FLEX Study Research Articles

Abstract P3-14-11: Mammaprint and Blueprint identify genomic differences in HR+ HER2- breast cancers from young Black and White women

Abstract Introduction: Hormone receptor positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer, the most common immunohistochemical subtype, remains the dominant contributor to annual breast cancer deaths worldwide across all racial and ethnic groups. Black women are 41% more likely to die from breast cancer compared to White women, predominantly among women diagnosed ≤ 50 years of age. Yet, Black women remain underrepresented in clinical trials and population-based studies. Thus, it is critical to better characterize tumor molecular features from young Black women to identify factors contributing to the existing racial survival disparity. In the current study, we compared risk of distant recurrence signature, MammaPrint (MP), molecular subtyping signature, BluePrint (BP), and whole transcriptome differences between young Black women with HR+ HER2- breast cancer compared to matched White controls. Methods: This study included 156 Black women aged ≤ 50 with stage I-III, HR+ HER2- breast cancer of whom, 68 were recruited from 2009-2014 as part of the BEST study (5R01CA204819-04) with follow-up data available (median 114.5 months). The remaining 88 Black women were enrolled in the ongoing FLEX Study (NCT03053193) from 2017. White women (n=156) were randomly selected from FLEX and matched by age, tumor stage, and receptor status. Tumors were classified through MP as Low Risk (MP-LR) versus High Risk (MP-HR), with MP-HR further stratified into HR1 and HR2; HR2 tumors exhibit superior chemosensitivity as demonstrated in a prior large clinical trial of breast cancer patients (ISPY2). All women had MP and BP to classify tumors as Luminal A (MP-LR), Luminal B (MP-HR), HER2, or Basal, and full transcriptomic analyses. Differential gene expression analysis was performed with R package ‘limma’ to compare Black and White women and further compare within each molecular subtype. Differentially expressed genes (DEGs) with a false discovery rate <0.05 were significant. Results: Of 312 young women with localized, HR+ HER2- breast cancer, high grade tumors were more frequent among Black compared to White women (34.6% vs 25.6%; p=0.08). MP-HR tumors were significantly more frequent among Black compared to White women (67.3% vs. 50.0%; p=0.002). Among MP-HR tumors, more HR2 tumors were seen in Black (25.6%) compared to White women (14.1%). Among women with MP-HR tumors and known treatment information, most Black women (94.3%) and all White women received chemotherapy. There were more Luminal B tumors in Black compared to White women (51.9% vs. 41.7%; p=0.07). BP reclassified a larger proportion of ER+ tumors as Basal in Black compared to White women (14.1% vs. 8.3%). Of 68 Black women with available survival data, 7 had death and/or distant recurrence events, of whom 6 (85.7%) had MP-HR tumors (4 Luminal B, 1 HER2, and 1 Basal) and 1 had MP-LR Luminal A tumor. Compared to White women, Black women with: 1) Luminal B tumors had 192 DEGs with upregulation of suspected poor prognosis genes, PSPH and IGHG1; 2) Luminal A tumors had upregulation of PSPH; and 3) Basal tumors had downregulation of POTEH. Conclusion: Among young women with localized HR+ HER2- breast cancer, MP and BP molecular signatures more robustly identified racial disparities in risk and subtype distribution beyond that identified through clinical factors adjusted for age and tumor characteristics. The transcriptomic differences among Black compared to White women across all BP subtypes provide novel insights about tumor biological differences. These findings have tremendous translational potential to identify etiologic underpinnings of racial survival disparities which may guide therapeutic strategies to improve outcomes. Citation Format: Sonya Reid, Tuya Pal, Ingrid A. Mayer, Xiao-Ou Shu, Ann L. Tezak, Kent Hoskins, Dipali Sharma, Patricia Robinson, Jennifer Wei, Jake Ruby, Shiyu Wang, Josien Haan, Andrea Menicucci, William Audeh, FLEX Investigators Group. Mammaprint and Blueprint identify genomic differences in HR+ HER2- breast cancers from young Black and White women [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-11.

Abstract CT260: The FLEX real-world data platform explores new gene expression profiles and investigator initiated protocols in early stage breast cancer

Abstract Background: Genomic expression profiling has significantly improved the personalized treatment of breast cancer, by providing prognostic information about the metastatic potential of tumors and enabling the classification of breast cancers into molecular subtypes, beyond clinical and pathological factors. When combined with comprehensive clinical data, full genome expression data can precisely stratify tumors into clinically actionable subgroups. The FLEX Study is aggregating a large, real-world dataset, which will enable the discovery of novel genomic profiles to improve precision in the management of breast cancer, particularly in patient subsets that are underrepresented in traditional clinical trials. Methods: The FLEX Study (NCT03053193) is a multicenter network, prospective, observational trial for patients with stage I-III breast cancer whose primary tumor is analyzed by MammaPrint, with or without BluePrint. The primary objective of FLEX is to create a large scale, population-based registry that links comprehensive clinical data with full genome expression data to elucidate new prognostic and/or predictive gene associations in a real-world setting. The FLEX Study employs a shared study infrastructure to develop and investigate hypotheses for targeted subset analyses and/or clinical trials based on genomic data. The adaptable protocol is designed to be amended to include additional targeted sub-studies, allowing participating investigators to leverage data within the FLEX network to investigate research questions. Patients enrolled in the initial study are eligible for inclusion in sub-studies for which they meet all eligibility criteria and additional consent is not required. Data is collected on patients from diagnosis through 10 years of follow-up and any necessary additional clinical data will be collected as specified in the appendix protocols. The target enrollment of FLEX is a minimum of 10,000 patients; over 6,500 patients have enrolled since April 2017 at more than 85 sites, including eight National Cancer Institute-designated comprehensive cancer centers. The FLEX collaborative platform allows participating investigators the opportunity to author their own sub-study protocols, as approved by the FLEX Scientific Review Committee. Sub-study research categories include: Age and Breast Cancer, Optimizing Therapy Strategies, Breast Cancer and Metabolic Syndrome, ctDNA and Liquid Biopsy, Genomics and Subtypes, Social and Ancestry, and Neoadjuvant Therapy and Surgery. To date, 29 investigator-initiated sub-studies have been approved. Trial contact information: NCT03053193 FLEX@agendia.com Citation Format: Adam Brufsky, Jennifer A. Crozier, Nina D'Abreo, Mehran Habibi, Sami Diab, Joyce O'Shaughnessy, Douglas K. Marks, VK Gadi, Amy M. Truitt, Lisa Blumencranz, Erin Yoder, William Audeh, Sarah Untch, Bastiaan van der Baan, FLEX Investigators' Group. The FLEX real-world data platform explores new gene expression profiles and investigator initiated protocols in early stage breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT260.

Molecular profiles of genomically high risk ER+ HER2- breast cancer tumors classified as functionally basal or luminal B by the 80-gene signature.

563 Background: The 80-gene signature (BluePrint/BP) classifies early-stage breast cancers based on functional molecular pathways as luminal, HER2, or Basal-type. In the NBRST study, 13% of immunochemistry (IHC) defined ER+ HER2- cancers reclassified as Basal-type by the BP assay (ER+ Basal), and these had worse prognosis but responded better to neoadjuvant chemotherapy than ER+ HER2- cancers classified as genomically luminal-type. The 70-gene risk of recurrence signature (MammaPrint/MP) further stratifies luminal-type cancers into low risk luminal A or high risk (HR) luminal B. HR cancers can be further stratified into High 1 (H1) or High 2 (H2), and the I-SPY2 trial has shown higher pCR rates in ER+ cancers classified as H2. Here, we investigated biological differences among ER+ Basal, ER- Basal, H1 luminal B, and H2 luminal B cancers by full transcriptome analysis. Methods: From the FLEX Study (NCT03053193), 1501 breast cancers with known IHC ER status were classified by MP and BP: 103 ER+ Basal, 210 ER- Basal and 1188 luminal B (H1 n=1034, H2 n=154). Clinical factors were assessed by either the Chi-square or Fisher’s exact tests; ANOVA or t test were used to analyze age. Differentially expressed genes (DEGs) were detected using Limma and pathway analyses were performed with GSEA. DEGs with a fold change >2 and FDR < 0.05 were considered significant. Results: Basal-type cancers (ER+/ER-) were larger and higher grade than luminal B cancers. Clustering analysis showed similar transcriptional profiles between ER+ Basal and ER- Basal cancers, distinct from luminal B cancers. Few DEGs were detected between ER+ Basal and ER- Basal cancers, and significantly more DEGs were found between ER+ Basal and luminal B cancers. Only three upregulated genes were detected in ER+ Basal compared to ER- Basal cancers: ESR1 and two immune-related genes ( FDCSP and LTF). Enrichment analysis of DEGs indicated increased immune activation and cell proliferation in ER+ Basal and ER- Basal cancers, and decreased estrogen response between ER+ Basal and luminal B cancers. Enrichment analysis between luminal B H1 and H2 cancers showed H2 cancers had higher immune activation and cell proliferation and lower estrogen response. Conclusions: Reclassification by BP of IHC defined ER+ HER2- cancers identified a subgroup of ER+ cancers that are biologically closer to ER- Basal than luminal-type cancers. Significant differences in response to neoadjuvant chemotherapy that have been seen between ER+ Basal and luminal B breast cancers lend support to the clinical importance of these findings. These data explain the poor prognosis observed in patients with ER+ Basal cancers and suggest that optimized chemotherapy, such as that for triple negative cancer, might be of benefit. BP provides clinically actionable information beyond pathological subtyping, which may guide neoadjuvant treatment recommendations. Clinical trial information: NCT03053193.

Gene expression associated with lymphovascular invasion and genomic risk in early-stage breast cancer.

559 Background: Lymphovascular invasion (LVI), the passage of carcinoma cells through lymphatic and blood vessels, is an important early step in metastasis; however, LVI is excluded from most breast cancer (BC) clinical risk assessments. Previous studies assessed the prognostic value of LVI to estimate clinical outcomes. To gain understanding of the molecular basis of LVI, we evaluated differentially expressed genes (DEGs) between tumors with LVI versus those without LVI, stratified by the 70-gene signature (MammaPrint/MP) and 80-gene molecular subtyping signature (BluePrint/BP). Methods: The prospective, observational FLEX Study (NCT03053193) includes stage I-III BC patients who receive MP/BP testing and consent to full transcriptome and clinical data collection. Patients with LVI (n=581) and without LVI (n=600, randomly selected), enrolled from 2017 to present, were included. LVI was assessed by local pathology laboratories. Differential gene expression analysis of 44k Agilent microarray data was performed with R limma package. DEGs were compared within all samples, BP Luminal subtype, MP risk groups (Low Risk [LR]/Luminal A and High Risk [HR]/Luminal B), and by lymph node (LN) status. DEGs with FDR<0.05 were considered significant. Results: Of tumors with LVI (LVI+), 66% were MP HR; notably, 51% of tumors without LVI (LVI-) were MP HR. LVI was associated with larger T stage, LN involvement, high grade, negative ER status by IHC, and younger patient age (LVI+ vs. LVI-, p<0.05 for all comparisons). Patient ethnicity, obesity, and tumor type did not differ by LVI status; however, prevalence of type 2 diabetes trended higher in patients with LVI+ HR tumors (21%), compared with LVI- HR (15%, p=0.09) and LVI+ LR (11%, p=0.004). There were significant transcriptomic differences between LVI+ and LVI, with most DEGs evident in the Luminal B subset. DEGs in LVI+, LN-negative (LN-) tumors overlapped substantially with the overall Luminal group analysis. Functional enrichment analysis showed dysregulation of cell cycle, extracellular matrix (ECM) organization, cell adhesion, and cytokine receptor pathways. Gene sets related to insulin growth factor pathways were also enriched in LVI+ tumors. Conclusions: DEGs associated with LVI were primarily found in MP HR Luminal, LN-negative tumors; enrichment analysis suggested dysregulation of ECM organization and cell adhesion pathways, consistent with previous reports. DEGs were not associated with LVI presence in LN+ tumors, suggesting that LVI assessment may be less relevant in LN+ breast cancer. Future studies will assess clinical outcomes, as well as LVI-associated gene expression in BP Basal- and HER2-type tumors. However, the current analysis indicates few DEGs in LVI+ MP LR tumors; thus, the potential prognostic information gained from LVI-associated gene expression is likely already captured by the MP and BP signatures. Clinical trial information: NCT03053193.

Disparities within luminal breast cancer: Clinical and molecular features of African American and non-Hispanic white patients.

1009 Background: African American breast cancer patients (AA) are diagnosed younger, have more high-risk features, and poorer clinical outcomes than non-Hispanic White patients (NHW), despite similar treatments. Although comorbidities such as obesity and metabolic syndrome may contribute to differences, ancestry-specific factors and effects of structural violence that disproportionately afflict AA individuals may influence tumor biology and outcomes. We previously reported differentially expressed genes (DEGs) associated with tumor aggressiveness in Basal tumors from AA compared with NHW (Sharma et al., 2020). Here, we compare DEGs in luminal tumors between AA and NHW. Methods: The prospective, observational FLEX study (NCT03053193) includes stage I-III breast cancer patients who receive 70-gene signature (MammaPrint/MP)/80-gene signature (BluePrint/BP) testing and consent to full transcriptome and clinical data collection. AA (n=364) and NHW (n=400, random selection) with BP luminal tumors, enrolled from 2017 to present, were included. Race/ethnicity was self-reported. AA were younger than NHW (mean, 59 vs. 62 years, p=0.001); thus, an age-matched subset (n= 360 AA, NHW) was compared. Differential gene expression analysis was performed with R limma package. Comparisons were made between AA and age-matched or randomly selected NHW in: (1) all, (2) luminal A, (3) luminal B, and (4) luminal B, obese. DEGs with FDR<0.05 were significant. Different fold change (FC) thresholds were evaluated. Results: Compared with age-matched NHW, AA were similar in menopausal status, T stage, grade, and tumor type; obesity, T2DM status, and nodal stage were significantly different ( p<0.01). Tumors from AA were more often MP high risk ( p<0.001), regardless of age matching. Luminal B AA vs. age-matched NHW comparison resulted in more DEGs (n=1070) than other comparisons; however, most were FC<2. Notably, 5/6 DEGs ( PSPH, NOTCH2NL, POLR1A, MAP1LC3P and RPS26P10) in basal tumors (Nunes et al. 2019) were also identified here. Of 9 DEGs (FC>1.7) in the luminal B age-matched comparison, 2 ( PSPH and LINC01139) were also found in the luminal B, obese subset. Consistently upregulated DEGs in AA were associated with metabolism, translation, and cellular stress response pathways. Conclusions: We found significant transcriptomic differences between luminal tumors from AA and NHW, when controlling for age, obesity, and genomic classification. A subset of DEGs in luminal B tumors were consistent with those in Basal tumors, suggesting that similar race-associated factors drive DEGs regardless of tumor subtype. DEGs that may be unique to AA luminal tumors were also found. This study suggests that some biological differences in breast tumors may result from patient ancestry or shared adverse socioeconomic exposures and underscores the need for inclusion of diverse patient groups in clinical trials. Clinical trial information: NCT03053193.

University of Minnesota Medical School.

University of Minnesota Medical School.

Strongly Enhanced Superconductivity Due to Finite Energy Spin Fluctuations Induced by an Incipient Band: A FLEX Study on the Bilayer Hubbard Model with Vertical and Diagonal Interlayer Hoppings

We study the spin-fluctuation-mediated $s\pm$-wave superconductivity in the bilayer Hubbard model with vertical and diagonal interlayer hoppings. As in the two-leg ladder model with diagonal hoppings, studied previously by the present authors, superconductivity is strongly enhanced when one of the bands lies just below (or touches) the Fermi level, that is, when the band is incipient. The strong enhancement of superconductivity is because large weight of the spin fluctuations lies in an appropriate energy range, whereas the low energy, pair-breaking spin fluctuations are suppressed. The optimized eigenvalue of the linearized Eliashberg equation, a measure for the strength of superconductivity, is not strongly affected by the bare width of the incipient band, but the parameter regime where superconductivity is optimized is wide when the incipient band is narrow, and in this sense, the coexistence of narrow and wide bands is favorable for superconductivity.

P202 Examining Shifts in School-Time Physical Activity Patterns From Elementary to Middle School Among Lower-Income Schoolchildren

Background The elementary-to-middle school transition likely impacts the decline in school-time physical activity (PA) and may exacerbate disparities in these behaviors that exist for girls and overweight/obese children. Objective To assess changes in school-time moderate-to-vigorous physical activity (sMVPA) and school sedentary time (ST) from elementary to middle school among children and whether there are disparities by sex and weight status. Study Design, Settings, Participants Lower-income children who participated in the FLEX Study during grades 4 and 5 were re-recruited into this supplementary prospective cohort study, FLEX+ during middle school (grade 6). Measurable Outcome/Analysis Demographic data were collected via parent questionnaire. Child assessments included height, weight, and 7-day accelerometry (Actigraph GT3X+) in 4th, 5th, and 6th grade. The association between grade and changes in sMVPA and ST were examined using mixed effects models adjusting for sex, race, BMI status, free/reduced price lunch eligibility (FPRL), and mean weekly ambient temperature. Individual-level clustering was adjusted for as a random intercept. Interactions were tested to examine disparities by sex and BMI status. Results Eighty eight children (grade 4: 9.2 ± 0.5 years, 42% male, 38% non-white, 39% FRPL, 33% overweight/obese, sMVPA = 19.9 ± 8.5 min/day) had valid accelerometer wear-time (> 3 days, >10 hrs/day). There was a decline in children meeting the 30-minute sMVPA recommendation from grades 4 to 6 (14.8%, 7.5%, and 3.5%, respectively; P P > .05). Notably, the change in ST reflects an increase in grade 5 (β = 16.5 mins/day; 95% CI: 8.4, 24.6; P P P > .05). Conclusion Policies and programs that promote movement time during school hours are urgently needed to counteract the steep increase in sedentary behaviors that occur in middle school. Funding NIH.

A systematic review and meta-analysis of the effect of bisphosphonate drug holidays on bone mineral density and osteoporotic fracture risk.

We performed a systematic review and meta-analysis on the effect of drug holidays (discontinuation) on BMD and fracture risk. We searched PubMed, Embase, and Cochrane Library databases to locate controlled clinical trials and cohort studies evaluating the effect of drug holidays/discontinuation versus osteoporosis treatment continuation. We performed random-effects meta-analyses of hazard ratios of hip and any clinical osteoporotic fracture for individuals who discontinued bisphosphonates compared to persistent users. Thirteen records reporting results from eight different studies met inclusion criteria. The FLEX study found a reduced clinical vertebral fracture risk with 10years of alendronate therapy compared to 5 (RR 0.45, 95% CI 0.24-0.85), and the HORIZON extension studies found a reduced risk of morphometric vertebral fracture with 6years of zoledronic acid therapy compared to 3 (OR = 0.51, 95% CI 0.26-0.95); subgroup analyses showed that women with low hip BMD T-scores after the initial treatment period benefitted from continued treatment in terms of reduced vertebral fracture risk. Meta-analysis of adjusted hazard ratios of hip and any clinical osteoporotic fracture for women who discontinued bisphosphonates revealed no significant differences in the risk of hip fracture (summary estimate of HR 1.09, 95% CI 0.87-1.37) or any clinical fracture (summary estimate of HR 1.13, 95% CI 0.75-1.70) compared to persistent users. Bisphosphonate discontinuation may be considered for women who do not have low hip BMD after 3 to 5years of initial treatment, while women who have low hip BMD may benefit from treatment continuation.

Cilengitide (Cil) Combined with Cetuximab and Platinum-Based Chemotherapy As First-Line Treatment in Advanced Non-Small Cell Lung Cancer (Nsclc) Patients (Pts): Phase Ii Randomised Certo Study

ABSTRACT Aim: CIL is a selective, competitive inhibitor of avb3 and avb5 integrins designed to attack the tumour and its microenvironment. CERTO is a multicentre, controlled, open-label phase II study (NCT00842712) for pts with histologically confirmed newly diagnosed advanced NSCLC. Methods: Pts were initially randomised 1:1:1 to receive cetuximab plus platinum-based chemotherapy alone (CTRL), or combined with CIL 2000 mg 1x/week (CIL1W) or 2x/week (CIL2W). During the study, an amendment triggered by cetuximab data from the FLEX study led to enrolment only of pts with EGFR histoscore ≥200, and closure of CIL2W due to feasibility issues. Primary endpoint was progression-free survival (PFS; independent read); secondary endpoints included overall survival (OS), safety, and biomarker analyses. Results: Overall, 220 pts were randomised: 85 to CIL1W; 51 to CIL2W; and 84 to CTRL. Baseline characteristics were balanced. Median PFS (independent read, ITT) was 6.2 mo in CIL1W vs 5.0 mo in CTRL (HR 0.72 [95% CI, 0.49, 1.05]; p = 0.085). For pts with EGFR ≥200, median PFS was 6.8 mo in CIL1W vs 5.6 mo in CTRL (HR 0.57 [95% CI, 0.32, 0.99], p = 0.045). Investigator-read PFS was not different between CIL1W and CTRL in either population. Median OS was 13.6 vs 9.7 mo in CIL1W vs CTRL, respectively (ITT; HR 0.81 [95% CI, 0.56, 1.17]; p = 0.265); in pts with EGFR ≥200 no difference between CIL1W vs CTR for OS was found. Generally, no relevant differences between adverse events across treatment arms were reported; nausea (56%), neutropaenia (49%), and anaemia (37%) were the most frequent, and there was no increased incidence of thromboembolic events or haemorrhages in CIL-treated pts. Biomarker sample size was small, but avb3 and avb5 expression did not reveal a prognostic correlation to PFS or OS, and was not predictive of treatment outcome. Conclusions: A benefit for addition of CIL1W to cetuximab and chemotherapy was seen for independent-read PFS, and a HR 0.81 for OS, equivalent to a 3.9 mo median survival improvement in favour of the combination, but no consistent efficacy was shown with CIL vs CTRL. The safety profile did not reveal any safety concerns for CIL. Disclosure: J.F. Vansteenkiste: Advisory board: advisory functions for Merck-Serono; F. Barlesi: Corporate-sponsored research: Merck; J. Bennouna: Advisory Board: Roche, Boehringer Ingelheim and Novartis; E. Goekkurt: Advisory board: Merck; H. Lena: Advisory board: Merck; C. Hicking: Ownership: Stock Ownership Merck KGaA Other substantive relationships: Employee of trial sponsor; J. Straub: Other substantive relationships:: Employee of the trial Merck; M. Picard: Other substantive relationships: Employee of the trial sponsor; W. Schuette: Advisory Board: Merck, Amgen, AstraZeneca, Roche Corporate-sponsored research: Roche, Lilly, AstraZeneca, Amgen, Genentech, Daiichi Sankyo; K. O' Byrne: Advisory board: for cetuximab non small lung cancer Corporate-sponsored research: Grant received for clinical trial which was completed 5 years ago Other substantive relationships: Honoraria for speaker bureau lectures. All other authors have declared no conflicts of interest.

Cetuximab in advanced non-small cell lung cancer (NSCLC): the showdown?

Cetuximab in advanced non-small cell lung cancer (NSCLC): the showdown?

Relationship Between EGFR Expression, EGFR Mutation Status, and the Efficacy of Chemotherapy Plus Cetuximab in FLEX Study Patients with Advanced Non–Small-Cell Lung Cancer

The phase III FLEX study (NCT00148798) in advanced non-small-cell lung cancer indicated that the survival benefit associated with the addition of cetuximab to cisplatin and vinorelbine was limited to patients whose tumors expressed high levels of epidermal growth factor receptor (EGFR) (immunohistochemistry score of ≥200; scale 0-300). We assessed whether the treatment effect was also modulated in FLEX study patients by tumor EGFR mutation status. A tumor mutation screen of EGFR exons 18 to 21 included 971 of 1125 (86%) FLEX study patients. Treatment outcome in low and high EGFR expression groups was analyzed across efficacy endpoints according to tumor EGFR mutation status. Mutations in EGFR exons 18 to 21 were detected in 133 of 971 tumors (14%), 970 of which were also evaluable for EGFR expression level. The most common mutations were exon 19 deletions and L858R (124 of 133 patients; 93%). In the high EGFR expression group (immunohistochemistry score of ≥200), a survival benefit for the addition of cetuximab to chemotherapy was demonstrated in patients with EGFR wild-type (including T790M mutant) tumors. Although patient numbers were small, those in the high EGFR expression group whose tumors carried EGFR mutations may also have derived a survival benefit from the addition of cetuximab to chemotherapy. Response data suggested a cetuximab benefit in the high EGFR expression group regardless of EGFR mutation status. The survival benefit associated with the addition of cetuximab to first-line chemotherapy for advanced non-small-cell lung cancer expressing high levels of EGFR is not limited by EGFR mutation status.

Abstract C14: Squamous non-small cell lung cancer (NSCLC-SCC) patient-derived xenografts (PDX) from Asian patients have high response rate (RR) to cetuximab than those of adenocarcinoma.

Abstract Cetuximab was approved for advanced colorectal carcinoma (CRC), but yet for NSCLC, although some trials [e.g. FLEX (1) and BMS099 (2)] demonstrated benefit in subsets of patients. Recent retrospective study on Caucasian patients among the FLEX study cohort revealed that high EGFR expressors, uniquely defined by high-IHC score, are likely to benefit from the treatment (3). In particular, this was confirmed in a 50-patient derived xenografts model trial, mouse clinical trial (MCT or “Patient Avatar Trial”), in a similar format of FLEX, first time demonstrating relevance of human trial and MCT. The goal of the present study is identify the subset of Asian NSCLC patients that respond to cetuximab in a MCT. We have established a large panel of PDXs from NSCLC Asian patients of different histology (4): squamous cell carcinoma (SCC), adenocarcinoma (ADC), pleiomorphic carcinoma (PLC), and large cell carcinoma (LCC). We tested cetuximab activity in a randomly selected 32 PDX in a MCT. While NSCLC had overall engraftment rate of 25%, SCC interestingly, had higher rate of ∼31%, near-double ∼17% of ADC, which may have metastasis and prognosis implications. A subset of PDXs were found to respond to cetuximab (10/32) (31%, defined by %ΔT/ΔC < 25%). Surprisingly, all 10 responders exclusively belong to SCC and PLC (9/10 and 1/10 respectively), contrasting none to ADC (0/10). Non-/poor-responders (%ΔT/ΔC > 25%) include ADC (7/22), LCC (2/22) and SCC (12/22). Among the 12 non-/poor-responsive SCC, one has KRAS-G12D mutation, one has ALK-EML4 fusion and one has c-MET gene amplification. Among the SCC responders are ones with activating EGFR mutations. SCC has strikingly higher cetuximab response rate (RR) than ADC, with possible positive factors of EGFR mutation and negative factors of KRAS mutation, ALK fusion and c-met amplification. If proven true clinically, cetuximab could become a new therapy for NSCLC-SCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C14. Citation Format: Mengmeng Yang, Jie Cai, Sheng Guo, Xuesong Huang, Jie Yang, Xiaoming Song, Dawei Chen, Likun Zhang, Jiahua Jiang, Jean-Pierre Wery, Henry Li. Squamous non-small cell lung cancer (NSCLC-SCC) patient-derived xenografts (PDX) from Asian patients have high response rate (RR) to cetuximab than those of adenocarcinoma. [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 C14.

Evaluation of EGFR protein expression by immunohistochemistry using H-score and the magnification rule: Re-analysis of the SATURN study

IntroductionThe phase III SATURN study demonstrated that first-line maintenance erlotinib extended progression-free survival (PFS) and overall survival (OS) versus placebo in patients with advanced non-small cell lung cancer (NSCLC). Analysis of epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC) found no significant interaction between EGFR IHC status and PFS (p=0.63) or OS (p=0.52). The FLEX study of first-line cetuximab plus chemotherapy demonstrated that EGFR IHC expression was predictive of improved OS with cetuximab when assessed by H-score with a magnification rule. This novel method was used to reassess samples from SATURN. MethodsThe H-score method assigned a score of 0–300 to each patient, based on the percentage of cells stained at different intensities viewed at various magnifications. The discriminatory threshold was set at 200, per the FLEX study, and existing samples were re-read and classed as low (H-score<200) or high (≥200) EGFR expression. PFS and OS were re-analyzed based on these new classifications. ResultsIn the overall and EGFR wild-type populations, erlotinib provided a consistent survival benefit versus placebo. Hazard ratios (HRs) in the overall population were similar between EGFR IHC-positive and -negative patients for median PFS (HR 0.68 [95% confidence interval (CI) 0.53–0.86] and 0.76 [95% CI 0.62–0.93], respectively) and OS (HR 0.80 [95% CI 0.62–1.05] and 0.80 [95% CI 0.64–1.01] for IHC-positive and IHC-negative, respectively). In the EGFR wild-type population, HRs were again similar between EGFR IHC-positive and -negative subpopulations for PFS (HR 0.69 [95% CI 0.51–0.95] and 0.84 [95% CI 0.63–1.12], respectively) and OS (HR 0.78 [95% CI 0.55–1.10] and 0.76 [95% CI 0.55–1.05], respectively). ConclusionsThese data suggest that EGFR IHC does not have value as a marker to predict erlotinib benefit in the first-line maintenance setting for advanced NSCLC.

Abstract 2385: Identification of a molecular marker for cetuximab sensitivity and development of a novel combination therapy for non-small cell lung cancers.

Abstract The epidermal growth factor receptor (EGFR) is an important molecular target for non-small-cell lung cancers (NSCLCs). A recent phase III study (the FLEX study) revealed that the combination of the anti-EGFR antibody cetuximab with conventional chemotherapy improved the survival of patients with NSCLCs. However, the survival benefit was only about 5 weeks. Therefore, the identification of a molecular marker for cetuximab sensitivity is urgently needed. In this study, we investigated a new molecular marker for cetuximab sensitivity and developed a combination therapy to improve the effectiveness of cetuximab. First, we established a NSCLC cell line that showed resistance to cetuximab by culturing a parental sensitive NSCLC cell line in the presence of increasing concentrations of cetuximab. Next, we performed microarray analysis to compare the gene expression patterns of cetuximab-sensitive and -resistant cell lines. Using pathway analysis, we found that the Akt and Wnt pathways were significantly activated in the resistant cell line as compared to their activation in the sensitive cell line. We confirmed this result by performing western blot analysis, which showed Akt phosphorylation and beta-catenin dephosphorylation in the resistant cell line. Therefore, the activation status of either the Akt or Wnt pathway is the candidate pathway that influences cetuximab sensitivity. Finally, we focused on the Akt pathway and treated both resistant and parental sensitive cell lines with the PI3-K inhibitor LY294002 in combination with cetuximab. This combination therapy induced a synergistic growth-inhibiting effect in both cell lines. The data suggest that the activation status of the Akt pathway is one of the markers for cetuximab sensitivity and the combination of an Akt pathway inhibitor and cetuximab would be a promising anti-EGFR therapy for NSCLCs. Citation Format: Miyako Takata, Hiroki Chikumi, Kosuke Yamaguchi, Jun Kurai, Masaki Nakamoto, Naoto Burioka, Tadashi Igishi, Eiji Shimizu. Identification of a molecular marker for cetuximab sensitivity and development of a novel combination therapy for non-small cell lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2013-2385 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Treatment holidays in patients with postmenopausal osteoporosis: Whom and when?

The controversy about the ideal duration of treatment with bisphosphonates is not new. As early as in 2005, some experts suggested that treatment with alendronate for patients with postmenopausal osteoporosis could be discontinued after five years, provided no fractures had occurred during this period and bone mass had improved as compared to baseline. This recommendation was based on the lack of data showing an additional fracture rate reduction when treatment was continued beyond five years, and on the fact that remodeling markers continued to be suppressed despite treatment discontinuation, which suggested a residual effect. The final data of the FLEX study were reported in 2006. In this study, 1099 women from the Fracture Intervention Trial (conducted to assess the anti-fracture efficacy of alendronate) were randomized again to three treatment arms: placebo, alendronate 5 mg/day, and alendronate 10 mg/day, for five years. In the placebo group, a decrease in bone mineral density (BMD) associated with increased remodeling was seen, but values prior to the start of treatment were not reached. The patients who continued to receive alendronate had a lower risk of clinical vertebral fractures as compared to the placebo patients, but no differences

1272P - Relationship Between EGFR Expression Level, EGFR Mutation Status and the Efficacy of Chemotherapy Plus Cetuximab in Flex Study Patients with Advanced Non-Small Cell Lung Cancer (NSCLC)

ABSTRACT Background Analysis of prospectively collected immunohistochemistry (IHC) data from the phase III FLEX study in advanced NSCLC showed that high EGFR expression (IHC score ≥200 [scale of 0–300]) is a tumor biomarker that can predict survival benefit from the addition of cetuximab to first-line vinorelbine/cisplatin chemotherapy (CT). Treatment outcome was analyzed further in FLEX study EGFR expression groups according to EGFR mutation status. Methods Tumors from patients (pts) in the FLEX intention to treat (ITT) population were screened for the presence of EGFR exon 18–21 mutations (excluding T790M) using the therascreen EGFR PCR Kit (Qiagen). Outcome according to treatment was assessed across key efficacy endpoints in high and low EGFR expression groups (IHC score ≥200 vs Results Tumors from 970/1125 pts (86%) were evaluable for both EGFR expression and EGFR mutation status. Mutations were detected in 133/970 (14%) tumors (124/133 [93%] exon 19 deletions or L858R); 50/288 (17%) in the high EGFR expression group and 83/682 (12%) in the low EGFR expression group. In the high EGFR group a survival benefit for the addition of cetuximab to CT was demonstrated in pts with EGFR WT tumors (hazard ratio [HR] 0.76, 95% CI 0.57–1.00) with the magnitude of benefit similar to that of the high EGFR group of the ITT population (HR 0.73, 95% CI 0.58–0.93). Response was also higher in the CT + cetuximab arm for pts in this group with EGFR WT tumors (odds ratio 1.73, 95% CI 1.00–3.00). Pts in the high EGFR group with EGFR Mut tumors also seemed to benefit from CT + cetuximab in relation to survival (HR 0.86, 95% CI 0.40–1.84) and response (odds ratio 3.27, 95% CI 1.01–10.6) although numbers in this group were low. Outcome for the EGFR Mut subgroup with exon 19 deletion or L858R mutations will be presented. Conclusions In the high EGFR expression group pts with EGFR WT advanced NSCLC had a higher response rate and greater survival benefit from CT + cetuximab compared with CT alone. Benefit was also seen in pts with EGFR Mut tumors (exon 18–21) but firm conclusions cannot be drawn due to low pt numbers. Disclosure J. Douillard: AGENNIX: Advisor ASTRAZENECA: Advisor, speaker in symposia BOERHINGER-INGELHEIM: Advisor GSK: Advisor, speaker in symposia MERCK SERONO: Advisor, speaker in symposia, research grant PFIZER: Advisor ROCHE/GENENTECH: Advisor, speaker in symposia. R. Pirker: The author declares the following: Honoraria for Advisory Board and speaker's fee from AstraZeneca, Boehringer Ingelheim, Merck Serono and Roche. K.J. O'Byrne: The author declares: Payment from Merck Serono in relation to a protocol writing committee and advisory boards and travel costs in related to attendance. Honoraria from Merck Serono associated with presentation of data at satellite/company symposia. K.M. Kerr: Keith Kerr has acted on Advisory Boards and has received speaker's fees from Merck Serono. He has had similar roles with Astra Zeneca, Roche, Eli Lilly, Daichi Sankyo, Pfizer, Boeringher Ingelheim and Glaxo Smith Klein. S. Storkel: The author has been involved in sponsored research by Merck KGaA. I. Celik: The author is an employee of Merck KGaA. A. von Heydebreck: Teh author is an employee of Merck KGaA and declares stock ownership in this company. F.A. Shepherd: Honorarium from Merck KGaA for presentation at Scientific, Industry-sponsored Satellite Symposium. Provided compensated consultation services to Merck KGaA.

1271P - Analysis of PFS and OS from the Saturn Study According to EGFR IHC Status Using the H-Score Reading Method

ABSTRACT Background In the phase III SATURN study, maintenance erlotinib significantly prolonged progression-free survival (PFS) vs placebo in patients (pts) with advanced non-small-cell lung cancer (NSCLC) and non-progressive disease after first-line chemotherapy (Cappuzzo et al, Lancet Oncol 2010). Epidermal growth factor receptor (EGFR) expression analysis by immunohistochemistry (IHC) found no significant difference in PFS (p = 0.63) or overall survival (OS; p = 0.52) with erlotinib by EGFR IHC status (Brugger et al, J Clin Oncol 2011). Recently, Pirker at al. (Lancet Oncol 2012) presented data on EGFR expression as a predictor of OS for first-line chemotherapy plus cetuximab in the phase III FLEX study, using a novel method (H-score) to assign EGFR IHC status. We used this method to reassess samples from the SATURN study. Methods The H-score method assigns an IHC score to each pt on a continuous scale of 0–300, based on the percentage of cells at different staining intensities (Pirker et al, Lancet Oncol 2012). As per this method, the outcome-based discriminatory threshold IHC H-score for our analysis was set at 200 and existing samples were re-read and classed as low (H-score Results The original EGFR IHC analysis used samples from 370 and 372 pts in the erlotinib and placebo arms, respectively. This analysis examined existing samples from 351 and 361 pts in the erlotinib and placebo arms, respectively. PFS and OS according to EGFR IHC by different methods are shown in the Table. Table: 1271P . SATURN protocol-defined EGFR IHC+ SATURN protocol-defined EGFR IHC– EGFR IHC by H-score ≥ 200 (high) EGFR IHC by H-score EGFR IHC by H-score ≥ 10% EGFR IHC by H-score n erlotinib: 308 placebo: 313 erlotinib: 62 placebo: 59 erlotinib: 146 placebo: 157 erlotinib: 205 placebo: 204 erlotinib: 267 placebo: 287 erlotinib: 84 placebo: 74 PFS 0.69 (0.58–0.82) log-rank p 0.77 (0.51–1.14) log-rank p = 0.1768 0.68 (0.53–0.86) log-rank p = 0.0010 0.76 (0.62–0.93) log-rank p = 0.0076 0.71 (0.59–0.84) log-rank p 0.79 (0.57–1.10) log-rank p = 0.1627 OS 0.77 (0.64–0.93) log-rank p = 0.0063 0.91 (0.59–1.38) log-rank p = 0.6482 0.80 (0.62–1.05) log-rank p = 0.1099 0.80 (0.64–1.01) log-rank p = 0.0639 0.78 (0.64–0.96) log-rank p = 0.0161 0.85 (0.60–1.22) log-rank p = 0.3901 n (EGFR WT) erlotinib: 170 placebo: 157 erlotinib: 28 placebo: 27 erlotinib: 86 placebo: 84 erlotinib: 103 placebo: 97 erlotinib: 147 placebo: 144 erlotinib: 42 placebo: 37 PFS in WT 0.79 (0.63–0.99) log-rank p = 0.0336 0.65 (0.37–1.13) log-rank p = 0.1146 0.69 (0.51–0.95) log-rank p = 0.0188 0.84 (0.63–1.12) log-rank p = 0.2166 0.78 (0.62–0.99) log-rank p = 0.0400 0.69 (0.44–1.10) log-rank p = 0.1126 OS in WT 0.77 (0.60–0.99) log-rank p = 0.0402 0.64 (0.35–1.20) log-rank p = 0.1608 0.78 (0.55–1.10) log-rank p = 0.1563 0.76 (0.55–1.05) log-rank p = 0.0964 0.78 (0.60–1.02) log-rank p = 0.0698 0.72 (0.43–1.19) log-rank p = 0.2004 Note: HR Conclusions Re-scoring of EGFR IHC status in SATURN by H-score found similar benefit of erlotinib treatment in PFS or OS for subsets with high or low EGFR expression, in overall or EGFR WT populations. Disclosure W. Brugger: Financial Interest: Advisory Board. F. Cappuzzo: Financial Interest: Advisory board, F. Hoffmann-La Roche Ltd. I. Bara: Employed by F. Hoffmann-La Roche Ltd. G. Klingelschmitt: Employed by F. Hoffmann-La Roche Ltd. B. Klughammer: Stock Ownership: F. Hoffmann-La Roche Ltd Employed by F. Hoffmann-La Roche Ltd. All other authors have declared no conflicts of interest.

Prognostic factors in patients with advanced non-small cell lung cancer: Data from the phase III FLEX study

The FLEX study demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). In the FLEX intention to treat (ITT) population, we investigated the prognostic significance of particular baseline characteristics. Individual patient data from the treatment arms of the ITT population of the FLEX study were combined. Univariable and multivariable Cox regression models were used to investigate variables with potential prognostic value. The ITT population comprised 1125 patients. In the univariable analysis, longer median survival times were apparent for females compared with males (12.7 vs 9.3 months); patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 compared with 1 compared with 2 (13.5 vs 10.6 vs 5.9 months); never smokers compared with former smokers compared with current smokers (14.6 vs 11.1 vs 9.0); Asians compared with Caucasians (19.5 vs 9.6 months); patients with adenocarcinoma compared with squamous cell carcinoma (12.4 vs 9.3 months) and those with metastases to one site compared with two sites compared with three or more sites (12.4 months vs 9.8 months vs 6.4 months). Age (<65 vs ≥65 years), tumor stage (IIIB with pleural effusion vs IV) and percentage of tumor cells expressing EGFR (<40% vs ≥40%) were not identified as possible prognostic factors in relation to survival time. In multivariable analysis, a stepwise selection procedure identified age (<65 vs ≥65 years), gender, ECOG PS, smoking status, region, tumor histology, and number of organs involved as independent factors of prognostic value. In summary, in patients with advanced NSCLC enrolled in the FLEX study, and consistent with previous analyses, particular patient and disease characteristics at baseline were shown to be independent factors of prognostic value. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798.

Abstract IA14: Cetuximab in NSCLC and predictive markers

Abstract The role of the EGFR humanized monoclonal antibody cetuximab (Erbitux®, Bristol-Myers Squibb) in the treatment of patients with advanced non-small cell lung cancer (NSCLC) has until recently been controversial. While it is clear that the EGFR pathway plays an important role in NSCLC development and progression, the clinical data with cetuximab in NSCLC has not been consistent from the different studies including the two large phase III studies (BMS -099 and FLEX), despite the overall survival in the FLEX study was statistically significant in favor the combination of chemotherapy plus cetuximab (HR 0.87 [0.76–1.0]) (1, 2). The FLEX study comparing gemcitabine/cisplatin with and without cetuximab required EGFR positive tumor (IHC &amp;gt; 1% positive cells) as inclusion criteria, while the non-significant results from BMS -099 was based on “all comers”. However, most recently a more detailed analysis of the EGFR protein expression in the FLEX study, by using the H-score (intensity:0–3 x percentage of cells within each intensity category:0–100% for a final H-score of 0–300) was presented (3). Patients having tumors with EGFR H-score≥ 200 (about 30% of the patients) had a significant better survival from chemotherapy plus cetuximab compared to cetuximab alone (HR=0.73 [0.58–0.93]), and even better for the largest subgroup of patients; the Caucasians with H-score ≥ 200 (HR=0.64 [0.49–0.83]) with a 3.5 months difference in median survival (11.4 months vs. 7.9 months HR= 0.49 [0.29–0.81]). Patients with high EGFR expression and adenocarcinoma had a median survival of 20.2 months on the combined therapy versus 8.0 months for chemotherapy alone (3). A reproducibility study of the H-score classification showed a high concordance (91%) among 10 pathologists after specific assessment training (4). A previous reported randomized phase II study comparing chemotherapy with concomitant cetuximab versus sequential cetuximab from SWOG (0342) demonstrated a doubling of PFS and OS in the EGFR FISH positive patients compared to the FISH negative patients (5), while he EGFR FISH assay did not show a significant impact on outcomes in the large randomized BMS -099 study or the FLEX study. Thus, very encouraging biomarker results are achieved retrospectively for selection of patients to chemotherapy plus cetuximab therapy, and these biomarkers are currently being prospectively validated in the large prospective randomized phase III study SWOG 0819, which compare chemotherapy (+/− bevacizumab) with and without cetuximab.