Amaya Gasco

Nondisruptive p53 Mutations Are Associated with Shorter Survival in Patients with Advanced Non–Small Cell Lung Cancer

Purpose: TP53 mutations in early-stage non–small cell lung cancer (NSCLC) may be associated with worse survival but their prognostic role in advanced NSCLC is controversial. In addition, it remains unclear whether mutated patients represent a clinically homogeneous group. Experimental Design: We retrospectively examined TP53 mutations and outcome in a training cohort of 318 patients with stage IIIB–IV NSCLC: 125 epidermal growth factor receptor (EGFR) wild-type (wt) and 193 EGFR mutated (mut). An independent validation cohort of 64 EGFR-mut patients was subsequently analyzed. Mutations were classified as “disruptive” and “nondisruptive” according to their predicted degree of disturbance of the p53 protein structure and function. Results: In the training cohort, TP53 mutations were found in 43 of the 125 EGFR-wt patients (34.4%). Of these, 28 had nondisruptive TP53 mutations and a median overall survival (OS) of 8.5 months, compared with 15.6 months for the remaining 97 patients (P = 0.003). In the EGFR-mut group, TP53 mutations were found in 50 of the 193 patients (25.9%). The OS for the 26 patients with TP53 nondisruptive mutations was 17.8 months versus 28.4 months for the remaining 167 patients (P = 0.04). In the validation cohort, the 11 patients with nondisruptive TP53 mutations had a median OS of 18.1 months compared with 37.8 months for the 53 remaining patients (P = 0.006). In multivariate analyses, nondisruptive TP53 mutations had an independent, significant association with a shorter OS. Conclusions: Nondisruptive mutations in the TP53 gene are an independent prognostic factor of shorter survival in advanced NSCLC. Clin Cancer Res; 20(17); 4647–59. ©2014 AACR.

Adaptive resistance to targeted therapies in cancer

It is widely acknowledged that there is a need for molecular profiling in non-small-cell lung cancer. For example, treatment based on EGFR mutation status has attained successful results. However, in spite of excellent initial response to oral EGFR tyrosine kinase inhibitors (TKIs), progression-free survival is still limited. Current research has focused mostly on acquired resistance mechanisms, such as overexpression of AXL and loss of the Mediator MED12. In this review, in contrast, we discuss adaptive, rather than acquired, resistance. Adaptive resistance can occur almost immediately after starting targeted therapy through a rapid rewiring of cancer cell signaling. By losing ERK negative feedback on receptor tyrosine kinase (RTK) expression, cancer cells are exposed to the stimuli of several ligands, and the ensuing activation of several RTKs reprograms all the canonical signaling pathways. The overexpression of several RTKs was observed in breast cancer cell lines treated with a MEK inhibitor and in BRAF(V600E) melanoma cell lines treated with BRAF inhibitors. This rebound effect of overexpression of several RTKs, including ERBB3, also occurs in lung cancers driven by Kras or EGFR mutations when treated with MEK, PI3K or dual PI3K/mTOR inhibitors. Synthetic lethality can be effectively induced by co-targeting these overexpressed RTKs. We speculate that in patients with EGFR mutations, adaptive resistance occurs in a significant proportion of patients. Rebiopsies performed hours after starting treatment with EGFR TKIs can identify which RTKs are overexpressed after treatment. Efficient co-targeting of these RTKs can induce synthetic lethality and help overcome the limited effect of EGFR TKI monotherapy.

BRCA1, LMO4, and CtIP mRNA Expression in Erlotinib-Treated Non–Small-Cell Lung Cancer Patients with EGFR Mutations

Introduction: Lung adenocarcinoma patients harboring EGFR activating mutations attain improved progression-free survival (PFS) with treatment with epidermal growth factor receptor tyrosine kinase inhibitors. However, patients ultimately relapse, indicating that other genetic factors could influence outcome in such patients. We hypothesized that PFS could be influenced by the expression of genes in DNA repair pathways. Methods: We examined the mRNA expression of C terminus-binding protein–interacting protein and Lin11, Isl-1, and Mec-3 domain only 4 (LMO4) in pretreatment tumor samples from 91 erlotinib-treated advanced non–small-cell lung cancer patients with EGFR mutations in whom breast cancer gene 1 (BRCA1) expression and the concomitant presence of the EGFR T790M mutation had previously been assessed. Gene expression was analyzed by polymerase chain reaction, using &bgr;-actin as endogenous gene. Results were correlated with PFS and overall survival. Results: In patients with low LMO4 levels, PFS was 13 months, whereas it was not reached for those with high LMO4 levels (p = 0.03). In patients with low levels of both BRCA1 and LMO4, PFS was 19 months whereas it was not reached in those with low BRCA1 and high LMO4 mRNA levels (p = 0.04). In patients with high BRCA1 and low LMO4 levels, PFS was 8 months, whereas it was 18 months in those with high levels of both genes (p = 0.03). Conclusions: Low BRCA1 and high LMO4 levels were associated with longer PFS to erlotinib. Baseline assessment of BRCA1 and LMO4 mRNA expression can help predict outcome to erlotinib.

CK-coated magnetic-based beads as a tool to isolate circulating tumor cells (CTCs) in human tumors

Circulating tumor cells (CTCs) can be detected in the blood of many cancer patients and play a key role in metastasis. In addition, after the development of technologies with the necessary sensitivity and reproducibility, the diagnostic potential of these cells is being actively explored. Recently, the U.S. Food and Drug Administration has approved the CellSearch(®) System, based on magnetic beads coated with epithelial cell-adhesion molecule (EpCAM) antibody. Despite its usefulness, this system can miss CTCs that lose epithelial antigens due to the epithelial-mesenchymal transition and, in the case of advanced NSCLC, CTCs positivity can be demonstrated only in 30-50% of patients. In an effort to overcome these drawbacks, new methods are being developed. In this study, we have evaluated CK-coated beads as a system to isolate CTCs from lung cancer patients in the clinical setting, and have evaluated if they can be a useful source of material for genetic testing. We were able to identify CTCs in 17 of the 30 patients included in the study (57%), with a range of 1 to 7 cells. In two of them, we found only CTCs with an EMT pattern. CTC positivity seemed to correlate with the clinical history of the malignancy. CTCs could be detected in more than 80% of stage III-IV lung cancer patients at presentation or in blood samples taken immediately after surgery. The percentage dropped to 13% in patients responding to chemotherapy or TKIs, raising again to 57% after tumor progression. Finally, we tested the CTCs isolated from 8 patients for EGFR and k-ras mutations, but gene amplification was successful only in the 3 patients with 4 or more CTCs.

肿瘤患者循环肿瘤细胞(CTCs)的分离工具：细胞角蛋白包被磁珠

多种癌症患者的外周血中均可检测到循环肿瘤细胞(circulating tumourcells，CTCs)，它在肿瘤 转移过程中发挥关键作用。此外，伴随CTCs检测技术的逐步发展，其敏感度和可重复性逐步 提高，进而可充分发挥其在肿瘤诊断中的潜能。最近，美国食品和药物管理局(food and drug administration，FDA)已批准CellSearch®检测系统用于检测外周血CTCs，该系统采用的是上皮细胞 粘附分子(EpCAM)抗体包被的磁珠。尽管它能有效检测出CTCs，但同样可因为上皮—间质转化 中上皮抗原的丢失而错失CTCs，数据显示只有30%~50%的晚期NSCLC外周血CTCs检测阳性。为 了克服这些缺点，研究人员正探索新的检测方法。本研究建立了细胞角蛋白(CK)包被的磁珠检测 系统来分离肺癌临床病例外周血中的CTCs，并评估该系统所分离的CTCs是否可作为基因检测的 有效材料来源。纳入研究的30例患者中，17例检测到CTCs (57%)，细胞数从1~7个，其中2例患者 中，仅存在EMT形式的CTCs。CTCs检测阳性率与恶性肿瘤的临床病史相关。在III-IV期肺癌患者 首诊时或术后血标本中，超过80%可检测到CTCs。对化疗或TKIs治疗有效的患者，其检测阳性率 下降到13%，肿瘤进展后又上升至57%。最后，我们对其中8例患者的CTCs进行EGFR和K-ras突变 检测，但只有3例CTCs≥4个的患者基因扩增成功。

Abstract 53: FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung.

SRY-related HMG-box (SOX2) gene is a key transcription factor that coordinates development, differentiation and self-renewal of normal non-alveolar epithelium of the airway. SOX2 amplification has been reported in lung squamous cell carcinoma (SCC). Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that, upon activation, promotes cell proliferation, survival and apoptotic resistance through PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is also amplified in 15-25% of lung SCC and pre-clinical data with targeted inhibitors showed a growth dependency on FGFR1 amplification either in vitro and in vivo. A clinical trial with BIBF1120 (which also inhibits FGFR1), will be developed in the Netherlands and Spain in second line SCC of the lung with FGFR1 amplified by FISH. Genetic heterogeneity in solid tumors is a major research area and therefore its assessment in SCC of the lung is of great relevance. We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC by multiplex ligation-dependent probe amplification (MLPA). Analysis of mutations in DDR2, PIK3CA, BRAF, EGFR, KRAS, and TP53 is ongoing. Genomic DNA (gDNA) was isolated from enriched tumor cells by laser captured microdisection from formalin-fixed paraffin embedded (FFPE) tumor tissue sections. 50-100 ng of gDNA was analyzed in each of the three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN value comparisons, the tumor results from each patient was normalized against the GCN values derived from FFPE peripheral blood leukocytes control. We also used MLPA technique to study intra-tumor heterogeneity (TH) by analyzing FGFR1 and SOX2 in different tumor areas. In particular, in 4 cases FGFR1 and SOX2 were co-amplified. Also, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression in two patients. Of the 76 patients evaluable for FGFR1 and SOX2 GCN, FGFR1 high amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%). SOX2 presented high amplification in 38/63 (60.32%) and low amplification in 9/63 (14.28%). Interestingly, 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. An important degree of TH was found in 2 of the 4 tumors analyzed. GCN changes in FGFR1, SOX2, PIK3CA, PDGFRa, KDR, EGFR and MET over 10 years follow-up will be presented for one surgically resected SCC lung cancer patient. Frequencies of FGFR1 and SOX2 gene amplifications detected are in agreement with previously published data. Survival according to FGFR1 and SOX2 status will also be presented. FGFR1 and SOX2 co-amplification, together with the TH, warrants further research and could represent a novel therapeutic target. Citation Format: Pedro Mendez, Jose L. Ramirez, Amaya Gasco, Teresa Moran, Enric Carcereny, Miquel Taron, Jose Luis Mate, Irene Sansano, Maria Perez, Monica Botia, Montserrat Tierno, Harry J.M Groen, Rafael Rosell. FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung. [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 53. doi:10.1158/1538-7445.AM2013-53

Abstract 1727: Correlation between BRCA1 and LMO4 mRNA expression levels in Erlotinib-treated non small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background Advanced NSCLC p harboring EGFR mutations show impressive progression-free survival (PFS) when treated with erlotinib. However, presence of EGFR mutations only imperfectly predicts outcome. We demonstrated that co-existence of the EGFR T790M pretreatment mutation was detected in one-third of p and, in conjunction with elevated BRCA1 mRNA levels, dramatically influenced PFS to erlotinib. Though this model is a breakthrough in outcome of EGFR-mutated NSCLC p treated with TKIs, we choose to analyze the impact of CTIP and LMO4 gene expression levels on p outcome and their correlation with BRCA1. CtIP binds to BRCA1 and LMO4 forming a complex. LMO4 is a negative regulator of BRCA1 function in sporadic breast cancers. Methods mRNA expression of LMO4 and CTIP was examined by RT-PCR in pretreatment tumor biopsies of 72 NSCLC p with EGFR mutations treated with erlotinib. Expression levels were correlated with outcome to erlotinib and BRCA1 expression levels. Results BRCA1 significantly correlated with CtIP (r=0.31;P=0.01) and LMO4 (r=0.32;P=0.02). There was no correlation between CtIP and LMO4 (r=0.09;P=0.49).PFS was not reached for p with high levels of LMO4 vs 13 months for p with low levels (P=0.006). Overall survival (OS) was not reached for p with high levels of LMO4 vs 29 months for p with low levels (P=0.10). Expression levels of CtIP did not correlate with PFS and OS. PFS was not reached for p with low levels of BRCA1 and high levels of LMO4 vs 19 months for p with low levels of both genes (P=0.04). PFS was 8 months for p with high levels of BRCA1 and low levels of LMO4 vs 18 months for p with high levels of both genes (P=0.03). Conclusions BRCA1 and LMO4 mRNA levels examined by RT-PCR could predict PFS to erlotinib in p with EGFR mutations and could be useful for development of new therapeutic strategies. ![Figure][1] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1727. doi:1538-7445.AM2012-1727 [1]: pending:yes

Differential expression of BRCA1 and genes involved in the nuclear factor kappa B (NFκB) and notch signalling pathways in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients (p).

e21025 Background: Genetic diversity in lung cancer according to histological subtype has not been fully explored. BRCA1 and RAP80 influence response to chemotherapy. Musashi 2 activates HES-1 in the Notch pathway, and HES-1 can abrogate CYLD. A20, AEG-1, EZH2 and TRAF6 are also involved in NFkB activation. We have examined mRNA expression of BRCA1, RAP80 and components of the NFkB and Notch pathways in lung cancer p. METHODS mRNA expression of Musashi 2, CYLD, HES-1, A20, EZH2, AEG-1, TRAF6, NFKBIA, RelA, BRCA1 and RAP80 was analyzed by quantitative RT-PCR in tumor samples from 85 lung cancer p (77 NSCLC, 8 SCLC). RESULTS p characteristics: 51 males; median age, 59; 33 smokers, 26 ex-smokers; 49 adenocarcinoma, 10 large cell carcinoma (LCC), 18 squamous cell carcinoma (SCC), 8 SCLC. Source of tumor sample: 36 primary tumor, metastasis, 15. 11 p had K-ras mutations. There were differences in the expression levels of Musashi 2, BRCA1, EZH2 and RAP80 according to histological subtype. The median Musashi 2 mRNA expression was 4 times higher in SCLC than in NSCLC (adenocarcinoma, SCC, LCC) (P<0.001); BRCA1 expression was 3 times higher (P<0.001); EZH2 was 7 times higher (P<0.001); RAP80 was 2 times higher (P=0.011). There were no differences in expression levels of any of the 11 genes analyzed according to p age, smoking history or source of the tumor sample. NSCLC p with K-ras mutations had higher expression of both AEG-1 and NFKBIA than p with wild-type K-ras (P=0.04). CONCLUSIONS Musashi 2, BRCA1, EZH2 and RAP80 expression was significantly higher in SCLC p. Further investigation is warranted to examine the potential association between expression of these genes and response to platinum regimens and radiotherapy in SCLC. Elevated AEG-1 levels associated with K-ras-mutant tumors could identify a subgroup of NSCLC p with poor prognosis.

The nuclear factor kB (NFkB) and Notch signaling pathways and BRCA1 mRNA expression in stage IV non-small cell lung cancer (NSCLC) patients (p).

7586 Background: Little is known about the potential effect of genetic alterations in the NFkB and Notch pathways on NSCLC p. Musashi 2 activates HES-1 in the Notch pathway, and HES-1 can abrogate CYLD. A20, AEG-1, EZH2 and TRAF6 are also involved in NFkB activation. BRCA1 and RAP80 are modulators of cisplatin-based chemotherapy. Mutations in NFKBIA and DUSP22, which prevent NFkB activation, were described in the sequencing exome of a single NSCLC p, together with K-ras mutations. METHODS mRNA expression of Musashi 2, CYLD, HES-1, A20, EZH2, AEG-1, TRAF6, NFKBIA, RelA, BRCA1 and RAP80 was analyzed by quantitative RT-PCR in tumor samples from 60 advanced NSCLC p. Expression levels by terciles were correlated with clinical characteristics and outcome to chemotherapy. Mutations in NFKBIA and DUSP22 were sequenced in 28 and 21 patients, respectively, and in 12 cancer cell lines. RESULTS p characteristics: 36 male; 39 adenocarcinomas; 22 smokers; 23 bone metastases; 9 EGFR mutations; 10 K-ras mutations. No NFKBIA or DUSP22 mutations were observed in any of the p or cell lines. PFS was 12.3 months (m) for p in the lowest tercile of AEG-1 expression vs 9.3 m for p in the intermediate tercile and 4.8 for p in the highest tercile (P=0.002). The multivariate analysis showed that only AEG-1 expression was associated with shorter PFS (HR, 1.43; P=0.006). Expression levels of the other genes did not correlate with outcome. However, we had previously generated a two-gene risk model based on AEG-1 and BRCA1 expression: p with high levels of both genes are considered high-risk, p with low levels of both genes are low-risk, and p with high levels of one and low levels of the other gene are intermediate-risk. In the present study, PFS was 13 m in the low-risk group, while it was 7.6 m for the intermediate-risk group and 5.3 m for the high-risk group (P=0.02). CONCLUSIONS NSCLCs have variegated gene expression. AEG-1 and BRCA1 mRNA expression is a genetic signature that can be used as a prognostic model for the management of NSCLC p.

Astrocyte elevated gene 1 (AEG-1) mRNA expression in non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations.

10541 Background: NSCLC p with EGFR mutations respond to the oral EGFR tyrosine kinase inhibitors erlotinib and gefitinib, although the duration of response in individual p is unpredictable. In our previous study, we found that erlotinib-treated p with low BRCA1 mRNA levels had a progression-free survival (PFS) of 27 months (m) vs 10 m for p with high levels (P=0.02). AEG-1 is a multifunctional oncogene that plays a role in several carcinogenic processes. Through PI3K/Akt, AEG-1 activates IKK, leading to phosphorylation and destabilization of the NF-kB inhibitor NFKBIA, which is a gatekeeper for EGFR signaling. METHODS Tumor mRNA expression levels of BRCA1 and AEG-1 were assessed by quantitative PCR in 77 erlotinib-treated p with EGFR mutations. Expression levels were dichotomized at the median. RESULTS PFS was longer in p with low AEG-1 expression (27 vs 12 m; P=0.003). Median survival (MS) was not reached for p with low AEG-1 levels and was 24 m for p with high levels (P=0.08). Based on these results, we generated an AEG-1/BRCA1 risk model: p with high levels of both genes were considered high-risk, p with low levels of both genes were low-risk, and p with high levels of one and low levels of the other gene were intermediate-risk. PFS was not reached in the low-risk group, while it was 18 m for the intermediate-risk group and 8 m for the high-risk group (P=0.00006) (HR for high- vs low-risk groups, 6.6; 95%CI, 2-4-18; P<0.00001). MS was not reached in the low-risk group, while it was 31 m for the intermediate-risk group and 18 m for the high-risk group (P=0.05). In the multivariate analysis for PFS, the only independent prognostic variables were bone metastases (HR, 2.7; 95%CI, 1.1-6.5; P=0.03) and the AEG-1/BRCA1 risk groups (HR for high-risk group, 7.7 (95%CI, 2.8-21.3; P<0.00001). CONCLUSIONS The two-gene risk model based on AEG-1 and BRCA1 mRNA expression was strongly associated with clinical outcome, with significantly shorter PFS and MS in the high-risk group. This model can be useful for the proper individualized management of p with EGFR mutations.