Multidisciplinary tumor board conferences foster collaboration among health care providers from a variety of specialties and help to facilitate optimal patient care. Typical cases from thoracic tumor board conferences include patients with known or suspected bronchogenic and esophageal carcinomas, as well as less common diseases such as thymomas and mesotheliomas. In most instances, the clinical questions revolve around the best options for establishing a diagnosis, staging the disease and directing treatment. This article describes and illustrates the clinical scenarios of three patients who were presented at our tumor board, focusing on management issues and the role of imaging. These patients had non-small cell lung cancer and mediastinal lymph node metastases; a small, growing ground glass nodule; and oligometastatic non-small cell lung cancer, respectively.
Lung cancer; tumor board; multidisciplinary care; stage IIIA; ground glass nodule
Multidisciplinary tumor board conferences foster collaboration among health care providers from a variety of specialties and help to facilitate optimal patient care. Generally, the clinical questions revolve around the best options for establishing a diagnosis, staging the disease and directing treatment. This article describes and illustrates the clinical scenarios of three patients who were presented at our thoracic Tumor Board, focusing on management issues and the role of imaging. These patients had invasive thymoma; concurrent small cell lung cancer and non-small cell lung cancer; and esophageal cancer with celiac lymph node metastases, respectively.
Lung cancer; small cell lung cancer; non-small cell lung cancer; esophageal cancer; invasive thymoma; tumor board
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.
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.
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.
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
The L-type amino acid transporter-1 (LAT-1) has been associated with tumor growth. Using cDNA microarrays, overexpression of LAT-1 was found in 87.5% (7/8) of esophageal adenocarcinomas relative to 12 Barrett's samples (33% metaplasia and 66% dysplasia) and was confirmed in 100% (28/28) of Barrett's adenocarcinomas by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed LAT-1 staining in 37.5% (24/64) of esophageal adenocarcinomas on tissue microarray. LAT-1 also transports the amino acid-related chemotherapeutic agent, melphalan. Two esophageal adenocarcinoma and one esophageal squamous cell line, expressing LAT-1 on Western blot analysis, were sensitive to therapeutic doses of melphalan (P < .001). Simultaneous treatment with the competitive inhibitor, BCH [2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid], decreased sensitivity to melphalan (P < .05). In addition, confluent esophageal squamous cultures were less sensitive to melphalan (P < .001) and had a decrease in LAT-1 protein expression. Tumors from two esophageal adenocarcinoma cell lines grown in nude mice retained LAT-1 mRNA expression. These results demonstrate that LAT-1 is highly expressed in a subset of esophageal adenocarcinomas and that Barrett's adenocarcinoma cell lines expressing LAT-1 are sensitive to melphalan. LAT-1 expression is also retained in cell lines grown in nude mice providing a model to evaluate melphalan as a chemotherapeutic agent against esophageal adenocarcinomas expressing LAT-1.
Esophageal adenocarcinoma; L-type amino acid transporter-1; amino acid transporters; melphalan; chemotherapy
Cytokeratins (CK) are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, and 19 occurred at significantly higher levels (P<.05) in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms (nos. 2165 and 2091), one of eight CK8 isoforms (no. 439), and one of three CK19 isoforms (no. 1955) were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.
lung adenocarcinoma; isoforms; 2D PAGE; mass spectrometry; microarrays
The prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to exert a chemopreventive effect in esophageal and other gastrointestinal tumors. The precise mechanism by which this occurs, however, is unknown. While the inhibition of COX-2 as a potential explanation for this chemopreventive effect has gained a great deal of support, there also exists evidence supporting the presence of cyclooxygenase-independent pathways through which NSAIDs may exert their effects. In this study, immunohistochemical analysis of 29 Barrett's epithelial samples and 60 esophageal adenocarcinomas demonstrated abundant expression of the COX-2 protein in Barrett's epithelium, but marked heterogeneity of expression in esophageal adenocarcinomas. The three esophageal adenocarcinoma cell lines, Flo-1, Bic-1, and Seg-1, also demonstrated varying expression patterns for COX-1 and COX-2. Indomethacin induced apoptosis in all three cell lines, however, in both a time- and dose-dependent manner. In Flo-1 cells, which expressed almost undetectable levels of COX-1 and COX-2, and in Seg-1, which expressed significant levels of COX-1 and COX-2, indomethacin caused upregulation of the pro-apoptotic protein Bax. The upregulation of Bax was accompanied by the translocation of mitochondrial cytochrome c to the cytoplasm, and activation of caspase 9. Pre-treatment of both cell lines with the specific caspase 9 inhibitor, z-LEHD-FMK, as well as the broad-spectrum caspase inhibitor, z-VAD-FMK, blocked the effect of indomethacin-induced apoptosis. These data demonstrate that induction of apoptosis by indomethacin in esophageal adenocarcinoma cells is associated with the upregulation of Bax expression and mitochondrial cytochrome c translocation, and does not correlate with the expression of COX-2. This may have important implications for identifying new therapeutic targets in this deadly disease.
esophageal adenocarcinoma; Barrett's esophagus; NSAIDs; chemoprevention; apoptosis
The expression of cytochromes P450 (CYP) in Barrett's esophagus and esophageal squamous mucosa was investigated. Esophagectomy specimens from 23 patients were examined for CYP expression of CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 by immunohistochemical analysis, and the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 in these tissues was further confirmed by reverse transcription polymerase chain reaction. Immunohistochemical analysis of esophageal squamous mucosa (n = 12) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 proteins, but it was noted that cells within the basal proliferative zone did not express CYPs. Immunohistochemical analysis of Barrett's esophagus (n = 13) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 that was prominent in the basal glandular regions, which are areas containing a high percentage of actively proliferating cells. Immunohistochemical staining for both proliferating cell nuclear antigen and the CYPs further supported the colocalization of CYP expression to areas of active cell proliferation in Barrett's esophagus, whereas in the esophageal squamous epithelium, CYP expression is limited to cells that are not proliferating. RT-PCR with amplification product sequence analysis confirmed CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA expression in Barrett's esophagus. These data suggest that the potential ability of cells in Barrett's esophagus to both activate carcinogens and proliferate may be important risk factors affecting carcinogenesis in this metaplastic tissue.
malignancy; esophagus; carcinogen; carcinogenesis; PCNA
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.