Next-generation sequencing of Chinese stage IV lung cancer patients reveals an association between EGFR mutation status and survival outcome

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1 Clin Genet 2017: 91: Printed in Singapore. All rights reserved Short Report 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Next-generation sequencing of Chinese stage IV lung cancer patients reveals an association between EGFR mutation status and survival outcome Li F., Du X., Zhang H., Ju T., Chen C., Qu Q., Zhang X., Qi L., Lizée G.. Next-generation sequencing of Chinese stage IV lung cancer patients reveals an association between EGFR mutation status and survival outcome. Clin Genet 2017: 91: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2016 Large-scale genomic characterization of non-small cell lung cancer (NSCLC) has revealed several putative oncogenic driver mutations that may constitute druggable therapeutic targets. However, there are little data to suggest that such gene alterations have clinical relevance. Over 12 consecutive months, tumor biopsy samples from 80 patients with stage IV NSCLC were analyzed for mutations in selected exons of 508 cancer-related genes using next-generation sequencing. From 85 specimens referred for genomic characterization, 80 (94%) specimens were successfully genotyped, and all had identifiable somatic alterations. Epidermal growth factor receptor (EGFR) andtp53 genes contained the highest frequency of observed mutations (65% and 40%, respectively) in the stage IV NSCLC cases. Notably, patients with EGFR mutations showed a significantly shorter survival time compared with patients expressing wild-type EGFR (p = ). Moreover, of the 32 patients harboring EGFR mutations, EGFR-L858R mutant patients showed a significantly shorter survival time compared with patients with other EGFR mutations (p = 0.036). In conclusion, tumors from stage IV NSCLC patients harbor characteristic gene alterations, of which EGFR L858R in particular appears to be a poor prognostic factor for overall survival. Conflict of interest Nothing to declare. F. Li a,b,,x.du c,,h.zhang d,,t. Ju c,c.chen c,q.qu a,x. Zhang e,l.qi f and G. Lizée g,h a Department of Gynecology, Tianjin First Center Hospital, Tianjin, China, b Department of Oncology Research, Tianjin HengJia Biotechnology Development Co, Ltd., Tianjin, China, c Department of Oncology, Tianjin Beichen Hospital, Tianjin, China, d Department of Integrated Chinese and Western Medicine Treatment Center, Henan Cancer Hospital, Zhengzhou, China, e Department of Lymphoma & Myeloma, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, f Laboratory of Molecular Neuro-Oncology, Texas Children s Cancer Center, Houston, TX, USA, g Department of Melanoma Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, and h Department of Immunology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA These authors equally contributed. Key words: epidermal growth factor receptor (EGFR) gene mutation next-generation sequencing non-small cell lung cancer (NSCLC) prognosis survival Corresponding authors: Fenge Li, Department of Gynecology, Tianjin First Center Hospital, Tianjin, China. Tel.: ; fax: ; rosetea85@163.com and 488

2 Next-generation sequencing of Chinese stage IV lung cancer patients Xueming Du, Department of Oncology, Tianjin Beichen Hospital, Beiyi Road, Beichen District, Tianjin , China. Tel.: ; fax: ; Received 30 March 2016, revised and accepted for publication 20 May 2016 Lung cancer is the second most diagnosed cancer in both men and women, and is the leading cause of cancer-related deaths (1). It has been reported that lung cancer is the most common malignancy worldwide and has the highest mortality rate in comparison to all other cancers (2). According to biological characteristics and prognosis, lung cancer is mainly divided into non-small cell lung cancer (NSCLC, about 85%) and small cell lung cancer (SCLC, about 15%) (1). NSCLC can be further divided into three subtypes, which are adenocarcinoma, squamous cell carcinoma (SCC) and large cell lung carcinoma. In recent years, it has been shown that somatic gene alterations play a very important role in the occurrence and development of lung cancer (2). The advent of new drugs targeting such mutations is driving efforts to incorporate tumor genotyping into clinical decision-making. For NSCLC patients, it is now standard to assess tumor-associated somatic alterations in the epidermal growth factor receptor (EGFR) gene to identify mutations sensitive to EGFR inhibitors erlotinib or gefitinib (3). The unique spectra of gene mutations that occur within each patient necessitate a personalized approach to treating NSCLC. However, a number of commonly shared mutations have also been identified, including TP53 and EGFR, with the latter gene showing a considerably higher prevalence among Asian lung cancer patients (4, 5). However, very few studies have examined whether these gene alterations are associated with clinical prognosis. Therefore, in this study, we performed mutational profiling of tumor specimens derived from 80 consecutive stage IV Chinese NSCLC patients to analyze potential associations between gene alterations and overall survival (OS). Materials and methods Patients and tumor specimen analysis Eighty consecutive patients with stage IV NSCLC underwent 508-tumor-related gene detection from 1 March 2015 until 1 March 2016 at Tianjin Beichen Hospital. The protocol followed the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Tianjin Anti-Cancer Association. Patient selection used the following inclusion criteria: (i) clinical assessments were classified with NSCLC stage IV (according to NCCN Clinical Practice Guidelines in Oncology, Version , NSCLC), which have no stage restrictions for gene testing; (ii) had not received any prior treatments for NSCLC; (iii) showed good or moderate performance status (PS, PS 2); (iv) received four to six cycles of systemic chemotherapy with vinorelbine and/or ciplatin; (v) the diagnosis of NSCLC was confirmed by biopsy. Patients were excluded if they received any targeted therapies (e.g. gefitinib, afatinib, erlitinib) or immune treatment (e.g. thymosin, lamivudine). Tumor samples were all obtained by biopsy from the tumor site within the lung. No patients received any other treatments during the follow-up period. All the patients data were collected from the hospital medical records, which were described precisely after confirmation of accurate specimen analysis. Baseline characteristics of patients are shown in Table S1, Supporting Information. Next-generation sequencing analysis A genotyping panel was used to detect 508 cancer-associated genes (Table 1) based on the biobanking system and in conjunction with the clinic and pathology lab. Tumor sample DNA was extracted immediately after the biopsy following the instruction of GenElute Mammalian Genomic DNA Kits (Sigma-Aldrich, Missouri, USA), and mutations were detected by Illumina Hiseq 2500 (Illumina, California, USA), which profiled them using exon capture by hybridization followed by next-generation sequencing. Follow-up assessments and statistical analysis All patients were followed from the date of sequencing analysis up to March 2016, or time of death. All statistical analyses were performed using the GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Survival curves and survival rates were calculated using the log-rank (Mantel Cox) test or Gehan Breslow Wilcoxon test, and survival was measured from the date of sequencing analysis until March 2016, or time of death. Probabilities with p < 0.05 were defined as statistically significant. Results Pathological characteristics and mutational profiling analysis of stage IV NSCLC patients As shown in Fig. 1a, the pathological subtypes of all 80 patients were adenocarcinoma (75%), SCC (22%) and large cell lung cancer (3%). In conjunction with the clinic and pathology lab, tumor specimens from all

3 Li et al. Table 1. Five-hundred and eight cancer-related gene panel Path ways Mitogen-Activated Protein Kinase (MAPK) signal PI3K signal Signal Transducers and Activators of Transcription (STAT) signal transforming growth factor-β (TGF-β) signal Cell cycle/apoptosis Hedgehog (HH) signal Antigen Presenting Cell (APC) signal NOTCH ssignal Transcription regulators Chromatin modifications DNA damage control Receptor Tyrosine Kinase (RTK) signal Others Gene list KRAS NF1 NF2 MAP3K1 ARAF BRAF RAF1 NRAS HRAS MAP2K2 MAP2K4 MAPK8IP1 MAPK2K1 MAPK3K13 MAPK1 MAPK3 MAPK8 JUN ANGPT1 ANGPT2 DUSP6 FGF10 FGF12 FGF14 FGF19 FGF23 IGF1R IGF1 IGF2 PIK3CA PIK3CB PIK3CG PTEN PIK3R1 PIK3R2 PIK3C2A PIK3C2B PIK3C2G PIK3C3 TLR4 MTOR STK11 TSC1 TSC2 NAV3 AKT3 AKT1 AKT2 PDK1 PRKCA PRKCB PRKCG PRKDC AXL FLCN HGF TMEM127 JAK1 JAK2 JAK3 STAT4 STAT5B SOCS1 CBL IFNAR1 IFNAR2 CRLF2 SMAD4 TGFBR2 ACVR1B SMAD2 ACVR2A BMPR1A SMAD3 INHBA ITGB2 ELAC2 FNTA MED12 CDKN2A RB1 CDK2 CDK4 CDK8 CDK6 CDK12 CCND1 CCND2 CCND3 CDKN1A CDKN1B CDKN2C CCNE1 CDKN2B MCL1 BCL2 BCL2A1 BCL2L1 BCL2L11 BCL2L2 AURKA AURKB CDC25C BAK1 BTG1 CASP8 CDC73 CFLAR CYLD EPCAM KIF1B PML PMS1 PMS2 RAD50 RAD51 RAD51B RAD51C RAD51D RAD52 RAD54L XIAP RPA1 NEK11 NPM1 PTP4A3 SMO PTCH1 PTCH2 SPOP APC CTNNA1 CTNNB1 TBL1XR1 SOX17 SOX10 AXIN1 AXIN2 GSK3B APCDD1 FAM123B NOCTH1 NOCTH2 NOTCH3 NOTCH4 VHL GATA1 GATA3 TSHZ EP300 CTCF TAF1 TSHZ2 RUNX1 RUNX1T1 MECOM TBX3 SIN3A EIF4A2 PHF6 CBFB SOX9 ELF3 VEZF1 CEBPA FOXA1 FOXA2 MITF NKX2-1 SOX-2 ERG ETV1 ETV6 CDX2 MYC NKX3-1 NFE2L2 NFE2L3 MED12 SF3B1 U2AF1 ZRSR2 SRSF2 PCBP1 BCL6 ARHGAP35 BACH1 BCOR BCORL1 C11orf30 DAXX DNMT1 EGR3 ESR1 EWSR1 FOXL2 FUBP1 GID4 HIF1A HNF1A IKZF1 IRF4 MAX MEF2B CIC KLF4 NCOA1 NCOA2 NCOR1 SF1 TOP1 TOP2A TOP2B HIST1H1C H3F3A H3F3C HIST1H2BD HIST1H3B MLL2 MLL3 MLL4 ARID1A PBRM1 SETD2 NSD1 SETBP1 KDM5A KDM5C KDM6A ARID5B ASXL1 EZH2 DNMT3A TET2 MLL CREBP SMARCA1 SMARCA4 SMARCB1 SMARCD1 ARID2 ARID1B CHD1 CHD2 CHD4 HDAC1 HDAC2 HDAC3 HDAC4 HDAC6 HDAC8 EP300 DOT1L KAT6A SPOP TP53 ATM ATRX ATR STAG2 BAP1 BRCA1 BRCA2 SMC1A SMC3 CHEK1 CHEK2 RAD21 ERCC2 ERCC3 MDM2 MADM4 MSH2 MSH3 MSH4 MSH5 MSH6 MLH1 MLH3 BLM BRIP1 CUL4A CUL4B FANCA FANCC FANCD2 FANCE FANCF FANCG FANCI FANCL FANCM MRE11A MUTYH NBN PARP1 PARP2 PARP3 PARP4 XPA XPC XRCC3 PALB2 EGFR FLT1 FLT4 FLT3 EPHA2 EPHA3 EPHA5 EPHB1 EPHB2 ERBB2 ERBB3 ERBB4 PDGFRA PDGFRB EPHB6 FGFR1 FGFR2 KIT FGFR3 FGFR4 MET ALK RET ROS1 CRKL VEGFA VEGFB ABL1 ABL2 AR DDR1 DDR2 FGF3 FGF4 FGF6 FGF7 IRS2 KDR LCK LYN TYR TEK MYD88 IKBKB IKBKE CHUK CDH1 AJUBA B2M BTK C1QA C1R C1S CARD11 CD79A CD79B CDC42 CTLA4 FCGR1A FCGR2A FCGR2B FCGR2C FCGR3A FCGR3B FYN HCK IL7R CSF1R KLHL6 SH2B3 SYK FBXW7 KEAP1 SPOP WWP1 FAM46C DIS3 BARD1 CBLB CRBN RNF43 TRAF7 EDNRA GNA11 GNA13 GNAQ GNAS GNRHR GPR124 GRM3 HRH2 MC1R TSHR LRRK2 PRKAA1 GRIN2A PRSS8 EML4 KIF5B TUBA1A TUBB TUBD1 TUBE1 TUBG1 FH RAC1 RAC2 RPL22 RPL5 TNFAIP3 TNFRSF14 TNFRSF8 TNFSF11 TNFSF13B IDH1 IDH2 CBR1 CYP17A1 FPGS ALOX12B HSD17B3 CROT SDHAF2 SDHB SDHC SDHD PPP2R1A PTPN11 B4GALT3 EXT1 EXT2 ARFRP1 BCR LIMK1 GAB2 CRIPAK EPPK1 HSD3B2 HSP90AA1 HSPA4 HSD3B2 PIGF PNRC1 POLQ PRKAR1A PRKAR1A PSMB1 PSMB2 PSMB5 PTPRD RARA RARB RARG ROBO1 ROBO2 SSTR2 NUP93 MALAT1 RNASEL SRC SUZ12 XPO1 NSCLC patients were analyzed for mutations in the 508 cancer-associated genes listed in Table 1. Accounting for all patients, the top 21 mutated genes were TP53 (65%), EGFR (40%), KMT2D (15%), KRAS (12.5%), CDKN2A (12.5%), FAT3 (10%), PTEN (10%), DDR2 (10%), SMAD4 (10%), ARIDA (10%), RB1 (10%), PIK3CA (10%), STK11 (7.5%), MAP3K1 (7.5%), MPL (7.5%), TNFRSF8 (7.5%), FBXW7 (7.5%), FGFR3 (7.5%), NOTCH1 (7.5%), GRIN2A (7.5%) and ROS1 (7.5%) (Fig. 1b). For the top two most frequently mutated genes TP53 and EGFR, we summarized the detailed mutation types. The top four EGFR alterations were: L858R point mutation in exon 21, 43.75%; EGFR copy number again, 37.5%; deletion in exon 19 (746_750delELREA), 25%; and T790M point mutation, 12.5% (Fig. 1c). This mutational distribution is consistent with the results of the Catalogue of Somatic Mutations in Cancer (COSMIC) data base (Fig. 1d), in which >95% of samples with EGFR mutations are lung cancers. Unlike the EGFR 490

4 Next-generation sequencing of Chinese stage IV lung cancer patients Fig. 1. Overall characteristics of stage IV non-small cell lung cancer (NSCLC) patients and detailed analysis of epidermal growth factor receptor (EGFR) andtp53 mutation types. (a) Pathological subtypes of all 80 NSCLC patients in this study. (b) Top 21 mutation genes of all 80 patients with 508 cancer-associated gene analyses. Different mutation type rates of EGFR in this study (c) compared to the Catalogue of Somatic Mutation in Cancer (COSMIC) database (d). Different mutation type rates of TP53 in this study (e) compared to the COSMIC database (f). gene, which shows a more limited number of mutational hotspots characteristic of oncogenic driver (i.e. gain-of-function) mutations, the tumor suppressor gene TP53 has a much more diverse array of inactivating (i.e. loss-of-function) mutations documented. Therefore, the mutant type, rates and diversity of TP53 found in this study were generally consistent with the results of TP53 mutations documented in the COSMIC database (Fig. 1e,f). Notably, the overall frequency of both EGFR and TP53 mutations was significantly higher in our NSCLC patients compared with patients in the COS- MIC database, which can largely be attributed to racial differences between the two cohorts (4, 5). EGFR mutations are associated with NSCLC patient prognosis We next performed a survival analysis to determine whether EGFR or TP53 mutations had any prognostic significance in our stage IV NSCLC patient cohort. The 3-, 9-, and 11-month OS rates of all 80 patients were 96%, 59% and 25%, respectively, and the median survival time of all patients was 10.0 months (Fig. 2a). At the end of follow-up time, 34 patients had died because of tumor progression and/or multiple organs failure. As shown in Fig. 2b, The 9- and 11-month OS rates in patients harboring EGFR mutant tumors were 36% and 18%, respectively, while the rates in EGFR wild-type patients were 70% and 29%, respectively (p = ). The median survival time of EGFR-mutated patients and EGFR wild-type patients were 9.0 and 10.0 months [95% confidence interval (CI): ]. The 9- and 11-month OS rates in TP53 mutant patients were 57% and 28%, and the rates in TP53 wild-type patients were 61% and 22%, respectively, as shown in Fig. 2c (p = ). The median survival time of the two groups of TP53 mutant patients and TP53 wild-type patients were both 10.0 months 491

5 Li et al. Fig. 2. Survival analysis of patients in this study using log-rank (Mantel Cox) test. (a) Overall survival curve of all 80 patients. (b) Survival comparison of epidermal growth factor receptor (EGFR) mutant patients and EGFR wild-type patients. (c) Survival comparison of TP53 mutant patients and TP53 wild-type patients. (d) Survival comparison of L858R mutation patients and not-l858r mutation patients of all 32 EGFR mutant patients. (e) Survival comparison of 746_750delELREA mutation patients and not-746_750delelrea mutation patients of all 32 EGFR mutant patients. [95% (CI): ]. Thus, while EGFR mutations were associated with significantly shorter survival, TP53 status was not associated with significant survival differences. Moreover and interestingly, we found that smoking status in this patient cohort is related to EGFR mutation incidence, but not to incidence of TP53 mutations, as shown in Table S2 (p = and p = 0.566, respectively). For the most frequently observed somatic EGFR mutation L858R and 746_750delELREA, we performed a separate survival analysis. As shown in Fig. 2d, patients with the L858R mutation showed a significantly worse survival time than patients with tumors harboring other EGFR mutations. The median OS time of EGFR-L858R patients and EGFR-not L858R patients were 7.0 and 9.0 months, respectively [95% (CI): ] (p = 0.036). By contrast, patients with the 746_750delELREA mutation showed a significantly longer OS, as the median survival time of EGFR-746_750delELREA patients and EGFR-not 746_750delELREA patients were 11.0 and 7.0 months, respectively [95% (CI): ] (p = 0.040) (Fig. 2e). Discussion Lung cancer is the leading cause of cancer death worldwide and late diagnosis is a major obstacle to improving lung cancer outcomes (6, 7). With the development of next-generation sequencing technology, the landscape in lung cancer is rapidly changing, allowing for the discovery and identification of new molecular targets and new therapies. In our stage IV NSCLC cohort, EGFR and TP53 were the most frequently mutated genes, a result consistent with a recent study by Wu et al. (8, 9). Furthermore, the most commonly observed EGFR mutations were L858R, 746_750delELREA and T790M, which was consistent with the COSMIC database. The molecular characteristics of lung tumors play an important role in clinical decisions, which ultimately affect patients survival. We found that patients with EGFR mutations had a significant shorter OS time compared with patients without EGFR mutation. This result was consistent with previous studies (10, 11). However, other studies have suggested that in NSCLC patients who are not receiving EGFR-inhibitor therapy, expression of EGFR is not a prognostic factor (12, 13). The latter studies included all four stages of NSCLC patients and only studied a very small population of stage IV patients, which may explain the differences between our findings. Interestingly, we also found that patients with L858R mutations had a significantly shorter survival time compared with patients harboring other EGFR mutations. Although this has not been previously reported, EGFR L858R mutation is known to be predictive of treatment benefit from EGFR tyrosine kinase inhibitors (14). By contrast, patients with EGFR-746_750delELREA mutations had a longer OS time compared with patients with other EGFR mutations. We could not find any studies corroborating this finding in the literature; however, this study is based on a small population, and larger population studies are needed to confirm this result. TP53 mutations occur frequently in lung cancer (15). However, we found no significant survival differences 492

6 Next-generation sequencing of Chinese stage IV lung cancer patients between patients bearing TP53 mutant or wild-type tumors, confirming results of recent studies (16, 17). Some studies have reported that TP53 mutations are significantly associated with a poor prognosis in NSCLC patients (18, 19). However, this worsening of OS was reported for stages I III NSCLC patients and not for stage IV patients. Furthermore, the role of TP53 mutations in response to therapies remains unclear. For example, patients with wild-type TP53 showed significantly better OS and response to cisplatin doublet therapy compared with TP53 mutant patients (20). Large-scale studies need to be designed to confirm its prognostic role in NSCLC. In conclusion, patients with NSCLC showed multiple cancer-associated somatic mutations, with EGFR and TP53 being the most frequently mutated genes observed. EGFR mutations, but not TP53 mutations, were shown to be a poor prognostic factor for OS of stage IV NSCLC patients. This finding indicates that EGFR mutations play an important role in the tumorigenesis of stage IV NSCLC and likely drives tumor aggressiveness, suggesting that treatment with EGFR inhibitors or other EGFR-targeted therapies may be most beneficial for NSCLC patients. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. Acknowledgements This work was supported by Tianjin HengJia Biotechnology Development Co., Ltd. and Tianjin Beichen Hospital. The authors declare that they have no competing interests. References 1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008: 359: American Cancer Society. Cancer facts & figures, 2011, from documents/document/acspc pdf. Accessed on June 24, Sequist LV, Heist RS, Shaw AT et al. Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice. Ann Oncol 2011: 22: Mitsudomi T. Molecular epidemiology of lung cancer and geographic variations with special reference to EGFR mutations. Transl Lung Cancer Res 2014: 3 (4): Parikh P, Puri T. Personalized medicine: Lung Cancer leads the way. Indian J Cancer 2013: 50 (2): Carney DN. Lung cancer-time to move on from chemotherapy. N Engl J Med 2002: 346: Chute JP, Chen T, Feigal E et al. Twenty years of phase III trials for patients with extensive-stage small cell lung cancer: perceptible progress. J Clin Oncol 1999: 17: Wu K, Zhang X, Li F et al. Frequent alterations in cytoskeleton remodeling genes in primary and metastatic lung adenocarcinomas. Nat Commun 2015: 6: Wang Z, Potter CS, Sundberg JP et al. SHARPIN is a key regulator of immune and inflammatory responses. J Cell Mol Med 2012: 16: Veale D, Ashcroft T, Marsh C et al. Epidermal growth factor receptors in non-small-cell lung cancer. Br J Cancer 1987: 55: Lee JS, Ro JY, Sahin A et al. Expression of epidermal growth factor receptor (EGFR): a favorable prognostic factor for surgically resected non-small cell lung cancer. Proc Am Soc Clin Oncol 1989: 8: A Meert AP, Martin B, Delmotte P et al. The role of EGFR expression on patient survival in lung cancer: a systematic review with meta-analysis. Eur Respir J 2002: 20: Tsao MS, Sakurada A, Cutz JC et al. Erlotinib in lung cancer molecular and clinical predictors of outcome. N Engl J Med 2005: 353 (2): Cheng L, Alexander RE, Maclennan GT et al. Molecular pathology of lung cancer: key to personalized medicine. Mod Pathol 2012: 25 (3): Viktorsson K, De Petris L et al. The role of p53 in treatment responses of lung cancer. Biochem Biophys Res Commun 2005: 331: Ma X, Le Teuff G, Lacas B et al. Prognostic and predictive effect of TP53 mutations in non-small-cell lung cancer patients from adjuvant cisplatin-based therapy randomized trials: a LACE-bio pooled analysis. J Thorac Oncol 2016: 11 (6): Scoccianti C, Vesin A, Martel G et al. Prognostic value of TP53, KRAS and EGFR mutations in non-small-cell lung cancer: the EUELC cohort. Eur Respir J 2012: 40: Mogi A, Kuwano H. TP53 mutations in non-small-cell lung cancer. J Biomed Biotechnol 2011: 2011: Ahrendt SA, Hu Y, Buta M et al. P53 mutations and survival in stage I non-small-cell lung cancer: results of a prospective study. J Natl Cancer Inst 2003: 95: Tsao MS, Aviel-Ronen S, Ding K et al. Prognostic and predictive importance of p53 and RAS for adjuvant chemotherapy in non-small-cell lung cancer. J Clin Oncol 2007: 25: