Supplementary Figures 1-3 from Novel D761Y and Common Secondary T790M Mutations in Epidermal Growth Factor Receptor–Mutant Lung Adenocarcinomas with Acquired Resistance to Kinase Inhibitors

Supplementary Methods and Figure Legends from Novel D761Y and Common Secondary T790M Mutations in Epidermal Growth Factor Receptor–Mutant Lung Adenocarcinomas with Acquired Resistance to Kinase Inhibitors

Supplementary Figure S1; Supplementary Table S1.

Background: Patient-reported symptom and health-related quality of life (HRQoL) benefit of afatinib, a novel, irreversible, ErbB Family Blocker, was investigated in a double-blind, randomized, phase IIb/III trial (LUX-Lung 1).Methods: Five hundred and eighty-five patients with lung adenocarcinoma (stage IIIb/IV), who had progressed after chemotherapy (1-2 lines) and at least 12 weeks of erlotinib or gefitinib, were randomized (2: 1) to receive either afatinib plus best supportive care (BSC) or placebo plus BSC. Symptom and HRQoL benefit were measured using the lung cancer-specific European Organisation for Research and Treatment of Cancer (QLQ-C30/LC13) and EuroQol (EQ-5D) questionnaires. Non-small-cell lung cancer-related symptoms (cough, dyspnea, and pain) were prespecified using three preplanned analyses (percentage of patients improved/worsened/stable, change in scores over time, and time to deterioration of scores).Results: Compared with patients on placebo, a significantly higher proportion of afatinib-treated patients showed an improvement in cough (p < 0.0001), dyspnea (p = 0.006), and pain (p < 0.0001). Afatinib also significantly improved the mean scores over time for cough (p < 0.0001), dyspnea (p = 0.0161), and pain (p = 0.0056); significantly delayed the time to deterioration for cough (p < 0.001); and showed a trend in delaying dyspnea (p = 0.170) and pain (p = 0.287). Consistent with the adverse-event profile of afatinib, a significantly (p < 0.05) higher proportion of afatinib-treated patients showed worsening of diarrhea, sore mouth, dysphagia, and appetite scores. However, compared with placebo, afatinib significantly (p < 0.05) improved QoL assessed with the EQ-5D questionnaire and global health status/QoL, physical functioning, and fatigue, which were assessed with the European Organisation for Research and Treatment of Cancer questionnaires.Conclusion: In the LUX-Lung 1 trial, the addition of afatinib to BSC significantly improved non-small-cell lung cancer-related symptoms (cough, dyspnea, and pain), fatigue, physical functioning, and HRQoL and significantly delayed time to deterioration of cough.

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Background: The epidermal growth factor receptor (EGFR) mutation T790M is reported in approximately 50% of lung cancers with acquired resistance to EGFR inhibitors and is a potential prognostic and predictive biomarker. Its assessment can be challenging due to limited tissue availability and underdetection at low mutant allele levels. Here, we sought to determine the feasibility of tumor rebiopsy and to more accurately assess the prevalence of the T790M using a highly sensitive locked nucleic acid (LNA) PCR/ sequencing assay. MET amplification was also analyzed. Methods: Patients with acquired resistance were rebiopsied and samples were studied for sensitizing EGFRmutations. Positive cases were evaluated for T790M using standard PCR-based methods and a subset were re-evaluated with an LNA-PCR/sequencing method with an analytical sensitivity of approximately 0.1%. MET amplification was assessed by FISH. Results: Of 121 patients undergoing tissue sampling, 104 (86%) were successfully analyzed for sensitizing EGFR mutations. Most failures were related to low tumor content. All patients (61/61) with matched pretreatment and resistance specimens showed concordance for the original sensitizing EGFR mutation. Standard T790M mutation analysis on 99 patients detected 51(51%) mutants. Retesting of 30 negative patients by the LNA-based method detected 11 additional mutants for an estimated prevalence of 68%. MET was amplified in 11% of cases (4/37). Conclusions: The re-biopsy of lung cancer patients with acquired resistance is feasible and provides sufficient material for mutation analysis in most patients. Using high sensitivity methods, the T790M is detected in up to 68% of these patients. Clin Cancer Res; 17(5); 1169–80. 2011 AACR.

strategies. models based on the characteristics of diverse cancer cell types could offer clues for designing optimal treatment utility of evolutionary mathematical modeling in designing cancer treatments. If they prove beneficial, individual will have to come from patients. Clinical trials based on these alternative dosing strategies will be the true test of the resistance to prevent fast overgrowth by the sensitive cells, a result also born out in vitro and in vivo. Ultimate proof prediction. Modeling also indicated the wisdom of prolonging treatment with the EGFR TKI after the development of with high-dose pulses of an EGFR TKI should delay the onset of resistance. Subsequent cellular studies bore out this combined different treatment regimes. Their models predicted that continuous administration of a low-dose EGFR TKI under Understanding the growth dynamics of how tumors behave allowed the authors to calculate what would happen based on the differential growth rates of TKI-sensitive and TKI-resistant cells in heterogeneous tumor cell populations. therapy after a drug holiday. The authors then constructed an evolutionary mathematical model of tumor behavior disease progression, and patients with acquired resistance have re-responded to tyrosine kinase inhibitor (TKI) observations corroborated these findings. For example, patients with resistant tumors showed a slow course of their sensitive counterparts, and resistance was not maintained in the absence of selection. Multiple clinical which is activated in a subset of NSCLCs. To the authors' surprise, the drug-resistant cells grew more slowly than drugs used to treat NSCLC that are directed against the epidermal growth factor receptor (EGFR) tyrosine kinase, −− cancer cells. The authors made paired isogenic cell lines that were sensitive and resistant to afatinib and erlotinib mathematical model that predicted alternative therapeutic strategies to delay the development of drug-resistant growth characteristics of these cells were consistent with patient tumor behavior, enabling construction of a cancer (NSCLC) before and after the cells acquire resistance to targeted therapy, which inevitably they do. The small cell lung − how to better fight the potent forces of evolution, Chmielecki et al. examined the behavior of non on characteristically compliant cancer cells, as the common recurrence of drug-resistant cancers testifies. To learn molecular changes that strengthen the clan's chances of survival. Therapeutic drugs exert a powerful selective force Like any organism under severe evolutionary pressure, a few select members of a cancer cell population acquire

Introduction:Adenocarcinoma is the most common histologic type of lung cancer. To address advances in oncology, molecular biology, pathology, radiology, and surgery of lung adenocarcinoma, an international multidisciplinary classification was sponsored by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society. This new adenocarcinoma classification is needed to provide uniform terminology and diagnostic criteria, especially for bronchioloalveolar carcinoma (BAC), the overall approach to small nonresection cancer specimens, and for multidisciplinary strategic management of tissue for molecular and immunohistochemical studies.Methods:An international core panel of experts representing all three societies was formed with oncologists/pulmonologists, pathologists, radiologists, molecular biologists, and thoracic surgeons. A systematic review was performed under the guidance of the American Thoracic Society Documents Development and Implementation Committee. The search strategy identified 11,368 citations of which 312 articles met specified eligibility criteria and were retrieved for full text review. A series of meetings were held to discuss the development of the new classification, to develop the recommendations, and to write the current document. Recommendations for key questions were graded by strength and quality of the evidence according to the Grades of Recommendation, Assessment, Development, and Evaluation approach.Results:The classification addresses both resection specimens, and small biopsies and cytology. The terms BAC and mixed subtype adenocarcinoma are no longer used. For resection specimens, new concepts are introduced such as adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) for small solitary adenocarcinomas with either pure lepidic growth (AIS) or predominant lepidic growth with ≤5 mm invasion (MIA) to define patients who, if they undergo complete resection, will have 100% or near 100% disease-specific survival, respectively. AIS and MIA are usually nonmucinous but rarely may be mucinous. Invasive adenocarcinomas are classified by predominant pattern after using comprehensive histologic subtyping with lepidic (formerly most mixed subtype tumors with nonmucinous BAC), acinar, papillary, and solid patterns; micropapillary is added as a new histologic subtype. Variants include invasive mucinous adenocarcinoma (formerly mucinous BAC), colloid, fetal, and enteric adenocarcinoma. This classification provides guidance for small biopsies and cytology specimens, as approximately 70% of lung cancers are diagnosed in such samples. Non-small cell lung carcinomas (NSCLCs), in patients with advanced-stage disease, are to be classified into more specific types such as adenocarcinoma or squamous cell carcinoma, whenever possible for several reasons: (1) adenocarcinoma or NSCLC not otherwise specified should be tested for epidermal growth factor receptor (EGFR) mutations as the presence of these mutations is predictive of responsiveness to EGFR tyrosine kinase inhibitors, (2) adenocarcinoma histology is a strong predictor for improved outcome with pemetrexed therapy compared with squamous cell carcinoma, and (3) potential life-threatening hemorrhage may occur in patients with squamous cell carcinoma who receive bevacizumab. If the tumor cannot be classified based on light microscopy alone, special studies such as immunohistochemistry and/or mucin stains should be applied to classify the tumor further. Use of the term NSCLC not otherwise specified should be minimized.Conclusions:This new classification strategy is based on a multidisciplinary approach to diagnosis of lung adenocarcinoma that incorporates clinical, molecular, radiologic, and surgical issues, but it is primarily based on histology. This classification is intended to support clinical practice, and research investigation and clinical trials. As EGFR mutation is a validated predictive marker for response and progression-free survival with EGFR tyrosine kinase inhibitors in advanced lung adenocarcinoma, we recommend that patients with advanced adenocarcinomas be tested for EGFR mutation. This has implications for strategic management of tissue, particularly for small biopsies and cytology samples, to maximize high-quality tissue available for molecular studies. Potential impact for tumor, node, and metastasis staging include adjustment of the size T factor according to only the invasive component (1) pathologically in invasive tumors with lepidic areas or (2) radiologically by measuring the solid component of part-solid nodules.

Pemetrexed is an antifolate agent approved for the treatment of advanced lung cancer. Major side effects include myelosuppression and neutropenia. Three patients developed kidney disease while being treated with maintenance pemetrexed. Kidney biopsy specimens showed tubulointerstitial injury with tubular simplification, shrinkage, loss of brush border, and tubular atrophy in a more advanced case. Kidney function remained impaired, but stable, after discontinuation of pemetrexed therapy in all cases. Am J Kidney Dis. 58(5):817-820. (C) 2011 by the National Kidney Foundation, Inc.

oncoprotein-targeted therapeutics. clinically for distinguishing those cancer patients who are most likely to derive clinical benefit from benefit to the field of oncology. Finally, quantitative imaging combined with classifier algorithms may be useful sufficient given the complexity and heterogeneity of tumors, so the ability to quantify complex systems will be of could be a useful tool for helping guide the management of cancer patients. Qualitative approaches alone are not . study demonstrates that mathematical modeling et al not. Although much more work needs to be done, the Tran tumors that were oncogene-addicted and hence were most likely to benefit from this treatment from those who did had been treated with oncoprotein-targeted therapy. Their model was able to differentiate patients who had lung mice that did not respond. They then extended this quantitative imaging approach to humans with lung cancer who support vector machine to accurately differentiate mice that responded to oncoprotein-targeted therapy from those survival and death pathways. The group then used quantitative imaging of mouse tumors to train an algorithm called for oncogene addiction could predict the distinct temporal relationships of signaling molecules from converging lymphoma cells in mice treated with oncoprotein-targeted therapies. They also showed that their mathematical model and cell death pathways in the tumors, Tran and colleagues were able to predict the clinical behavior of lung and premise that oncogene addiction could be deduced from the balance between converging signals from cell survival mathematically model the behavior of oncogene-addicted lung and lymphoma tumors. Starting from the simple . use a systems approach to et al cancer patients are likely to respond best to such drugs. In a new study, Tran addiction would be useful not only for developing new oncoprotein-targeted therapies but also for selecting which tumors that exhibit this behavior of oncogene addiction. The ability to predict when a tumor will exhibit oncogene after treatment with drugs targeting oncoproteins. However, only a distinct subset of human cancer patients have addiction.'' Marked clinical responses have been reported in some cancer patients, particularly those with lung cancer, that target these oncoproteins induce the addicted cancer cells to die rapidly, a phenomenon called ''oncogene Some cancer cells are dependent or ''addicted'' to the continued activity of oncoproteins such as Myc. Drugs Predicting Oncogene Addiction with Math

7526 Background: The ATLAS study (n=768) was designed to evaluate B (15 mg/kg q3w)+ E (150 mg qd) vs. B + placebo (PL) following B + platin-containing doublet chemo in patients (pts) with stage IIIb/IV NSCLC. The trial met its primary (1°) endpoint of improving PFS. Median PFS was 4.8 mo for B + E vs. 3.7 mo for B + PL (HR=0.72, 95% CI: 0.59–0.88, p=0.0012). The safety profile for B + E was consistent with known profiles for B and E. Here we report on OS, a secondary endpoint. Methods: Enrolled pts were B-eligible (including pts with treated brain metastases and pts receiving low MW heparins); peripheral and/or extrathoracic squamous tumors allowed. Pts received 4 cycles of B (15 mg/kg q3w) + platinum-containing doublet chemo. Pts who did not progress after 4 cycles of B + chemo and had no significant toxicity were randomized to receive B + E or B + PL as maintenance therapy. OS was defined as time from randomization to death. The study was not powered to detect OS difference. The 1° OS analysis was prespecified to occur at the time of primary PFS analysis (July 18, 2009 cutoff). At the time of primary OS analysis, few death events had occurred (31% of pts had died). Two post-hoc analyses of OS, after more death events had occurred, were performed (data cutoffs, January 28 and June 19, 2009). Results: See Table. An equal percentage (39.7%) of patients in the B + E and B + PL arms received subsequent therapy with an EGFR tyrosine kinase inhibitor. Conclusions: For pts treated in the first-line setting for locally advanced, recurrent, or metastatic NSCLC, the addition of E to B after chemotherapy significantly improved PFS and may also provide OS benefit. Clinical and biomarker subset data will be presented. Data Cutoff Patients with events n/N (%) Median OS (mo) B+E vs. B HR (95% CI) p value July 18, 2008 (prespecified) 228/743 (31) 14.4 vs. 13.3 0.92 (0.70–1.21) 0.5604 January 28, 2009 357/768* (46) 14.4 vs. 13.6 0.90 (0.73–1.12) 0.3574 June 19, 2009 439/768* (57) 15.9 vs. 13.9 0.90 (0.74–1.09) 0.2686 * 25 pts were randomized after cutoff for main analysis of PFS. ** HR and p value based on prespecified, stratified analysis. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech AstraZeneca, Boehringer Ingelheim, Genentech, KEW Group (Diagnostics), Millennium, Pfizer, Roche Celgene, Roche Genentech, Roche Genzyme

Abstract Mutations within exons encoding the kinase domain of EGFR confer increased sensitivity of non-small cell lung carcinomas to the tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. Unfortunately, all patients whose disease responds initially to these therapies eventually develop acquired resistance. In ∼50% of cases, resistance is associated with acquisition of a second-site ‘gatekeeper’ mutation within EGFR, T790M. More potent ‘2nd generation’ EGFR inhibitors theoretically able to overcome T790M-mediated resistance have displayed limited clinical success. In preclinical models of acquired resistance, many of these TKIs (HKI-272, BIBW2992, PF00299804) actually select for the emergence of EGFR T790M-harboring clones. To determine if EGFR TKIs in general lead to the emergence of T790M, we modeled acquired resistance in vitro with the reversible TKI, XL647 (Symphony Evolution, Inc., Rockville, MD), the irreversible EGFR TKI, BIBW2992 (synthesized at MSKCC), and erlotinib. EGFR mutant TKI-sensitive PC-9 cells (lung adenocarcinoma, del E746-A750) were grown with increasing concentrations of XL647 for ∼160 days. Polyclonal resistant (XL-R) cells were derived that proliferated in drug concentrations ∼50-fold higher than the initial IC50. No other secondary mutations within EGFR were detected by direct sequencing, and the T790M mutation was not found using more sensitive mutation detection methods (e.g. SNaPShot; limit of detection ∼5%). By contrast, both BIBW2992- and erlotinib-resistant PC-9 cells (PC-9 BI-R and PC-9 E-R) harbored T790M, as shown by direct sequencing. No resistant cells displayed MET amplification, another known resistance mechanism. Crystal structure data revealed that XL647 binds reversibly within the ATP binding pocket of EGFR T790M with a similar orientation to erlotinib. Therefore, structural differences alone do not readily explain the differential selective pressure for EGFR T790M with gefitinib/erlotinib vs. XL647. Interestingly, PC-9 XL-R cells remained intermediately sensitive to erlotinib. Consistent with the in vitro observations, early clinical trial data revealed that only 1 of 6 patients with EGFR mutant lung cancer harbored T790M after developing acquired resistance to XL647. Furthermore, at least 2 of these patients subsequently derived clinical benefit from single-agent erlotinib. Collectively, these data suggest that the propensity to develop T790M-mediated resistance varies among different TKIs. We are currently exploring why the selective pressure for resistance conferred by XL647 is different from that of other EGFR TKIs. 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 1774.

8013 Background: EGFR mutations are associated with exquisite sensitivity to EGFR TKIs in NSCLC. A phase II trial evaluating the efficacy of BIBW 2992 (Tovok), a novel, potent, irreversible, dual EGFR and HER2 TKI with preclinical activity in cell lines harboring activating (H3255, IC50=0.7 nM) and resistant (H1975, IC50=99 nM) EGFR mutations, is reported. METHODS Objective response rate is the primary endpoint of this 2-stage trial. Based on 16 or more unconfirmed PRs in an interim analysis of the first 40 2nd line patients (pts) completing 1 course (28 days), accrual will continue to a total of 120 1st and 2nd line pts (expected completion of accrual by May 2009. Data on 2nd line pts only are presented). Eligible pts have stage IIIB/IV lung adenocarcinoma, EGFR mutation in exons 18-21 (tested by direct sequencing), measurable disease, ECOG PS 0-2 and adequate end organ function. Pts receive 50 mg BIBW 2992 qd until progression. Tumor assessments are performed every 4 weeks for 12 weeks, then every 8 weeks. RESULTS Since Oct 2007, samples from 289 pts (222 from Taiwan and 67 from the US) have been sequenced. 100 had detectable EGFR mutations including del19 (n=39), L858R (n=45) and others (n=16). 69 pts have started treatment. The trial was moved to stage 2 after 21 of the first 38 treated pts had objective response at 28 days. Of 55 evaluable 2nd line pts, 29 (53%) had PR, and 23 (42%) had SD. Median follow up is 5.1 months. Most common related AEs were diarrhea and skin-related AEs, reported in 87% and 88% of pts, respectively. 27 pts (42.9 %) had dose reduction to 40 mg and 7 pts (11%) to 30 mg but only 1 pt permanently discontinued due to AEs. Diarrhea and rash were main causes of dose reduction. CONCLUSIONS In the 2nd line setting, BIBW 2992 shows efficacy in NSCLC harboring EGFR activating mutations. Diarrhea and skin disorders, the most frequently observed AEs, are manageable with supportive care and dose reduction. Updated response and disease control rates and preliminary progression-free survival data will be presented. An international Phase III trial program investigating BIBW 2992 in NSCLC, LUX-Lung, is now recruiting. [Table: see text].