The potential utility of circulating tumor cells (CTCs) to guide clinical care in oncology patients has gained momentum with emerging micro- and nanotechnologies. Establishing the role of CTCs in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of CTCs for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. Recent attempts in the literature to culture CTCs isolated from metastatic patients using monoculture have had limited success rates of less than 20%. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully expanded CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis.
expansion of CTCs; early stage lung cancer; microfluidic co-culture
Esophageal adenocarcinomas (EAC) are aggressive cancers that are increasing in incidence and associated with a poor prognosis. The identification of highly expressed genes in EAC relative to metaplastic Barrett’s esophagus (BE) may provide new targets for novel early cancer detection strategies using endoscopically administered, fluorescently labeled peptides.
Gene expression analysis of BE and EACs were used to identify the cell surface marker transglutaminase 2 (TGM2) as overexpressed in cancer. The expression of two major isoforms of TGM2 was determined by qRT-polymerase chain reaction in an independent cohort of 128 EACs. Protein expression was confirmed by tissue microarrays and immunoblot analysis of EAC cell lines. TGM2 DNA copy number was assessed using single nucleotide polymorphism microarrays and confirmed by qPCR. TGM2 expression in neoadjuvantly treated EACs and following small interfering RNA-mediated knockdown in cisplatin-treated EAC cells was used to determine its possible role in chemoresistance.
TGM2 is overexpressed in 15 EACs relative to 26 BE samples. Overexpression of both TGM2 isoforms was confirmed in 128 EACs and associated with higher tumor stage, poor differentiation, and increased inflammatory and desmoplastic response. Tissue microarrays and immunohistochemistry confirmed elevated TGM2 protein expression in EAC. Single nucleotide polymorphism and qPCR analysis revealed increased TGM2 gene copy number as one mechanism underlying elevated TGM2 expression. TGM2 was highly expressed in resistant EAC after patient treatment with neoadjuvant chemotherapy/radiation suggesting a role for TGM2 in chemoresistance.
TGM2 may be a useful cell surface biomarker for early detection of EAC.
Esophageal adenocarcinoma; TGM2; Cell surface biomarker
The microRNA-34b/c (miR-34b/c) has been considered a tumor suppressor in different tumor types and it is a known transcriptional target of the tumor suppressor gene TP53. The main objectives of this study were to investigate the clinical implications of miR-34b/c methylation in early stage lung adenocarcinoma (AC) patients and to determine the functional role of miR-34b/c re-expression in lung AC cell lines.
Aberrant methylation and expression of miR-34b/c were assessed in 15 lung AC cell lines and a cohort of 140 early stage lung AC. Lung AC cell lines were transfected with miR-34b/c and the effects upon cell proliferation, migration, invasion and apoptosis were investigated.
Aberrant methylation of miR-34b/c was detected in 6 (40%) of 15 lung AC cell lines and 64 out of 140 (46%) primary lung adenocarcinomas. Expression of miR-34b/c was significantly reduced in all methylated cell lines and primary tumors, especially in those harboring a TP53 mutation. Patients with high levels of miR-34b/c methylation had significantly shorter disease-free survival and overall survival as compared to patients with unmethylated miR-34b/c or low level of miR-34b/c methylation. Ectopic expression of miR-34b/c in lung AC cell lines decreased cell proliferation, migration and invasion.
Epigenetic inactivation of miR-34b/c by DNA methylation has independent prognostic value in early stage lung AC patients with surgically resected tumors. Re-expression of miR-34b/c leads to a less aggressive phenotype in lung AC cell lines.
microRNA; DNA methylation; microRNA-34b/c; lung adenocarcinoma; TP53
Overexpression of checkpoint kinase 1 (CHK1) is associated with poorer patient outcome and therapeutic resistance in multiple tumor models. Inhibition of CHK1 has been proposed as a strategy to increase the effectiveness of chemotherapeutic agents, especially in p53-deficient tumors. In this study, we evaluated the effects of a novel CHK1 inhibitor, MK-8776, in combination with pemetrexed (PMX) on cell proliferation and survival in a panel of p53 mutant non-small cell lung cancer (NSCLC) cell lines.
We examined CHK1 expression in 442 resected lung adenocarcinoma specimens using Affymetrix U133A gene expression arrays. We correlated CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. We evaluated CHK1 levels in NSCLC cell lines and identified four p53 mutant cell lines with variable CHK1 expression (H1993, H23, H1437 and H1299) based on publicly available gene expression data. We confirmed differential CHK1 mRNA and CHK1 protein levels by qRT-PCR, ELISA, Western Blot analysis (WB) and immunohistochemistry. We examined cell line sensitization to PMX in response to CHK1 inhibition with MK-8776 using WST-1 and clonogenic survival assays.
We found that elevated CHK1 expression in primary lung adenocarcinomas correlates with poor tumor differentiation and significantly worse patient survival. Tumors with elevated CHK1 mRNA levels have a higher number of gene mutations and DNA copy number gain or amplifications. CHK1 inhibition by MK-8776 enhances sensitivity of NSCLC cell lines to PMX. CHK1 mRNA and protein expression are variable among NSCLC cell lines, and cells expressing higher levels of CHK1 protein are more sensitive to the CHK1 inhibition by MK-8776 as compared to low CHK1 expressing cells.
These findings suggest that CHK1 levels may not only serve as a biomarker of poor prognosis in surgically-resected lung adenocarcinomas, but could also be a predictive marker for CHK1 inhibitor sensitivity, pending in vivo and clinical confirmation.
CHK1; Lung; Chemosensitivity; NSCLC; Patient survival; Genomic complexity
The chemopreventive effects of selenium have been extensively examined but its role in cancer development or as a chemotherapeutic agent have only recently been explored. Because Selenium Binding Protein 1 (SELENBP1, SBP1, hSP56) has been shown to bind selenium covalently and selenium deficiency has been associated with esophageal adenocarcinoma (EAC), we examined its role in EAC development and its potential effect on chemosensitivity in the presence of selenium.
SELENBP1 expression level and copy number variation were determined by oligonucleotide microarrays, real-time RT-PCR, tissue microarrays, immunoblotting and SNP arrays. Bisulfite sequencing and sequence analysis of RT-PCR-amplified products explored epigenetic and post-transcriptional regulation of SELENBP1 expression, respectively. WST-1 cell proliferation assays, senescence-associated β-galactosidase staining, immunoblotting, and flow cytometry were performed to evaluate the biological significance of SELENBP1 overexpression in selenium-supplemented EAC cells.
SELENBP1 expression decreased significantly in Barrett's esophagus to adenocarcinoma progression. Both epigenetic and post-transcriptional mechanisms appeared to modulate SELENBP1 expression. Stable overexpression of SELENBP1 in methylseleninic acid-supplemented Flo-1 cells resulted in enhanced apoptosis, increased cellular senescence, and enhanced cisplatin cytotoxicity. Although inorganic sodium selenite similarly enhanced cisplatin cytotoxicity, these 2 forms of selenium elicited different cellular responses.
SELENBP1 expression may be an important predictor of response to chemoprevention or chemosensitization with certain forms of selenium in esophageal tissues.
The transcription factor, nuclear factor κB (NF-κB), plays a central role as a key mediator of cell survival and proliferation, and its activation may confer increased tumor chemoresistance. Curcumin, an orally available naturally occurring compound, has been shown to inhibit NF-κB and has a potential role in cancer chemoprevention. We investigated the effects of curcumin on NF-κB activity, on cell viability, and as a chemosensitizing agent with 5-fluorouracil (5-FU) or cisplatin (CDDP) in esophageal adenocarcinoma (EAC). Oligonucleotide microarray analysis of 46 cases, consisting of Barrett metaplasia, low-grade dysplasia, high-grade dysplasia and EAC, showed increased expression of NF-κB and IκB kinase subunits and decreased effector caspase expression in EAC compared with Barrett metaplasia. Stromal expression of both IκB and phospho-IκB was detected in several EAC samples by tissue microarray analysis. Curcumin alone inhibited NF-κB activity and induced apoptosis in both Flo-1 and OE33 EAC cell lines as determined by Western blot analysis, NF-κB reporter assays, and Caspase-Glo 3/7 assays. It also increased 5-FU- and CDDP-induced apoptosis in both cell lines. These data suggest that activation of NF-κB and inhibition of apoptosis may play a role in the progression from Barrett metaplasia to EAC. In addition, curcumin, a well-known inhibitor of NF-κB activity, was shown to increase apoptosis and enhance both 5-FU- and CDDP-mediated chemosensitivity, suggesting that it may have potential application in the therapy of patients with EAC.
This prospective study aimed to develop a robust and clinically-applicable method to identify high-risk early stage lung cancer patients and then to validate this method for use in future translational studies.
Patients and Methods
Three published Affymetrix microarray data sets representing 680 primary tumors were used in the survival-related gene selection procedure using clustering, Cox model and random survival forest (RSF) analysis. A final set of 91 genes was selected and tested as a predictor of survival using a qRT-PCR-based assay utilizing an independent cohort of 101 lung adenocarcinomas.
The RSF model built from 91 genes in the training set predicted patient survival in an independent cohort of 101 lung adenocarcinomas, with a prediction error rate of 26.6%. The mortality risk index (MRI) was significantly related to survival (Cox model p < 0.00001) and separated all patients into low, medium, and high-risk groups (HR = 1.00, 2.82, 4.42). The MRI was also related to survival in stage 1 patients (Cox model p = 0.001), separating patients into low, medium, and high-risk groups (HR = 1.00, 3.29, 3.77).
The development and validation of this robust qRT-PCR platform allows prediction of patient survival with early stage lung cancer. Utilization will now allow investigators to evaluate it prospectively by incorporation into new clinical trials with the goal of personalized treatment of lung cancer patients and improving patient survival.
Lung cancer; qRT-PCR; Prognosis
Esophagectomy is indicated occasionally for the treatment of patients with refractory gastroesophageal reflux disease (GERD) or recurrent hiatus hernia. The purpose of this study was to evaluate the impact of previous gastroesophageal operations on outcomes after esophagectomy for recurrent GERD or hiatus hernia.
Using a prospectively accumulated database, a retrospective review was performed to identify patients undergoing esophagectomy for complicated GERD or hiatus hernia. Mortality, perioperative and functional outcomes, and need for reoperation were evaluated, assessing esophagectomy patients who had undergone prior operations for GERD or hiatus hernia.
Of 258 patients with GERD or hiatus hernia undergoing esophagectomy, 104 had undergone a previous operation, with a median interval to esophagectomy of 28 months. Transhiatal resection was accomplished in fewer patients undergoing reoperation (87 of 104 versus 151 of 154; p < 0.005). A gastric conduit was used as an esophageal replacement in fewer patients with previous operation(s) (89 of 104 versus 150 of 154; p < 0.005). Esophagectomy patients with a history of prior gastroesophageal surgery, as compared with those without, sustained more blood loss and were more likely to require reoperation, and fewer reported good to excellent swallowing function (p < 0.05). There was no difference in the occurrence of anastomotic leak.
Esophagectomy in patients who have undergone prior operations for either GERD or hiatus hernia can be accomplished without thoracotomy and with satisfactory intermediate-term quality of life. Such patients should be evaluated and prepared for the use of alternative conduits should the remobilized stomach prove to be an unsatisfactory esophageal substitute at the time of esophagectomy.
This article will focus on the impact of patient age on outcomes following esophageal resection as well as potential strategies to improve perioperative management of geriatric patients undergoing esophagectomy for cancer.
Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.
Although prognostic gene expression signatures for survival in early stage lung cancer have been proposed, for clinical application it is critical to establish their performance across different subject populations and in different laboratories. Here we report a large, training-testing, multi-site blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. The hypotheses proposed examined whether microarray measurements of gene expression either alone or combined with basic clinical covariates (stage, age, sex) can be used to predict overall survival in lung cancer subjects. Several models examined produced risk scores that substantially correlated with actual subject outcome. Most methods performed better with clinical data, supporting the combined use of clinical and molecular information when building prognostic models for early stage lung cancer. This study also provides the largest available set of microarray data with extensive pathological and clinical annotation for lung adenocarcinomas.
Somatic alterations in cellular DNA underlie almost all human cancers1. The prospect of targeted therapies2 and the development of high-resolution, genome-wide approaches3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumors (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ~12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.
Angiogenesis is crucial for tumor biology. There are many mechanisms by which tumors induce angiogenesis. We hypothesize that each individual tumor develops a unique mechanism to induce angiogenesis, and that activation of a particular angiogenic pathway suppresses the evolution of alternative pathways. We characterized 168 human non–small cell lung cancer (NSCLC) specimens for levels of angiogenic factors (angiogenic CXC chemokines, basic fibroblast growth factor, and vascular endothelial growth factor). We also induced lung tumor formation in A/J mice by injecting the tobacco carcinogen NNK. We dissected individual lung tumors and measured expression of angiogenic factors from three distinct families using real-time PCR. Finally, we controlled the angiogenic milieu using in vivo models to determine the resultant phenotype of the angiogenic factors expressed by NSCLC cells. Human tumors displayed marked variation in the expression of angiogenic factors. Individual mouse tumors, even from within the same mouse, displayed variability in their pattern of expression of angiogenic factors. In a sponge model of angiogenesis using murine lung cancer cells, implanting LLC cells with an angiogenic factor suppressed the expression of other angiogenic factors in implanted sponges. This suppressive effect was not seen in vitro. We conclude that lung cancer tumors evolve a unique and dominant angiogenic phenotype. Once an angiogenic pathway is activated, it may allow for tumor growth to proceed in the absence of a selection pressure to activate a second pathway.
angiogenesis; mouse model; chemokines; cytokines; carcinogen
Ubiquitin-dependent proteolysis of cyclins plays a critical role in cell cycle progression and tumorigenesis. We examined the expression of ubiquitin-conjugating enzyme E2C (UBE2C) during progression from Barrett's metaplasia to esophageal adenocarcinoma (EA) and the effects of targeting this enzyme on EA-derived cell lines. Using oligonucleotide microarrays UBE2C expression was elevated in 73% (11 of 15) of EAs relative to Barrett's metaplasia. Tissue microarray showed elevated UBE2C in 70% (7 of 10) of dysplastic samples and in 87% (58 of 67) of tumors relative to metaplastic samples. Transfection of dominant-negative UBE2C into Seg-1 cells decreased proliferation (P = .04) and increased mitotic arrest compared to vector controls (63.5% vs 6.8%; P < .001). Transfection of UBE2C small interfering RNA also caused inhibiton of cell proliferation and distortion of the cell cycle, with maximal increase of G2 cells (155% of mock cells) at 72 hours and of S-phase cells (308% of mock cells) at 24 hours. Treatment of Seg-1 cells with the proteasome inhibitor MG-262 (1 nM-1 µM) showed decreased proliferation (P = .02). EA-derived cells expressing UBE2C are sensitive to treatment with MG-262 and to silencing of UBE2C, suggesting that patients with EAs overexpressing UBE2C may benefit from agents targeting this ubiquitin-conjugating enzyme.
Esophageal adenocarcinoma; ubiquitin; UBE2C; proteasome inhibitor; siRNA
In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin-
2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of
two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this
proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus
size and number of thymocytes compared to normal littermates. These mice also exhibited
apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy
thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-,
CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further
examination of the DN population for CD44 and CD25 expression showed a remarkable
decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow
cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the
capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed
normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal
and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development
by delivering cell survival and differentiation signals at specific stages of development
in young adult mice.
apoptosis; dystrophic; integrins; laminin; thymocytes
The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated.
INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2′ deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression.
Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin βA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2′ deoxycytidine.
INHBA is overexpressed in AD relative to controls. Inhibin βA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs.