Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

Background

Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be ‘major’, characterized by a single tumor showing two different histologic types, and ‘minor’, due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas.

Methods

590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR.

Results

10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components.

Conclusion

Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes.     

MMP11在肺鳞癌和腺癌组织中的表达及与预后的关系

MMP11属于MMPs家族,它与肿瘤的浸润转移密切相关.探讨MMP11在肺鳞癌和腺癌组织中的表达,及其与浸润转移和预后的关系.应用免疫组化检测50例肺鳞癌和38例肺腺癌组织中MMP11蛋白的表达.结果显示,MMP11在肺鳞癌和腺癌组织中的阳性率分别为68.0%(34/50)和73.7%(28/38)(P=0.563).MMP11的阳性率随着肺癌T分期的增加、淋巴结转移和TNM分期的增加而增加(P<0.05).Kaplan-meier单因素生存分析显示,MMP11表达(P=0.001)、T分期(P=0.009)、淋巴结转移(P<0.001)和TNM分期(P<0.001)对生存期的影响有统计学意义.Cox多因素分析显示,只有TNM分期(P=0.028)是肺癌的独立预后危险因素,MMP11(P=0.105)不是肺癌的独立预后危险因素.MMP11的表达与肺癌的浸润、转移及生存期密切相关.     

Gene Expression Profiling in Human Lung Development: An Abundant Resource for Lung Adenocarcinoma Prognosis

A tumor can be viewed as a special “organ” that undergoes aberrant and poorly regulated organogenesis. Progress in cancer prognosis and therapy might be facilitated by re-examining distinctive processes that operate during normal development, to elucidate the intrinsic features of cancer that are significantly obscured by its heterogeneity. The global gene expression signatures of 44 human lung tissues at four development stages from Asian descent and 69 lung adenocarcinoma (ADC) tissue samples from ethnic Chinese patients were profiled using microarrays. All of the genes were classified into 27 distinct groups based on their expression patterns (named as PTN1 to PTN27) during the developmental process. In lung ADC, genes whose expression levels decreased steadily during lung development (genes in PTN1) generally had their expression reactivated, while those with uniformly increasing expression levels (genes in PTN27) had their expression suppressed. The genes in PTN1 contain many n-gene signatures that are of prognostic value for lung ADC. The prognostic relevance of a 12-gene demonstrator for patient survival was characterized in five cohorts of healthy and ADC patients [ADC_CICAMS (n = 69, p = 0.007), ADC_PNAS (n = 125, p = 0.0063), ADC_GSE13213 (n = 117, p = 0.0027), ADC_GSE8894 (n = 62, p = 0.01), and ADC_NCI (n = 282, p = 0.045)] and in four groups of stage I patients [ADC_CICAMS (n = 22, p = 0.017), ADC_PNAS (n = 76, p = 0.018), ADC_GSE13213 (n = 79, p = 0.02), and ADC_qPCR (n = 62, p = 0.006)]. In conclusion, by comparison of gene expression profiles during human lung developmental process and lung ADC progression, we revealed that the genes with a uniformly decreasing expression pattern during lung development are of enormous prognostic value for lung ADC.     

Comparison of Multi-Slice Computed Tomographic Angiography and Dual-Source Computed Tomographic Angiography in Resectability Evaluation of Pancreatic Carcinoma

The assessment of pancreatic cancer resectability is based mainly on the extent of the peripancreatic vasculature involvement with tumor mass. The 16-slice computed tomography (MSCT) and dual-source computed tomography (DSCT) were used in non-invasive imaging of the pancreas and the regional vessels in 48 pancreatic carcinoma patients. Both of these techniques were combined with contrast-enhanced angiography and post-scanning reconstruction of 2D and 3D images. Based on the degree of involvement revealed by these images, the pre-operative tumor resectability was determined. The CTA-based resectability was then correlated with the surgical and pathological findings for the evaluation of their sensitivity, specificity, negative and positive predictive values, and diagnostic accuracy. The study suggests that resectability based on dual-source CTA showed higher sensitivity, specificity, and diagnostic accuracy than that obtained from MSCTA scanning.     

Quantitative Mass Spectrometry to Interrogate Proteomic Heterogeneity in Metastatic Lung Adenocarcinoma and Validate a Novel Somatic Mutation CDK12-G879V

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Highlights

• •Serial biopsies and autopsy from a metastatic lung cancer patient over 7 years.
• •Tumor heterogeneity characterized by quantifying the proteome and phosphoproteome.
• •Patient-specific database built using whole genome sequencing data from tumors.
• •MRM assay and functional validation of a novel lung-specific CDK12-G879V mutant.

     

Ion Channel Gene Expression in Lung Adenocarcinoma: Potential Role in Prognosis and Diagnosis

Ion channels are known to regulate cancer processes at all stages. The roles of ion channels in cancer pathology are extremely diverse. We systematically analyzed the expression patterns of ion channel genes in lung adenocarcinoma. First, we compared the expression of ion channel genes between normal and tumor tissues in patients with lung adenocarcinoma. Thirty-seven ion channel genes were identified as being differentially expressed between the two groups. Next, we investigated the prognostic power of ion channel genes in lung adenocarcinoma. We assigned a risk score to each lung adenocarcinoma patient based on the expression of the differentially expressed ion channel genes. We demonstrated that the risk score effectively predicted overall survival and recurrence-free survival in lung adenocarcinoma. We also found that the risk scores for ever-smokers were higher than those for never-smokers. Multivariate analysis indicated that the risk score was a significant prognostic factor for survival, which is independent of patient age, gender, stage, smoking history, Myc level, and EGFR/KRAS/ALK gene mutation status. Finally, we investigated the difference in ion channel gene expression between the two major subtypes of non-small cell lung cancer: adenocarcinoma and squamous-cell carcinoma. Thirty ion channel genes were identified as being differentially expressed between the two groups. We suggest that ion channel gene expression can be used to improve the subtype classification in non-small cell lung cancer at the molecular level. The findings in this study have been validated in several independent lung cancer cohorts.     

DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations

The specific mechanisms how lung cancer cells harboring epidermal growth factor receptor (EGFR) activating mutations can survive treatment with EGFR-tyrosine kinase inhibitors (TKIs) until they eventually acquire treatment-resistance genetic mutations are unclear. The phenotypic diversity of cancer cells caused by genetic or epigenetic alterations (intratumor heterogeneity) confers treatment failure and may foster tumor evolution through Darwinian selection. Recently, we found DDX3X as the protein that was preferentially expressed in murine melanoma with cancer stem cell (CSC)-like phenotypes by proteome analysis. In this study, we transfected PC9, human lung cancer cells harboring EGFR exon19 deletion, with cDNA encoding DDX3X and found that DDX3X, an ATP-dependent RNA helicase, induced CSC-like phenotypes and the epithelial-mesenchymal transition (EMT) accompanied with loss of sensitivity to EGFR-TKI. DDX3X expression was associated with upregulation of Sox2 and increase of cancer cells exhibiting CSC-like phenotypes, such as anchorage-independent proliferation, strong expression of CD44, and aldehyde dehydrogenase (ALDH). The EMT with switching from E-cadherin to N-cadherin was also facilitated by DDX3X. Either ligand-independent or ligand-induced EGFR phosphorylation was inhibited in lung cancer cells that strongly expressed DDX3X. Lack of EGFR signal addiction resulted in resistance to EGFR-TKI. Moreover, we found a small nonadherent subpopulation that strongly expressed DDX3X accompanied by the same stem cell-like properties and the EMT in parental PC9 cells. The unique subpopulation lacked EGFR signaling and was highly resistant to EGFR-TKI. In conclusion, our data indicate that DDX3X may play a critical role for inducing phenotypic diversity, and that treatment targeting DDX3X may overcome primary resistance to EGFR-TKI resulting from intratumor heterogeneity.     

人原发性肺腺癌转移相关分子的定量蛋白质组学研究

癌细胞转移是人原发性肺腺癌(lung adenocarcinoma, AdC)死亡率高和预后差的主要原因．为了筛选潜在的肺腺癌转移相关分子标志物,依据临床诊断选取无转移的肺腺癌组织和有转移的肺腺癌组织作为研究对象,首先采用激光捕获显微切割技术(laser capture microdissection, LCM)对两组肺腺癌组织中的癌细胞进行纯化,再利用荧光差异凝胶电泳技术(two-dimensional differential in-gel electrophoresis, 2D-DIGE)分离无转移肺腺癌组和有转移肺腺癌组的癌细胞总蛋白,通过Decyder软件分析两组差异表达的蛋白质点,质谱(mass spectrometry, MS)对差异表达的蛋白质点进行鉴定,Western blot验证部分差异蛋白annexin A1, annexin A2, annexin A3, B23和 S100A9的表达．建立了LCM 纯化的无转移和有转移的肺腺癌组织癌细胞的2D-DIGE图谱,质谱鉴定了20个非冗余差异蛋白质,其中12个蛋白质在有转移肺腺癌组中较无转移肺腺癌组表达上调,8个蛋白质在有转移肺腺癌组中表达下调．Western blot验证分析显示,差异蛋白annexin A1,annexin A2,annexin A3和 S100A9的表达水平在有转移肺腺癌中较无转移肺腺癌增高,B23的表达水平在有转移肺腺癌中较无转移肺腺癌降低．免疫组化进一步证实S100A9在有转移的肺腺癌中较无转移的肺腺癌中表达上调．首次应用LCM技术联合2D-DIGE及MS技术分析鉴定出肺腺癌转移相关蛋白质,为研究肺腺癌的转移分子机制、筛选预测肺腺癌转移的分子标志物奠定了基础．     

The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas.     

基于WGCNA和SVM-RFE算法挖掘肺腺癌诊断和预后基因标志物

目的 肺癌是世界上最常见的癌症之一,在众多肺癌患者中,肺腺癌(lung adenocarcinoma,LUAD)的死亡率最高.基因表达谱的变化与肿瘤的发生和发展过程有关,通过识别与LUAD患者相关的诊断和预后基因标志物,可以为肺腺癌的预防和治疗提供理论依据.方法 本研究以肿瘤基因组图谱(The Cancer Gene ...     

Prognostic Significance of Tumor Size of Small Lung Adenocarcinomas Evaluated with Mediastinal Window Settings on Computed Tomography

Background

We aimed to clarify that the size of the lung adenocarcinoma evaluated using mediastinal window on computed tomography is an important and useful modality for predicting invasiveness, lymph node metastasis and prognosis in small adenocarcinoma.

Methods

We evaluated 176 patients with small lung adenocarcinomas (diameter, 1–3 cm) who underwent standard surgical resection. Tumours were examined using computed tomography with thin section conditions (1.25 mm thick on high-resolution computed tomography) with tumour dimensions evaluated under two settings: lung window and mediastinal window. We also determined the patient age, gender, preoperative nodal status, tumour size, tumour disappearance ratio, preoperative serum carcinoembryonic antigen levels and pathological status (lymphatic vessel, vascular vessel or pleural invasion). Recurrence-free survival was used for prognosis.

Results

Lung window, mediastinal window, tumour disappearance ratio and preoperative nodal status were significant predictive factors for recurrence-free survival in univariate analyses. Areas under the receiver operator curves for recurrence were 0.76, 0.73 and 0.65 for mediastinal window, tumour disappearance ratio and lung window, respectively. Lung window, mediastinal window, tumour disappearance ratio, preoperative serum carcinoembryonic antigen levels and preoperative nodal status were significant predictive factors for lymph node metastasis in univariate analyses; areas under the receiver operator curves were 0.61, 0.76, 0.72 and 0.66, for lung window, mediastinal window, tumour disappearance ratio and preoperative serum carcinoembryonic antigen levels, respectively. Lung window, mediastinal window, tumour disappearance ratio, preoperative serum carcinoembryonic antigen levels and preoperative nodal status were significant factors for lymphatic vessel, vascular vessel or pleural invasion in univariate analyses; areas under the receiver operator curves were 0.60, 0.81, 0.81 and 0.65 for lung window, mediastinal window, tumour disappearance ratio and preoperative serum carcinoembryonic antigen levels, respectively.

Conclusions

According to the univariate analyses including a logistic regression and ROCs performed for variables with p-values of <0.05 on univariate analyses, our results suggest that measuring tumour size using mediastinal window on high-resolution computed tomography is a simple and useful preoperative prognosis modality in small adenocarcinoma.     

IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

Background

Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas.Aim of the study was to investigate i) IL-12Rβ2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC.

Methodology/Principal Findings

Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rβ2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R⁺ neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/β2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/β2⁺ cells inhibited angiogenesis in vitro. Tumors formed by Calu6/β2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines.

Conclusions

This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial.     

FoxM1 Is Associated with Poor Prognosis of Non-Small Cell Lung Cancer Patients through Promoting Tumor Metastasis

Background

FoxM1 has been reported to be important in initiation and progression of various tumors. However, whether FoxM1 has any indication for prognosis in non-small cell lung cancer patients remains unclear.

Methodology/Principal Findings

In this study, FoxM1 expression in tumor cells was examined first by immunohistochemistry in 175 NSCLC specimens, the result of which showed that FoxM1 overexpression was significantly associated with positive smoking status (P = 0.001), poorer tissue differentiation (P = 0.0052), higher TNM stage (P<0.0001), lymph node metastasis (P<0.0001), advanced tumor stage (P<0.0001), and poorer prognosis (P<0.0001). Multivariable analysis showed that FoxM1 expression increased the hazard of death (hazard ratio, 1.899; 95% CI, 1.016–3.551). Furthermore, by various in vitro and in vivo experiments, we showed that targeted knockdown of FoxM1 expression could inhibit the migratory and invasive abilities of NSCLC cells, whereas enforced expression of FoxM1 could increased the invasion and migration of NSCLC cells. Finally, we found that one of the cellular mechanisms by which FoxM1 promotes tumor metastasis is through inducing epithelial-mesenchymal transition (EMT) program.

Conclusions

These results suggested that FoxM1 overexpression in tumor tissues is significantly associated with the poor prognosis of NSCLC patients through promoting tumor metastasis.