clinical relevance of VeriStrat in the advanced NSCLC oulation is defined by its ability to assist in the choice of the otimal treatment between chemotheray and targeted theray in the second-line setting. This is esecially imortant in EGFR wild-tye atients and in atients with unknown EGFR status. Our reliminary data resented at 13th World Conference on Lung Cancer give an indication that VeriStrat oor atients have longer OS when treated with chemotheray, rather than with gefitinib, suggesting that chemotheray might be referred in this subgrou. In the VeriStrat good grou, there was no statistically significant difference in survival between the chemotheray- and gefitinib-treated atients. 32 Taken together, these data suggest that VeriStrat oor classified atients have worse clinical outcome than VeriStrat good atients, esecially when treated with targeted agents, whereas chemotheray might imrove survival in this subgrou. This observation needs further validation and the ongoing rosective hase III trial Randomized Proteomic Stratified Phase III Study of Second Line Erlotinib versus Chemotheray in Patients with Inoerable Non-Small Cell Lung Cancer (PROSE) was designed to rovide further clarification of this question. The aim of this study was to investigate ossible changes in VeriStrat classification of lasma samles from NSCLC atients collected in the course of treatment with gefitinib, concomitantly with CT evaluations. Statistical reanalysis of baseline samles, now using the mature survival data, confirmed a significant searation in OS and PFS curves between VeriStrat good and oor atients. Seventy er cent of atients retained their baseline VeriStrat classification at treatment withdrawal, whereas 30% exerienced a change. The majority of the observed changes were from good to oor at rogression of the disease. About one-third of Coyright 2011 by the International Association for the Study of Lung Cancer 45

7 Lazzari et al. Journal of Thoracic Oncology Volume 7, Number 1, January 2012 TABLE 4. Characteristics Results of Cox Univariate and Multivariate Analyses for Progression-Free Survival (PFS) and Overall Survival (OS) PFS Univariate HR (95% CI) Multivariate HR (95% CI) OS Univariate HR (95% CI) Multivariate HR (95% CI) Sex Male Reference Not included Reference Not included Female 0.76 ( ) ( ) Smoking history Never smoke Reference Reference Reference Reference Ever smoke 1.50 ( ) ( ) ( ) ( ) ECOG-PS 0 Reference Reference ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) Histology Other Reference Not included Reference Not included Adenocarcinoma 1.11 ( ) ( ) Platinum-based I line CHT No Reference Reference Reference Not included Yes 1.51 ( ) ( ) ( ) Metastases No Reference Reference Reference Not included Yes 1.90 ( ) ( ) ( ) Line of treatment a 1.19 ( ) ( ) ( ) Not included Veristrat rofile b Poor Reference Reference Reference Reference Good 0.54 ( ) ( ) ( ) ( ) a Included as continuous variable. b Included as time-deendent variable. PFS, rogression-free survival; OS, overall survival; ECOG-PS, ECOG erformance status; CHT, chemotheray; HR, hazard ratio; CI, confidence interval; OR, odds ratio. baseline good classifications had changed to oor, and in 90% of these cases, rogression was associated with the develoment of new lesions, detected by radiological assessment, or atients died within 1 month from the start of treatment. The risk of new lesions in atients with the shift in classification from good to oor was significantly higher than in the rest of the oulation (OR, 2.9; 0.049). There were three distinct tyes of rogression in atients who remained good in the course of observation. Twenty-two er cent of cases had rogression with new lesions. Other atients did not develo new lesions, and disease rogression was defined as an increase of the target lesion diameter by RECIST criteria. This observation suorts criticism of the clinical imact of RECIST criteria, in articular, of the relation between critical dimensional increase of target lesions and OS. Finally, in a small grou of stably good atients, rogression was diagnosed as occurrence of new lesions in the brain only. Aarently, this tye of rogression is not associated with changes of VeriStrat molecular rofile, robably because of the secific nature of brain metastases. The majority of baseline oor atients maintained their classification in the course of treatment and at withdrawal; four atients had shifted to good and, interestingly, two of them had ronounced clinical benefits and long survival times. Of note, the change in the VeriStrat classification was observed in these atients at the time of the second blood draw (after 1 month of theray), whereas the timing of the baseline samle coincided with atient conditions associated with acute inflammation, i.e., surgery (femoral rosthesis) in one case and ulmonary thrombosis in the other case. In summary, the data obtained in this study indicate that the molecular secies resonsible for the VeriStrat mass sectral signature are time-deendent dynamic markers, robably related to some secific unknown rimary (in the case of baseline VeriStrat oor atients) or acquired (in the case of change of classification from good to oor ) EGFR TKI resistance mechanisms. Recent results resented at 2011 AACR conference showed that VeriStrat oor and good serum can have different biological effects on NSCLC cell lines: incubation of HCC4006 EGFR-TKI sensitive cell lines in a medium containing 10% serum from VeriStrat oor atients increased the resistance of these cells to gefitinib, whereas addition of VeriStrat good serum did not cause changes in the drug sensitivity. These data suort the hyothesis that the VeriStrat oor signature may be associated with some mechanisms that influence cell sensitivity to targeted agents. 33 The ongoing study aimed at the identification of the roteins constituting the VeriStrat mass-sectral signature and understanding the biological mechanism associated with it is suorted by Associazione Italiana sulla Ricerca del Cancro. 46 Coyright 2011 by the International Association for the Study of Lung Cancer

8 Journal of Thoracic Oncology Volume 7, Number 1, January 2012 Plasma Mass-Sectral Profile in Treatment NSCLC Although sensitivity to EGFR-TKIs is considered to be associated with activating mutations in tyrosine-kinase domain, the data on rimary resistance in NSCLC are conflicting, with some studies showing the involvement of KRAS, BRAF, and PI3K mutations. A secondary T790M mutation in the kinase domain of the EGFR 34 and activation of alternative athways allowing the byassing of inhibition of EGFR signaling, such as overexression of heatocyte growth factor 35 and MET amlification, 36 activation of AKT/mTOR, or changes in signaling via insulin-like growth factor-1 recetor, are known resistance mechanism to EGFR TKI theray. Available data on the VeriStrat test in relation to studied genetic markers obtained in the retrosective analysis of the BR.21 study 27 as well on several smaller studies 28,40 did not show any significant correlations between VeriStrat classification and EGFR mutational status, EGFR gene amlification, or KRAS mutations. In this study, we also did not find a correlation between EGFR gene amlification by FISH and VeriStrat. Possible associations with other genetic characteristics, esecially with T790M, need further investigation. Nevertheless, it has been shown that the T790M increases ATP affinity only for the EGFR mutant L858R and not for the wild-tye recetor, which is a redominant oulation in our study. 41 In addition, not fully understood mechanisms, robably involving concomitant activation of multile, often overlaing signaling athways, may also be involved in resistance to targeted agents. 42,43 It was shown that lung cancer develos in a host environment in which the deregulated inflammatory resonse romotes tumor rogression, and inflammatory mediators are derived from neolastic cells and from stromal and inflammatory cells surrounding the tumor. 44 A substantial role of inflammatory rocesses in the resistance mechanisms is suorted by a growing body of exerimental evidence. For examle, it has been shown that activation of the roinflammatory cytokine IL-6 reduced the sensitivity to erlotinib in NSCLC cells harboring EGFR mutations. 45 Activation of the NFkB, a major regulator of immune resonse, has been recently roosed as another resistance mechanism to EGFR TKIs. Low IkB exression ( high-nfkb activation state) was redictive of worse PFS and decreased OS in 52 NSCLC atients harboring EGFR activating mutations and treated with erlotinib. 46 Although the biological mechanism associated with the VeriStrat test is as yet unknown, the available body of evidence allows us to hyothesize that they may be related to host-resonse rocesses. The results of this study, and reviously ublished data, suggest that roteins involved with the oor signature may be associated both with the intrinsic resistance to anti-egfr agents and with the switch to a certain tye of acquired resistance, which is associated with metastases and the formation of new lesions. This noninvasive test may be esecially useful for the selection of second-line theray in the absence of available tumor tissue and in atients who do not harbor EGFR-activating mutations. The ongoing hase III PROSE study is rosectively validating this aroach. Possibly the test can be used as an additional tool for treatment monitoring and investigation of mechanisms of rimary and acquired resistance. ACKNOWLEDGMENTS Suorted by Associazione Italiana er la Ricerca sul Cancro (AIRC). REFERENCES 1. Marmor MD, Skaria KB, Yarden Y. Signal transduction and oncogenesis by ErbB/HER recetors. Int J Radiat Oncol Biol Phys 2004;58: Hirsch FR, Scagliotti GV, Langer CJ, et al. Eidermal growth factor family of recetors in reneolasia and lung cancer: ersectives for targeted theraies. Lung Cancer 2003;41: Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med 2008;358: Fukuoka M, Yano S, Giaccone G, et al. 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