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Purpose: Not all patients with metastatic castration-resistant prostate cancer (mCRPC) have sufficient tumor tissue available for multigene molecular testing. Furthermore, samples may fail because of difficulties within the testing procedure. Optimization of screening techniques may reduce failure rates; however, a need remains for additional testing methods to detect cancers with alterations in homologous recombination repair genes. We evaluated the utility of plasma-derived circulating tumor DNA (ctDNA) in identifying deleterious BRCA1, BRCA2 (BRCA), and ATM alterations in screened patients with mCRPC from the phase III PROfound study. Patients and Methods: Tumor tissue samples were sequenced prospectively at Foundation Medicine, Inc. (FMI) using an investigational next-generation sequencing (NGS) assay based on FoundationOne®CDx to inform trial eligibility. Matched ctDNA samples were retrospectively sequenced at FMI, using an investigational assay based on FoundationOne®Liquid CDx. Results: 81% (503/619) of ctDNA samples yielded an NGS result, of which 491 had a tumor tissue result. BRCA and ATM status in tissue compared with ctDNA showed 81% positive percentage agreement and 92% negative percentage agreement, using tissue as reference. At variant-subtype level, using tissue as reference, concordance was high for nonsense (93%), splice (87%), and frameshift (86%) alterations but lower for large rearrangements (63%) and homozygous deletions (27%), with low ctDNA fraction being a limiting factor. Conclusions: We demonstrate that ctDNA can greatly complement tissue testing in identifying patients with mCRPC and BRCA or ATM alterations who are potentially suitable for receiving targeted PARP inhibitor treatments, particularly patients with no or insufficient tissue for genomic analyses.

Purpose:The phase III PROfound study (NCT02987543) evaluated olaparib versus abiraterone or enzalutamide (control) in metastatic castration-resistant prostate cancer (mCRPC) with tumor homologous recombination repair (HRR) gene alterations. We present exploratory analyses on the use of circulating tumor DNA (ctDNA) testing as an additional method to identify patients with mCRPC with HRR gene alterations who may be eligible for olaparib treatment.Patients and Methods:Plasma samples collected during screening in PROfound were retrospectively sequenced using the FoundationOne®Liquid CDx test for BRCA1, BRCA2 (BRCA), and ATM alterations in ctDNA. Only patients from Cohort A (BRCA/ATM alteration positive by tissue testing) were evaluated. We compared clinical outcomes, including radiographic progression-free survival (rPFS) between the ctDNA subgroup and Cohort A.Results:Of the 181 (73.9%) Cohort A patients who gave consent for plasma sample ctDNA testing, 139 (76.8%) yielded a result and BRCA/ATM alterations were identified in 111 (79.9%). Of these, 73 patients received olaparib and 38 received control. Patients’ baseline demographics and characteristics, and the prevalence of HRR alterations were comparable with the Cohort A intention-to-treat (ITT) population. rPFS was longer in the olaparib group versus control [median 7.4 vs. 3.5 months; hazard ratio (HR), 0.33; 95% confidence interval (CI), 0.21–0.53; nominal P < 0.0001], which is consistent with Cohort A ITT population (HR, 0.34; 95% CI, 0.25–0.47).Conclusions:When tumor tissue testing is not feasible or has failed, ctDNA testing may be a suitable alternative to identify patients with mCRPC carrying BRCA/ATM alterations who may benefit from olaparib treatment.

Supplementary Figure from Detection of BRCA1, BRCA2, and ATM Alterations in Matched Tumor Tissue and Circulating Tumor DNA in Patients with Prostate Cancer Screened in PROfound

Supplementary Data from Detection of BRCA1, BRCA2, and ATM Alterations in Matched Tumor Tissue and Circulating Tumor DNA in Patients with Prostate Cancer Screened in PROfound

Purpose:Not all patients with metastatic castration-resistant prostate cancer (mCRPC) have sufficient tumor tissue available for multigene molecular testing. Furthermore, samples may fail because of difficulties within the testing procedure. Optimization of screening techniques may reduce failure rates; however, a need remains for additional testing methods to detect cancers with alterations in homologous recombination repair genes. We evaluated the utility of plasma-derived circulating tumor DNA (ctDNA) in identifying deleterious BRCA1, BRCA2 (BRCA), and ATM alterations in screened patients with mCRPC from the phase III PROfound study.Patients and Methods:Tumor tissue samples were sequenced prospectively at Foundation Medicine, Inc. (FMI) using an investigational next-generation sequencing (NGS) assay based on FoundationOne®CDx to inform trial eligibility. Matched ctDNA samples were retrospectively sequenced at FMI, using an investigational assay based on FoundationOne®Liquid CDx.Results:81% (503/619) of ctDNA samples yielded an NGS result, of which 491 had a tumor tissue result. BRCA and ATM status in tissue compared with ctDNA showed 81% positive percentage agreement and 92% negative percentage agreement, using tissue as reference. At variant-subtype level, using tissue as reference, concordance was high for nonsense (93%), splice (87%), and frameshift (86%) alterations but lower for large rearrangements (63%) and homozygous deletions (27%), with low ctDNA fraction being a limiting factor.Conclusions:We demonstrate that ctDNA can greatly complement tissue testing in identifying patients with mCRPC and BRCA or ATM alterations who are potentially suitable for receiving targeted PARP inhibitor treatments, particularly patients with no or insufficient tissue for genomic analyses.

PDF file - 78K, Mutation status of AZ samples acquired through Liverpool Heart and Chest Hospital.

Table S2 shows the categorization of local testing methods based on assay limit of detection compared to the cobas® EGFR Mutation Test v2

PDF file - 129K, Reduced nuclear PTEN in SCC compared with AC (TMA Set 2).

PDF file - 145K, Isoform specificity of PI3Kbeta antibody (AGG4888).

PDF file - 57K, Clinicopathological features of TMA Set 1.

PDF file - 221K, Validation of PTEN antibody across FFPE xenograft samples.

Suplement_Tracked changes

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kβ and its relation to PTEN in non–small cell lung cancer NSCLC remain unclear.Experimental Design: Antibodies directed against PI3Kβ and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kβ and PTEN were explored and colocalization of these markers in individual tumor cores were correlated.Results: PI3Kβ expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kβ and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2.Conclusions: We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kβ accompanied by a reduction/loss of PTEN, for whom selective PI3Kβ inhibitors may be predicted to achieve greater clinical benefit. Clin Cancer Res; 20(3); 595–603. ©2013 AACR.

PDF file - 133K,Reduced nuclear PTEN in SCC compared with AC (TMA Set 1).

PDF file - 61K, Clinicopathological features of TMA Set 2.

Purpose:To assess the utility of the cobas EGFR Mutation Test, with tissue and plasma, for first-line osimertinib therapy for patients with EGFR-mutated (EGFRm; Ex19del and/or L858R) advanced or metastatic non–small cell lung cancer (NSCLC) from the FLAURA study (NCT02296125).Experimental Design:Tumor tissue EGFRm status was determined at screening using the central cobas tissue test or a local tissue test. Baseline circulating tumor (ct)DNA EGFRm status was retrospectively determined with the central cobas plasma test.Results:Of 994 patients screened, 556 were randomized (289 and 267 with central and local EGFR test results, respectively) and 438 failed screening. Of those randomized from local EGFR test results, 217 patients had available central test results; 211/217 (97%) were retrospectively confirmed EGFRm positive by central cobas tissue test. Using reference central cobas tissue test results, positive percent agreements with cobas plasma test results for Ex19del and L858R detection were 79% [95% confidence interval (CI), 74–84] and 68% (95% CI, 61–75), respectively. Progression-free survival (PFS) superiority with osimertinib over comparator EGFR-TKI remained consistent irrespective of randomization route (central/local EGFRm-positive tissue test). In both treatment arms, PFS was prolonged in plasma ctDNA EGFRm-negative (23.5 and 15.0 months) versus -positive patients (15.2 and 9.7 months).Conclusions:Our results support utility of cobas tissue and plasma testing to aid selection of patients with EGFRm advanced NSCLC for first-line osimertinib treatment. Lack of EGFRm detection in plasma was associated with prolonged PFS versus patients plasma EGFRm positive, potentially due to patients having lower tumor burden.

Purpose: The phase III PROfound study (NCT02987543) evaluated olaparib versus abiraterone or enzalutamide (control) in metastatic castration-resistant prostate cancer (mCRPC) with tumor homologous recombination repair (HRR) gene alterations. We present exploratory analyses on the use of circulating tumor DNA (ctDNA) testing as an additional method to identify patients with mCRPC with HRR gene alterations who may be eligible for olaparib treatment. Patients and Methods: Plasma samples collected during screening in PROfound were retrospectively sequenced using the FoundationOne®Liquid CDx test for BRCA1, BRCA2 (BRCA), and ATM alterations in ctDNA. Only patients from Cohort A (BRCA/ATM alteration positive by tissue testing) were evaluated. We compared clinical outcomes, including radiographic progression-free survival (rPFS) between the ctDNA subgroup and Cohort A. Results: Of the 181 (73.9%) Cohort A patients who gave consent for plasma sample ctDNA testing, 139 (76.8%) yielded a result and BRCA/ATM alterations were identified in 111 (79.9%). Of these, 73 patients received olaparib and 38 received control. Patients’ baseline demographics and characteristics, and the prevalence of HRR alterations were comparable with the Cohort A intention-to-treat (ITT) population. rPFS was longer in the olaparib group versus control [median 7.4 vs. 3.5 months; hazard ratio (HR), 0.33; 95% confidence interval (CI), 0.21–0.53; nominal P < 0.0001], which is consistent with Cohort A ITT population (HR, 0.34; 95% CI, 0.25–0.47). Conclusions: When tumor tissue testing is not feasible or has failed, ctDNA testing may be a suitable alternative to identify patients with mCRPC carrying BRCA/ATM alterations who may benefit from olaparib treatment.

Purpose: To assess the utility of the cobas EGFR Mutation Test, with tissue and plasma, for first-line osimertinib therapy for patients with EGFR-mutated (EGFRm; Ex19del and/or L858R) advanced or metastatic non–small cell lung cancer (NSCLC) from the FLAURA study (NCT02296125). Experimental Design: Tumor tissue EGFRm status was determined at screening using the central cobas tissue test or a local tissue test. Baseline circulating tumor (ct)DNA EGFRm status was retrospectively determined with the central cobas plasma test. Results: Of 994 patients screened, 556 were randomized (289 and 267 with central and local EGFR test results, respectively) and 438 failed screening. Of those randomized from local EGFR test results, 217 patients had available central test results; 211/217 (97%) were retrospectively confirmed EGFRm positive by central cobas tissue test. Using reference central cobas tissue test results, positive percent agreements with cobas plasma test results for Ex19del and L858R detection were 79% [95% confidence interval (CI), 74–84] and 68% (95% CI, 61–75), respectively. Progression-free survival (PFS) superiority with osimertinib over comparator EGFR-TKI remained consistent irrespective of randomization route (central/local EGFRm-positive tissue test). In both treatment arms, PFS was prolonged in plasma ctDNA EGFRm-negative (23.5 and 15.0 months) versus -positive patients (15.2 and 9.7 months). Conclusions: Our results support utility of cobas tissue and plasma testing to aid selection of patients with EGFRm advanced NSCLC for first-line osimertinib treatment. Lack of EGFRm detection in plasma was associated with prolonged PFS versus patients plasma EGFRm positive, potentially due to patients having lower tumor burden.

Objective Maintenance olaparib provided a progression-free survival benefit in the phase III SOLO2 trial (NCT01874353) in patients with platinum-sensitive relapsed ovarian cancer and a BRCA mutation (BRCAm). However, questions remain regarding tumor versus germline BRCA testing and the impact of heterozygous versus bi-allelic loss of BRCA1 or BRCA2 in the tumor. Methods Blood and tumor samples were analyzed. A concordance analysis of germline BRCAm status (BRACAnalysis® CLIA test) and tumor BRCAm status (myChoice® CDx test) was conducted (Myriad Genetic Laboratories, Inc.). Bi-allelic loss of BRCA1 and BRCA2 and a genomic instability score (GIS) (myChoice® CDx test) were also determined. Results 289 of 295 enrolled patients had a germline BRCAm confirmed centrally and tumor BRCAm status was evaluable in 241 patients. There was 98% and 100% concordance between tumor and germline testing for BRCA1m and BRCA2m, respectively, with discordance found in four cases. Of 210 tumor samples evaluable for BRCA zygosity, 100% of germline BRCA1-mutated tumors (n = 144) and 98% of germline BRCA2-mutated tumors (n = 66) had bi-allelic loss of BRCA. One patient with a heterozygous BRCA2m had a GIS of 53, was progression free for 911 days and remained on olaparib at data cut-off. Conclusions Very high concordance was demonstrated between tumor and germline BRCA testing, supporting wider implementation of tumor BRCA testing in ovarian cancer. Near 100% rates of bi-allelic loss of BRCA in platinum-sensitive relapsed ovarian tumors suggest routine testing for BRCA zygosity is not required in this population and reflects BRCA loss being a driver of tumorigenesis.

Presence of a germline BRCA1 and/or BRCA2 mutation (gBRCAm) may sensitize tumors to poly(ADP-ribose) polymerase (PARP) inhibition via inactivation of the second allele, resulting in gene-specific loss of heterozygosity (gsLOH) and homologous recombination deficiency (HRD). Here we explore whether tissue sample testing provides an additional route to germline testing to inform treatment selection for PARP inhibition.In this prespecified exploratory analysis, BRCA1 and/or BRCA2 mutations in blood samples (gBRCAm) and tumor tissue (tBRCAm) were analyzed from patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer and known gBRCAm, enrolled in the phase III OlympiAD trial. The frequency and nature of tBRCAm, HRD score status [HRD-positive (score ≥42) versus HRD-negative (score42) using the Myriad myChoice® CDx test] and rates of gsLOH were determined, and their impact on clinical efficacy (objective response rate and progression-free survival) was explored.Tissue samples from 161/302 patients yielded tBRCAm, HRD and gsLOH data for 143 (47%), 129 (43%) and 125 (41%) patients, respectively. Concordance between gBRCAm and tBRCAm was 99%. gsLOH was observed in 118/125 (94%) patients [BRCA1m, 73/76 (96%); BRCA2m, 45/49 (92%)]. A second mutation event was recorded for two of the three BRCA1m patients without gsLOH. The incidence of HRD-negative was 16% (21/129) and was more common for BRCA2m (versus BRCA1m) and/or for hormone receptor-positive (versus triple-negative) disease. Olaparib antitumor activity was observed irrespective of HRD score.gBRCAm identified in patients with HER2-negative metastatic breast cancer by germline testing in blood was also identified by tumor tissue testing. gsLOH was common, indicating a high rate of biallelic inactivation in metastatic breast cancer. Olaparib activity was seen regardless of gsLOH status or HRD score. Thus, additional tumor testing to inform PARP inhibitor treatment selection may not be supported for these patients.

TPS9137 Background: The standard of care (SOC) for patients with oncogene driver subsets of metastatic NSCLC is guided by specific molecular characterization and includes immunotherapy, chemoimmunotherapy, and matched targeted therapies. Although targeting HER2 has transformed the care of patients with breast and gastric cancers, there is currently no approved HER2-targeted therapy for NSCLC. T-DXd is an antibody-drug conjugate consisting of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a topoisomerase I inhibitor payload. In a cohort of pretreated patients (median, 2 prior lines) with unresectable or metastatic HER2-mutant NSCLC, T-DXd demonstrated durable and robust anticancer activity, with a confirmed objective response rate (ORR) of 55% and median duration of response (DOR), progression-free survival (PFS), and overall survival (OS) of 9.3, 8.2, and 17.8 months, respectively (Li et al. N Engl J Med. 2022). Given the efficacy observed in later-line settings and the unmet need for targeted therapies in patients with HER2-mutant NSCLC, evaluating the efficacy of T-DXd in the first-line setting vs SOC is important to determine the optimal treatment. Here we describe the phase 3 DESTINY-Lung04 trial (NCT05048797) evaluating T-DXd as a first-line treatment in patients with NSCLC harboring HER2 mutations. Methods: DESTINY-Lung04 is an open-label, randomized, multicenter, phase 3 study evaluating the efficacy and safety of first-line T-DXd vs SOC in patients with unresectable, locally advanced (not amenable to curative therapy), or metastatic nonsquamous NSCLC with HER2 exon 19 or 20 mutations (detected in tissue or circulating tumor DNA). Patients must be naive to systemic therapy with palliative intent in the locally advanced or metastatic disease setting and must not have tumors with EGFR or other targetable oncogenic alterations. Patients with brain metastases must have previously completed local therapy. Patients will be randomized to receive T-DXd or SOC (platinum [investigator’s choice of cisplatin or carboplatin], pemetrexed, and pembrolizumab). The primary endpoint is PFS defined by RECIST version 1.1 per blinded independent central review (BICR). Secondary endpoints include OS, ORR, and DOR (by RECIST 1.1 per BICR and investigator), investigator-assessed PFS (by RECIST 1.1) and time to second progression or death (per local standard clinical practice), central nervous system PFS (by RECIST 1.1 per BICR), landmark PFS at 12 months (by RECIST 1.1 per BICR and investigator), and landmark OS at 24 months. Safety and tolerability, pharmacokinetics, immunogenicity, and patient-reported outcomes, including pulmonary symptoms and tolerability, will be assessed. Clinical trial information: NCT05048797.

Introduction Osimertinib is an oral, central nervous system–active, EGFR tyrosine kinase inhibitor (TKI) for the treatment of EGFR T790M–positive advanced NSCLC. Here we have evaluated EGFR mutation frequencies in two phase II studies of osimertinib (AURA extension and AURA2). Methods After progression while receiving their latest line of therapy, patients with EGFR mutation–positive advanced NSCLC provided tumor samples for mandatory central T790M testing for the study selection criteria. Tumor tissue mutation analysis for patient selection was performed with the Roche cobas EGFR Mutation Test (European Conformity–in vitro diagnostic, labeled investigational use only) (Roche Molecular Systems, Pleasanton, CA). Patients should not have been prescreened for T790M mutation status. The cobas test results were compared with those of the MiSeq next-generation sequencing system (Illumina, San Diego, CA), which was used as a reference method. Results Samples from 324 and 373 patients screened for AURA extension and AURA2, respectively, produced valid cobas test results. The T790M detection rates were similar between AURA extension and AURA2 (64% and 63%, respectively). The pooled T790M rate was 63%, with no difference by ethnicity (63% for Asian and non-Asian patients alike) or immediately prior treatment with an EGFR TKI (afatinib, 69%; erlotinib, 69%; and gefitinib, 63%). A higher proportion of patients had T790M detected against a background of exon 19 deletions versus L858R mutation (73% versus 58% [ p = 0.0002]). In both trials the cobas test demonstrated high sensitivity (positive percent agreement) and specificity (negative percent agreement) for T790M detection when compared with the next-generation sequencing reference method: positive percent agreement of 91% versus 89% and negative percent agreement of 97% versus 98%. Conclusions In both trials, the rate of detection of T790M mutation in patients with advanced NSCLC was approximately 63% and was unaffected by immediately prior treatment with an EGFR TKI or ethnicity.

Objectives Reliable epidermal growth factor receptor (EGFR) mutation testing techniques are required to identify eligible patients with EGFR mutation/T790M positive advanced non-small cell lung cancer (NSCLC), for treatment with osimertinib (AZD9291), an oral, potent, irreversible EGFR tyrosine kinase inhibitor (TKI) selective for EGFR-TKI-sensitizing and T790M resistance mutations over wild-type EGFR. There is no current consensus regarding the best method to detect EGFR T790M mutations. The aim of this study was to describe the concordance between local testing, which used a variety of methods, and central testing, using the cobas ® EGFR Mutation Test, for EGFR-sensitizing mutations and the T790M resistance mutation. Materials and methods Tumor samples were obtained from all patients screened for inclusion onto the osimertinib Phase I expansion component of the AURA Phase I/II study (NCT01802632). Samples underwent central laboratory testing for EGFR-sensitizing mutations and T790M resistance mutation using the cobas ® EGFR Mutation Test. Results were compared with local laboratory test results, based on other testing methodologies including Sanger sequencing, therascreen ® , PNAClamp™, and Sequenom MassARRAY ® . Results Central laboratory testing was successful in 99% of samples passing histopathology review and testing success rates were comparable across the three central laboratories. Concordance between central and local testing for common sensitizing mutations was high (>98%) and concordance for the T790M mutation was also high (>90%). Tumor heterogeneity, along with other technical factors may have influenced this result. Conclusions Within the osimertinib AURA Phase I study, EGFR mutation testing across three centralized laboratories using the cobas ® EGFR Mutation Test was feasible and successful, with strong concordance between local and central laboratory results, including for T790M. The cobas ® EGFR Mutation Test has subsequently been approved as the companion diagnostic test for osimertinib in the USA and Japan.

26 Background: Not all mCRPC patients have available or sufficient tissue for multigene molecular testing. In the Phase 3 PROfound study, olaparib significantly improved radiographic progression-free survival compared with physician’s choice of abiraterone or enzalutamide in men with homologous recombination repair (HRR)-gene-mutated mCRPC (de Bono et al. N Engl J Med 2020). Overall, 31% of patients’ tissue samples failed molecular screening during the study, showing the need for additional testing methods to detect patients with HRR-gene-mutated cancers. We evaluated the utility of plasma-derived ctDNA to identify deleterious BRCA and ATM mutations in screened patients from PROfound. Methods: Tumour samples were prospectively tested at Foundation Medicine, Inc (FMI) using an investigational next-generation sequencing test (based on FoundationOne CDx) to inform trial eligibility. Matched ctDNA samples were sequenced at FMI with the FoundationOne Liquid CDx assay. Tissue samples were clinically heterogeneous regarding location and timing of collection; plasma samples were collected as part of screening in PROfound. Results: 81% (503/619) of ctDNA samples tested yielded a result, of which 491 had a tumour result. BRCA and ATM status in tissue compared with ctDNA reported 81% (95% CI 75–87%) positive percentage agreement (PPA) and 92% (95% CI 89–95%) negative percentage agreement (NPA), with tissue as reference (Table). Further concordance and discordance measures will be presented. Conclusions: High concordance between tumour tissue and ctDNA supports the development of ctDNA testing as a minimally invasive method to identify patients with HRR-gene-mutated mCRPC and guide treatment decisions, particularly for those with insufficient tissue for genomic analyses. Clinical trial information: NCT02987543. [Table: see text]

27 Background: ctDNA testing offers additional opportunities for homologous recombination repair (HRR) gene alteration determination in patients who are not able to access tumor tissue testing. Alteration testing in ctDNA, for BRCA1, BRCA2 and ATM alterations was performed retrospectively in the PROfound study (phase 3 trial of olaparib versus physician’s choice of abiraterone or enzalutamide in men with HRR gene-mutated mCRPC [NCT02987543]). Methods: ctDNA samples were sequenced at FMI, using the FoundationOne Liquid CDx assay for alterations in BRCA1, BRCA2 (BRCA) and ATM, using plasma samples collected during screening in PROfound. Only patients who consented and provided plasma samples from Cohort A (BRCA/ ATM alteration positive by tissue testing) were tested for ctDNA alterations. Radiographic progression-free survival (rPFS) assessed by blinded independent central review (BICR) in patients positive for alterations in ctDNA was analysed via stratified log-rank test. Additional efficacy endpoints (Objective Response Rate [ORR], Overall Survival[OS]) were also assessed. Results: In total, 181/245 (73.9%) Cohort A patients consented and provided a plasma sample for ctDNA testing, of which 139/181 (76.8%) had a ctDNA result reported (either mutation positive or mutation negative) and 42 patient samples failed testing due to insufficient DNA yield or a technical failure of the test.BRCA/ ATM alterations were identified in111/139 (79.9%) patients and 28/139 patients did not report a BRCA/ATM mutation either due to lack of ctDNA shedding from the tumour or ctDNA levels below the sensitivity of the assay. Patients who carried BRCA/ ATM ctDNA alterations had comparable demography, baseline characteristics and proportions of BRCA1, BRCA2 and ATM mutated patients to the overall Cohort A patients. rPFS by BICR results, in patients with BRCA/ ATM alterations in ctDNA, are presented in the Table. Key secondary efficacy endpoints (ORR, OS) will also be reported for ctDNA alteration positive patients. Conclusions: ctDNA testing in patients with mCRPC is feasible and most, but not all patients have concordant results. Patients who were positive for alterations in BRCA1, BRCA2 or ATM showed a consistent rPFS improvement (hazard ratio [HR] 0.33 [0.21, 0.53]) when compared with the ITT population identified by tumor tissue testing (HR 0.34 [0.25, 0.47]). This suggests that using ctDNA to identify patients carrying BRCA1, BRCA2 or ATM alterations who will benefit from olaparib may be beneficial. Clinical trial information: NCT02987543. [Table: see text]

Head and neck squamous cell carcinoma (HNSCC) is a widely prevalent cancer globally with high mortality and morbidity. We report here changes in the genomic landscape in the development of HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Frequent likely pathological mutations are restricted to a relatively small set of genes includingTP53, CDKN2A,FBXW7,FAT1,NOTCH1andKMT2D; these arise early in tumour progression and are present in PPOLs withNOTCH1mutations restricted to cell lines from lesions that subsequently progressed to HNSCC. The most frequent genetic changes are of consistent somatic copy number alterations (SCNA). The earliest SCNAs involved deletions ofCSMD1(8p23.2),FHIT(3p14.2) andCDKN2A(9p21.3) together with gains of chromosome 20.CSMD1deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines (promoter hypermethylation ~63%, hemizygous deletions ~75%, homozygous deletions ~18%). Forced expression ofCSMD1in the HNSCC cell line H103 showed significant suppression of proliferation (p=0.0053) and invasioninvitro(p=5.98X10−5) supporting a role forCSMD1inactivation in early head and neck carcinogenesis. In addition, knockdown ofCSMD1in theCSMD1-expressing BICR16 cell line showed significant stimulation of invasionin vitro(p=1.82 x 10−5) but not cell proliferation (p=0.239). HNSCC with and without nodal metastases showed some clear differences including high copy number gains ofCCND1, hsa-miR-548k andTP63in the metastases group. GISTIC peak SCNA regions showed significant enrichment (adj PLay SummaryCancers affecting the head and neck region are relatively common. A large percentage of these are of one particular type; these are generally detected late and are associated with poor prognosis. Early detection and treatment dramatically improve survival and reduces the damage associated with the cancer and its treatment. Cancers arise and progress because of changes in the genetic material of the cells. This study focused on identifying such changes in these cancers particularly in the early stages of development, which are not fully known. Identification of these changes is important in developing new treatments as well as markers of behaviour of cancers and also the early or ‘premalignant’ lesions. We used a well-characterised panel of cell lines generated from premalignant lesions as well as cancers, to identify mutations in genes, and an increase or decrease in number of copies of genes. We mapped new and previously identified changes in these cancers to specific stages in the development of these cancers and their spread. We additionally report here for the first time, alterations inCSMD1gene in early premalignant lesions; we further show that this is likely to result in increased ability of the cells to spread and possibly, multiply faster as well.

Background We conducted tumour testing in pts with germline mutations in BRCA1 and/or BRCA2 (gBRCAm) and HER2-negative mBC in the OlympiAD study (NCT02000622). Methods Tumour tissue of gBRCAm pts was analysed with Myriad myChoice HRD Plus, including determination of tumor (t) BRCAm, HRD score and gene-specific loss of heterozygosity (LOH). Results Tissue was available from 161/302 pts: tBRCAm status n = 143 (47%); HRD score n = 129 (43%); LOH n = 125 (41%). Concordance of gBRCAm and tBRCAm was 99% (141/142). Absence of LOH was observed in 7/125 pts (6%): 4/49 (8%) BRCA2m, 3/76 (4%) BRCA1m; 2 had a 2nd mutation event (Table). Low HRD scores ( Table: 1936P . Pts with absence of LOH Treatment Biopsy Gene Variant HRD score Hormone receptor status 2nd event RECIST response PFS (months)† Ola Pri BRCA2 c.9097dupA (p.Thr3033Asnfs*11) 31 HR + PR 11.0‡ Ola Pri BRCA2 c.5946del (p.Ser1982Argfs*22) 28 HR + PD 1.3§ Ola Met BRCA2 c.658_659del (p.Val220Ilefs*4) 12 HR + SD 8.2§ Ola Pri BRCA2 c.7977-1G>C 34 HR + CR 11.0‡ Ola Met¶ BRCA1 c.188T>A (p.Leu63*) 56 HR + S1580fs*2 PR 6.5§ TPC Met¶ BRCA1 c.5266dupC (p.Gln1756Profs*74) 73 HR + 494dupT and 528_535delins25 PR 5.9§ TPC Pri BRCA1 c.1931del (p.Cys644Phefs*7) 41 TN SD 8.1§ †Overall median PFS was 7.0 months for ola vs 4.2 months for TPC; ‡Censor; §Event; ¶Diagnosis to sample interval was ≥7 years. CR, complete response; Met, metastatic; PD, progressive disease; PR, partial response; Pri, primary; SD, stable disease Conclusions Tumour testing detected almost all gBRCAm in the study. In this mBC population, absence of LOH was uncommon, indicating a high rate of biallelic inactivation. There was no evidence for a reduction in ola efficacy in the small number of pts with BRCAm HER2-negative mBC whose tumors lack LOH and/or have HRD score Clinical trial identification NCT02000622. Editorial acknowledgement Medical writing assistance was provided by Debbi Gorman, PhD, from Mudskipper Business, Ltd, funded by AstraZeneca and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, U.S.A. (MSD). Legal entity responsible for the study AstraZeneca. Funding This study was sponsored by AstraZeneca and is part of an alliance between AstraZeneca and Merck & Co, Inc. Disclosure M. Robson: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Uncompensated: Daiichi-Sankyo; Advisory / Consultancy: McKesson; Advisory / Consultancy, Uncompensated: Merck; Advisory / Consultancy, Research grant / Funding (institution), Consulting/advisory: Uncompensated; other transfer of value (editorial services): Pfizer; Research grant / Funding (institution): Abbvie; Research grant / Funding (institution): Invitae; Research grant / Funding (institution): Medivation; Research grant / Funding (institution): Myriad; Research grant / Funding (institution): Tesaro; Research grant / Funding (institution), Funded in part: Grant P30 CA008748: NIH/NCI Cancer Center Support. Z. Lai: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. S. Dearden: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. J.C. Barrett: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. E.A. Harrington: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. K. Timms: Shareholder / Stockholder / Stock options, Full / Part-time employment: Myriad. J. Lanchbury: Shareholder / Stockholder / Stock options, Full / Part-time employment: Myriad. W. Wu: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. A. Allen: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. C. Goessl: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. E. Senkus: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self): Clinigen; Honoraria (self), Travel / Accommodation / Expenses: Egis; Honoraria (self), Research grant / Funding (institution): Eli Lilly; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer; Honoraria (self): Pierre Fabre; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self): Sandoz; Honoraria (self), Research grant / Funding (institution): Samsung; Honoraria (self), Research grant / Funding (institution): Merck; Research grant / Funding (institution): Boehringer. S. Domchek: Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Clovis Oncology; Honoraria (self): Bristol-Myers Squibb; Research grant / Funding (institution): PharmaMar. D. Hodgson: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca.

Objectives To assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations, including T790M, from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients. Materials and methods A comparison of multiple platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples were collected from patients entering the ongoing AURA trial (NCT01802632), investigating the safety, tolerability, and efficacy of AZD9291 in patients with EGFR-sensitizing mutation-positive NSCLC. Plasma was collected prior to AZD9291 dosing but following clinical progression on a previous EGFR-tyrosine kinase inhibitor (TKI). Extracted ctDNA was analyzed using two non-digital platforms (cobas ® EGFR Mutation Test and therascreen™ EGFR amplification refractory mutation system assay) and two digital platforms (Droplet Digital™ PCR and BEAMing digital PCR [dPCR]). Results Preliminary assessment (38 samples) was conducted using all four platforms. For EGFR-TKI-sensitizing mutations, high sensitivity (78–100%) and specificity (93–100%) were observed using tissue as a non-reference standard. For the T790M mutation, the digital platforms outperformed the non-digital platforms. Subsequent assessment using 72 additional baseline plasma samples was conducted using the cobas ® EGFR Mutation Test and BEAMing dPCR. The two platforms demonstrated high sensitivity (82–87%) and specificity (97%) for EGFR-sensitizing mutations. For the T790M mutation, the sensitivity and specificity were 73% and 67%, respectively, with the cobas ® EGFR Mutation Test, and 81% and 58%, respectively, with BEAMing dPCR. Concordance between the platforms was >90%, showing that multiple platforms are capable of sensitive and specific detection of EGFR-TKI-sensitizing mutations from NSCLC patient plasma. Conclusion The cobas ® EGFR Mutation Test and BEAMing dPCR demonstrate a high sensitivity for T790M mutation detection. Genomic heterogeneity of T790M-mediated resistance may explain the reduced specificity observed with plasma-based detection of T790M mutations versus tissue. These data support the use of both platforms in the AZD9291 clinical development program.

Introduction In the phase IV, open-label, single-arm study NCT01203917, first-line gefitinib 250 mg/d was effective and well tolerated in Caucasian patients with epidermal growth factor receptor ( EGFR ) mutation-positive non–small-cell lung cancer (previously published). Here, we report EGFR mutation analyses of plasma-derived, circulating-free tumor DNA. Methods Mandatory tumor and duplicate plasma (1 and 2) baseline samples were collected (all screened patients; n = 1060). Preplanned, exploratory analyses included EGFR mutation (and subtype) status of tumor versus plasma and between plasma samples. Post hoc, exploratory analyses included efficacy by tumor and plasma EGFR mutation (and subtype) status. Results Available baseline tumor samples were 1033 of 1060 (118 positive of 859 mutation status known; mutation frequency, 13.7%). Available plasma 1 samples were 803 of 1060 (82 positive of 784 mutation status known; mutation frequency, 10.5%). Mutation status concordance between 652 matched tumor and plasma 1 samples was 94.3% (95% confidence interval [CI], 92.3–96.0) (comparable for mutation subtypes); test sensitivity was 65.7% (95% CI, 55.8–74.7); and test specificity was 99.8% (95% CI, 99.0–100.0). Twelve patients of unknown tumor mutation status were subsequently identified as plasma mutation-positive. Available plasma 2 samples were 803 of 1060 (65 positive of 224 mutation status-evaluable and -known). Mutation status concordance between 224 matched duplicate plasma 1 and 2 samples was 96.9% (95% CI, 93.7–98.7). Objective response rates are as follows: mutation-positive tumor, 70% (95% CI, 60.5–77.7); mutation-positive tumor and plasma 1, 76.9% (95% CI, 65.4–85.5); and mutation-positive tumor and mutation-negative plasma 1, 59.5% (95% CI, 43.5–73.7). Median progression-free survival (months) was 9.7 (95% CI, 8.5–11.0; 61 events) for mutation-positive tumor and 10.2 (95% CI, 8.5–12.5; 36 events) for mutation-positive tumor and plasma 1. Conclusion The high concordance, specificity, and sensitivity demonstrate that EGFR mutation status can be accurately assessed using circulating-free tumor DNA. Although encouraging and suggesting that plasma is a suitable substitute for mutation analysis, tumor tissue should remain the preferred sample type when available.

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kβ and its relation to PTEN in non–small cell lung cancer NSCLC remain unclear. Experimental Design: Antibodies directed against PI3Kβ and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kβ and PTEN were explored and colocalization of these markers in individual tumor cores were correlated. Results: PI3Kβ expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kβ and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2. Conclusions: We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kβ accompanied by a reduction/loss of PTEN, for whom selective PI3Kβ inhibitors may be predicted to achieve greater clinical benefit. Clin Cancer Res; 20(3); 595–603. ©2013 AACR.

Purpose The prognostic potential of KRAS mutations in advanced colorectal cancer (CRC) patients and the impact of KRAS mutation status on the effectiveness of chemotherapy or vascular endothelial growth factor (VEGF) signalling inhibitor therapy remain unclear. KRAS mutation status was evaluated retrospectively as a potential prognostic/predictive marker of clinical outcomes using tumour samples from patients with metastatic CRC receiving cediranib or placebo plus FOLFOX/XELOX in a Phase III trial (HORIZON II; NCT00399035). Methods KRAS codon 12 and 13 mutation analyses were performed using a commercially available, allele-specific, amplification refractory mutation system (ARMS)-based polymerase chain reaction (PCR) assay. Retrospective analyses of progression-free survival (PFS) and overall survival (OS) according to KRAS mutation status were performed for patients randomised to cediranib 20mg or placebo. Results KRAS status was determined in 599/1076 patients (cediranib 20mg, n =285/502; cediranib 30mg, n =110/216; placebo, n =204/358). Baseline characteristics were similar across KRAS mutant ( n =258; 24.0%), wild-type ( n =341; 31.7%) and status unknown ( n =477; 44.3%) groups. There was a trend towards improved PFS and OS in the wild-type versus mutant subgroups independent of treatment (cediranib 20mg and placebo: PFS hazard ratio (HR)=0.85 [median PFS: wild-type=8.5months; mutant=8.3months]; OS HR=0.71 [median OS: wild-type=20.9months; mutant=16.9months]). Treatment effects were similar between KRAS subgroups for cediranib 20mg versus placebo (PFS: wild-type HR=0.78, mutant HR=0.82; OS: wild-type HR=0.92, mutant HR=1.01). Conclusion Data from this large randomised Phase III study show that KRAS codon 12/13 mutations have negative prognostic value in metastatic CRC patients receiving treatment with FOLFOX/XELOX, but KRAS mutation status is not predictive of treatment benefit with cediranib, using PFS or OS.

BRCA1 protein measurement has previously been evaluated as a potential diagnostic marker without reaching a conclusive recommendation. In this study, we applied current best practice in antibody validation to further characterize MS110, a widely used antibody targeting BRCA1. Antibody specificity was investigated using different biochemical validation techniques. We found that BRCA1 could not be reliably detected using immunoprecipitation and Western blot in endogenously expressing cells. We used immunohistochemistry on formalin-fixed paraffin-embedded cell pellets to establish compatibility with formalin-fixed paraffin-embedded samples. We demonstrated that in transfected cells and cell lines with known genetic BRCA1 status, MS110 successfully detected BRCA1 giving the expected level of staining in immunohistochemistry. Following this, we investigated the use of BRCA1 protein measurement by immunohistochemistry in a cohort of triple negative breast and serous ovarian tumour samples to explore the use of BRCA1 protein measurement by immunohistochemistry for patient stratification. Using MS110 in repeated standardized experiments, on serial sections from a panel of patient samples, results demonstrated considerable run-to-run variability. We concluded that in formalin-fixed tissue samples, MS110 does detect BRCA1; however, using standard methodologies, BRCA1 expression levels in tissue samples is incompatible with the use of this protein as a statistically robust patient selection marker in immunohistochemistry. These results demonstrate the need for further development to deliver BRCA1 protein quantification by immunohistochemistry as a patient stratification marker.

AimsActivating mutations in the gene encoding epidermal growth factor receptor (EGFR) can confer sensitivity to EGFR tyrosine kinase inhibitors such as gefitinib in patients with advanced non-small-cell lung cancer. Testing for mutations in EGFR is therefore an important step in the treatment-decision pathway. We reviewed reported methods for EGFR mutation testing in patients with lung cancer, initially focusing on studies involving standard tumour tissue samples. We also evaluated data on the use of cytology samples in order to determine their suitability for EGFR mutation analysis.MethodsWe searched the MEDLINE database for studies reporting on EGFR mutation testing methods in patients with lung cancer.ResultsVarious methods have been investigated as potential alternatives to the historical standard for EGFR mutation testing, direct DNA sequencing. Many of these are targeted methods that specifically detect the most common EGFR mutations. The development of targeted mutation testing methods and commercially available test kits has enabled sensitive, rapid and robust analysis of clinical samples. The use of screening methods, subsequent to sample micro dissection, has also ensured that identification of more rare, uncommon mutations is now feasible. Cytology samples including fine needle aspirate and pleural effusion can be used successfully to determine EGFR mutation status provided that sensitive testing methods are employed.ConclusionsSeveral different testing methods offer a more sensitive alternative to direct sequencing for the detection of common EGFR mutations. Evidence published to date suggests cytology samples are viable alternatives for mutation testing when tumour tissue samples are not available.

12017Background: The PARP inhibitor olaparib is approved for maintenance treatment of platinum-sensitive relapsed ovarian cancer (PSR OC) in the USA based on pivotal studies (Study 19, SOLO2) that ...

Background: This study investigated the potential clinical utility of circulating free DNA (cfDNA) as a source of BRAF mutation detection in patients enrolled into a phase II study of AZD6244, a specific MEK1/2 inhibitor, in patients with advanced melanoma. Methods: BRAF mutations were detected using Amplification Refractory Mutation System allele-specific PCR. BRAF mutation status was assessed in serum-derived cfDNA from 126 patients enrolled into the study and from 94 matched tumour samples. Results: Of 94 tumour samples, 45 (47.9%) were found to be BRAF mutation positive (BRAF+). Serum-derived cfDNA was BRAF+ in 33 of 126 (26.2%) samples, including in five samples for which tumour data were unavailable. Of BRAF+ tumours, 25 of 45 (55.6%) were BRAF+ in cfDNA. In three cases in which the tumour was negative, cfDNA was BRAF+. Progression-free survival (PFS) of patients with BRAF+ tumour and cfDNA was not significantly different compared with tumour BRAF+ but cfDNA BRAF-negative patients, indicating that cfDNA BRAF detection is not associated with poorer prognosis on PFS in stage III/IV advanced melanoma. Conclusions: These data demonstrate the feasibility of BRAF mutation detection in cfDNA of patients with advanced melanoma. Future studies should aim to incorporate BRAF mutation testing in cfDNA to further validate this biomarker for patient selection.

Mutations in the epidermal growth factor receptor (EGFR) gene are commonly observed in non-small-cell lung cancer (NSCLC), particularly in tumors of adenocarcinoma (ADC) histology (NSCLC/ADC). Robust data exist regarding the prevalence of EGFR mutations in Western and Asian patients with NSCLC/ADC, yet there is a lack of data for patients of other ethnicities. This review collated available data with the aim of creating a complete, global picture of EGFR mutation frequency in patients with NSCLC/ADC by ethnicity. Worldwide literature reporting EGFR mutation frequency in patients with NSCLC/ADC was reviewed, to create a map of the world populated with EGFR mutation frequency by country (a ‘global EGFR mutMap’). A total of 151 worldwide studies (n=33162 patients with NSCLC/ADC, of which 9749 patients had EGFR mutation-positive NSCLC/ADC) were included. There was substantial variation in EGFR mutation frequency between studies, even when grouped by geographic region or individual country. As expected, the Asia-Pacific NSCLC/ADC subgroup had the highest EGFR mutation frequency (47% [5958/12819; 87 studies; range 20%-76%]) and the lowest EGFR mutation frequency occurred in the Oceania NSCLC/ADC subgroup (12% [69/570; 4 studies; range 7%-36%]); however, comparisons between regions were limited due to the varying sizes of the patient populations studied. In all regional (geographic) subgroups where data were available, EGFR mutation frequency in NSCLC/ADC was higher in women compared with men, and in never-compared with ever-smokers. This review provides the foundation for a global map of EGFR mutation frequency in patients with NSCLC/ADC. The substantial lack of data from several large geographic regions of the world, notably Africa, the Middle East, Central Asia, and Central and South America, highlights a potential lack of routine mutation testing and the need for further investigations in these regions.

Increased understanding of the genetic aetiology of advanced non-small-cell lung cancer (aNSCLC) has facilitated personalised therapies that target specific molecular aberrations associated with the disease. Biopsy samples for mutation testing may be taken from primary or metastatic sites, depending on which sample is most accessible, and upon differing diagnostic practices between territories. However, the mutation status concordance between primary tumours and corresponding metastases is the subject of debate. This review aims to ascertain whether molecular diagnostic testing of either the primary or metastatic tumours is equally suitable to determine patient eligibility for targeted therapies. A literature search was performed to identify articles reporting studies of mutations in matched primary and metastatic aNSCLC tumour samples. Clinical results of mutation status concordance between matched primary and metastatic tumour samples from patients with aNSCLC were collated. Articles included in this review (N =26) all reported mutation status data from matched primary and metastatic tumour samples obtained from adult patients with aNSCLC. Generally, substantial concordance was observed between primary and metastatic tumours in terms of EGFR, KRAS, BRAF, p16 and p53 mutations. However, some level of discordance was seen in most studies; mutation testing methodologies appeared to play a key role in this, along with underlying tumour heterogeneity. Substantial concordance in mutation status observed between primary and metastatic tumour sites suggests that diagnostic testing of either tumour type may be suitable to determine a patient’s eligibility for personalised therapies. As with all diagnostic testing, highly sensitive and appropriately validated mutation analysis methodologies are desirable to ensure accuracy. Additional work is also required to define how much discordance is clinically significant given natural tumour heterogeneity. The ability of both primary and metastatic tumour sites to accurately reflect the tumour mutation status will allow more patients to receive therapies personalised to their disease.

Background Meta-analyses were conducted to characterize patterns of mutation incidence in non small-cell lung cancer (NSCLC). Design Nine genes with the most complete published mutation coincidence data were evaluated. One meta-analysis generated a ‘mutMap’ to visually represent mutation coincidence by ethnicity (Western/Asian) and histology (adenocarcinoma [ADC] or squamous cell carcinoma). Another meta-analysis evaluated incidence of individual mutations. Extended analyses explored incidence of EGFR and KRAS mutations by ethnicity, histology, and smoking status. Results Genes evaluated were TP53, EGFR, KRAS, LKB1, EML4-ALK, PTEN, BRAF, PIK3CA, and ErbB2. The mutMap highlighted mutation coincidences occurring in ≥5% of patients, including TP53 with KRAS or EGFR mutations in patients with ADC, and TP53 with LKB1 mutation in Western patients. TP53 was the most frequently mutated gene overall. Frequencies of TP53, EGFR, KRAS, LKB1, PTEN, and BRAF mutations were influenced by histology and/or ethnicity. Although EGFR mutations were most frequent in patients with ADC and never/light smokers from Asia, and KRAS mutations were most frequent in patients with ADC and ever/heavy smokers from Western countries, both were detected outside these subgroups. Conclusions Potential molecular pathology segments of NSCLC were identified. Further studies of mutations in NSCLC are warranted to facilitate more specific diagnoses and guide treatment.

Objective AZD9291 is an oral, selective, irreversible EGFR-TKI, effective against EGFR-TKI-sensitizing (EGFRm) and resistance T790M mutations. Pre-study tumor samples from pts enrolled into expansion cohorts of a PhI study (AURA, NCT01802632) were tested centrally for T790M status. Pts with T790M positive tumor samples had higher objective response rate (ORR) than pts with no detectable T790M (T790M ‘negative’): 78/127 (61%) and 13/61 (21%), respectively (Jänne et al 2015). Exploratory analyses to characterize AZD9291 activity in pts whose tumors were T790M negative by central test were performed. Methods Pts with EGFRm aNSCLC and acquired resistance to EGFR-TKIs were enrolled into expansion cohorts of the AURA study. The T790M status of pre-study tumor samples was determined locally (any method allowed) and/or centrally using the cobasTM EGFR mutation test (Roche Molecular Systems, Inc.). Presence of T790M in plasma derived ctDNA was determined by digital PCR (BEAMing). Pts with a centrally determined tumor based T790M negative status were then classified into one of nine groups according to local tumor status (T790M positive, negative, or unknown) and ctDNA status (T790M positive, negative, or unknown). Clinical outcome is reported for each group. Results 69 pts with T790M negative tumor status were determined centrally, and received AZD9291; 35 and 21 were also negative by local test and ctDNA, respectively and 7 were negative by all 3 tests; despite a negative central test, T790M positive tumor status was identified in 11 pts by prior local test and 26 by ctDNA. Confirmed responses by RECIST 1.1, n/N (%) in centrally tested tissue samples with no detectable T790M (Data cut-off 01 May 2015)T790M status of plasma derived ctDNA (Method: BEAMing)PositiveNegativeUnknownTotalT790M status of tumor tissue determined by a local test (Method: Any)Positive2/6 (33)0/1 (0)4/4 (100)6/11 (55)Negative2/14 (14)0/7 (0)2/14 (14)4/35 (11)Unknown2/6 (33)3/13 (23)2/4 (50)7/23 (26)Total6/26 (23)3/21 (14)8/22 (36)17/69 (25) 17 pts with a central T790M negative status responded to AZD9291 treatment. ORR was 55% (6/11) in pts with a central T790M negative/local positive test result. No objective responses (0/7) were observed in pts with T790M negative tumor status by all three tests. Additional analysis of the sub-groups, by immediate prior TKI, EGFRm status, and duration of response will be available for presentation. Discussion In 69 pts with centrally identified T790M negative tumor status, 17 achieved a confirmed response. Of these, 12 were T790M positive by local test, ctDNA, or both. Different sensitivity and specificity of assays, tumor heterogeneity, and ctDNA shedding may explain positive results in this small group of pts classified as central T790M negative. The data support the hypothesis that T790M directed inhibitors are active against T790M driven resistance. Citation Format: Pasi A. Jänne, Enriqueta Felip, Dong-Wan Kim, Thomas John, Daniel E. Haggstrom, Myung-Ju Ahn, Kenneth S. Thress, Suzanne C. Jenkins, Helen Mann, Mireille V. Cantarini, Simon P. Dearden, James Chih-Hsin Yang. Characterization of the activity of AZD9291 in patients (pts) with T790M ‘negative’ advanced non-small cell lung cancer (aNSCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B105.

Aim: AZD9291 is in clinical development to address T790M-mediated drug resistance associated with treatment of EGFR-mutant NSCLC. Genotyping of circulating plasma DNA may provide an option to identify patients (pts) unable to provide tissue biopsies. We aimed to study plasma levels of EGFR T790M in relation to AZD9291 drug activity. Methods: 101 pts with advanced NSCLC consented to plasma analysis as part of the Phase I study of AZD9291(NCT01802632). Central tumour genotyping was performed when feasible using the cobas™ assay. After pre-amplification of plasma DNA, levels of pre-dose T790M, L858R and exon 19 del variants were quantified using an emulsion PCR assay (Sysmex). Cases with no detectable plasma-sensitising mutation were omitted from T790M analysis. Response rate (RR) as of 16 Jan 2014 was calculated as the proportion of pts with ≥1 follow-up tumour measurement having a diameter decrease ≥30%. Results: Plasma assays for L858R and 19 del were studied in 44 pts with 19 del and 28 pts with L858R centrally confirmed in their tumour. A threshold of 0.05% mutant had 0% false positive rate (FPR) and 83% sensitivity for these mutations. In 64 pts with central T790M results, the same threshold applied to plasma T790M had 50% FPR, 89% sensitivity. Notably, of 6 cases falsely T790M+ in plasma, 5 were also T790M+ using a second plasma assay (cobas™). RR was 61% (25/41) and 24% (5/21) in 62 pts with central T790M+ and T790M- tumour genotyping (p = 0.008). Similarly, RR was 65% (33/51) and 38% (8/21) in 72 pts with T790M+ and T790M- plasma genotyping (p Conclusions: Plasma levels of EGFR-TKI-sensitising mutations correlate more closely with tumour genotype than T790M, likely due to the greater genomic heterogeneity of resistance. RR is higher in T790M+ pts than in T790M- pts, either using tumour or plasma genotyping. Plasma T790M genotyping using emulsion PCR may be an attractive alternative to rebiopsy for tumour genotyping of NSCLC pts with acquired EGFR resistance. Disclosure: K. Thress: Employment and stock ownership: AstraZeneca; J.C. Yang: Advisory boards: Boehringer Ingelheim, Eli Lilly, Pfizer, Novartis, Roche/Genentech, AstraZeneca, Merck, Bayer, Clovis Oncology. Corporate-sponsored research: Boehringer Ingelheim; D. Planchard: Advisory boards: AstraZeneca, Boehringer Ingelheim, Lilly, Novartis, Pfizer, Roche, BMS; R. Brant: Employment and stock ownership: AstraZeneca; H. Carr: Employment and stock ownership: AstraZeneca; S. Dearden: Employment and stock ownership: AstraZeneca; S. Jenkins: Employment and stock ownership: AstraZeneca; M. Cantarini: Employment and stock ownership: AstraZeneca; S. Ghiorghiu: Employment and stock ownership: AstraZeneca; J.C. Barrett: Employment and stock ownership: AstraZeneca; P.A. Janne: Consultant or advisory role: AstraZeneca, Boehringer Ingelheim, Clovis Oncology, Pfizer, Merrimack Pharmaceuticals, Chugai, Immunogen. Stock ownership: Gatekeeper. Other: Lab Corp; G.R. Oxnard: Compensated consultancy: Astra-Zeneca, Boehringer Ingelheim, Clovis, Genentech, Novartis, Sanofi. All other authors have declared no conflicts of interest.

e19051 Background: EGFR tyrosine kinase inhibitors (TKIs) have demonstrated clinical benefit in patients (pts) with advanced EGFR mutation-positive NSCLC. Eventually pts develop acquired resistance...

8092 Background: EGFR tyrosine kinase inhibitors are highly effective in treating EGFR mutant+ NSCLC. Patients invariably relapse and resistance is most often associated with an EGFR T790M mutation...

Emerging evidence suggests that circulating tumor DNA (ctDNA) provides an alternative diagnostic sample when surgical biopsies are inaccessible, and could enable disease diagnosis, frequent assessment of disease progression, and almost real-time monitoring of the emergence of resistance mutations. We hypothesized that novel highly sensitive technologies enable EGFR T790M mutation detection in ctDNA from patients that had previously been treated with and become resistant to EGFR-TKIs. We have used 3 technologies to assess T790M mutation detection rate. Samples comprised patients with sensitizing EGFR mutations and wild type EGFR patients who had been previously treated with EGFR TKI therapies. In approximately 50% of EGFR mutation positive cases, acquired resistance to EGFR-TKIs is mediated by an EGFR T790M mutation. Reference data for T790M mutations from tumor material was unavailable.We undertook a comprehensive cross-technology comparison of three technology platforms: ARMS based detection using the Roche cobas EGFR mutation detection kit; digital droplet PCR using the BioRad ddPCR instrument (by MolecularMD) and bead based digital PCR using the Inostics BEAMing technology for the detection of T790M mutations. In total, 135 frozen plasma samples, obtained from 2 clinical studies, were used to estimate T790M detection rates in ctDNA. Within the study group, 72 samples were taken from EGFR mutation positive patients, of which around 36 (expected range 28-44) would be expected to have T790M mutations, the remaining samples would be expected to be T790M negative. Each individual patient plasma sample was split and evaluated across multiple platforms. In addition, the suitability of 3 ctDNA preparation kits (cobas Circulating DNA Isolation kit, QIAamp Circulating Nucleic Acid kit and QIAamp DNA Mini kit) in terms of DNA yield was evaluated. The QIAamp Circulating Nucleic Acid kit and cobas Circulating DNA Isolation kit were equally effective in preparing ctDNA from plasma samples. Overall, concordance of EGFR T790M mutation status was high between all 3 methods. In addition, the false positive rate in the known EGFR mutation negative cases was low for all 3 methods. However, differences were seen in the rate of false negative results (assay sensitivity) between methods. DNA preparation kits specifically designed for use with ctDNA samples are preferable to other methods. Current technologies for the detection of T790M mutations in ctDNA, where patient tumor material is not available, still require further development to increase detection rates in these samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C196. Citation Format: Helen Brown, Roz Brant, Simon Dearden, Suzanne Jenkins, Kenneth Thress, Alain Horvais, Ruth March, J. Carl Barrett. EGFR mutation detection in ctDNA isolated from NSCLC patient plasma; a cross-platform comparison of leading technologies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C196.

Background This study comprises a meta-analysis of publicly available and newly generated mutation data in non-small-cell lung cancer patients to identify differences between histological subtypes and geographical populations. Gene mutation frequencies, along with two-gene coincidence data have been collated. We call this analysis a MutMap. Methods Publications reporting mutation data on two or more genes, in histologically and geographically defined populations, were identified. Assessments of single-gene mutation frequencies and two-gene mutation co-occurrences were carried out in Western and Eastern adenocarcinoma and squamous cell carcinoma populations. Results In total, 21 studies were identified. Data from AstraZeneca and the Guangdong Lung Cancer Institute were included in the meta-analysis. Mutation data were collated for eight genes (EGFR, KRAS, TP53, LKB1, BRAF, PIK3CA, PTEN and ERBB2). Adenocarcinoma cases contained more mutations than squamous cases. The most commonly mutated gene was TP53; however, mutation frequencies varied between geographical regions and between histologies. Mutations in EGFR were common (46.3%) in Eastern adenocarcinoma patients but rarer in Western cases (14.8%). Conversely, KRAS mutations were more common in Western adenocarcinoma patients (25.7%) than Eastern cases (11.2%). Mutations in LKB1 were also more common in Western patients. Mutations in PTEN were common in Eastern squamous samples (9.8%). Mutation co-occurrence in two genes was also examined. Mutations in EGFR and KRAS were exclusive to each other. In addition, in Western adenocarcinoma patients, mutations in both EGFR and LKB1 occurred significantly less often than would be expected by chance. There were several two-gene mutation co-occurrences which were detected at levels ≥5% of the NSCLC population, e.g. TP53 with either KRAS or EGFR in adenocarcinoma. Conclusions Overall, this study expands on the view that mutation prevalence in NSCLC is complex, with variation between histological subtypes and geographical populations. Mutation co-occurrence may also be a factor in future treatment paradigms as segments ≥5% of NSCLC possess both activating oncogenic driver mutations and mutations in tumour suppressor genes.

Background: Early results from Phase II trials have shown that treatment with the PARP inhibitor olaparib (AZD2281, KU-0059436) can induce tumour specific synthetic lethality in patients with BRCA-mutated breast and ovarian cancer, with little effect on normal tissues. Pre-clinical studies have indicated that sensitivity can also result from other perturbations affecting homologous recombination (HR), suggesting broader patient populations may benefit from this drug. We aimed to generate pre-clinical hypotheses as to the key biological mechanisms regulating response to this drug; and identify associated biomarkers by which responding patient subsets could be stratified. Methods: A panel of 95 cell lines representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D-clonogenic survival assays. Baseline (untreated) gene expression profiles (Affymetrix U133A Plus2) were determined for each cell line, alongside protein expression and mutational status of core HR genes. Dynamic pathway gene expression signatures were collated from the literature, and additional signatures generated using RNAi against core HR components. Statistical and bioinformatic approaches were then applied to prioritise correlated networks of genes displaying consistent pathway overlay and predictive of response in cell line subsets. Results: 30 cell lines were highly sensitive to olaparib treatment (IC50 4 µM). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines, highlighting the presence of other factors able to modulate olaparib responsiveness. Transcriptome analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib response. Based on these results, a candidate predictive baseline gene expression profile was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts predictive of in vitro sensitivity to olaparib were identified which may have utility as predictive biomarkers in the clinic. Although primarily linked to mechanisms expected to influence response (DNA repair, cell-cycle checkpoints and proliferation), novel roles were suggested for other pathways such as Aurora A kinase signalling. Analyses also highlighted that some classical HR pathway components may prove to be more robust mRNA markers of pathway activity than others. Studies are already underway to determine whether this baseline transcript tumour profile can be correlated with patient responses to olaparib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3497.