The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of
their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of
Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared
to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations
accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited
hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant
tumors. Twenty-four genes that were significantly and differentially expressed between
WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients.
Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent
than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to
eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of
Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to
efficiently kill tumor cells in vitro. Our results identify a prognostic signature
for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC
with elevated AKT signaling.
eEF2K; p53; prognosis; Pten; triple-negative breast cancer
The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non–small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay.
Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made.
Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively.
On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.
Non–small-cell lung cancer; Anaplastic lymphoma kinase; Immunohistochemistry; Fluorescence in situ hybridization; Companion diagnostics; Biomarkers; Crizotinib
Apoptosis plays an important role in neoplastic processes. Bcl-B is an antiapoptotic Bcl-2 family member, which is known to change its phenotype upon binding to Nur77/TR3. The expression pattern of this protein in human malignancies has not been reported.
We investigated Bcl-B expression in normal human tissues and several types of human epithelial and nonepithelial malignancy by immunohistochemistry, correlating results with tumor stage, histologic grade, and patient survival.
Bcl-B protein was strongly expressed in all normal plasma cells but found in only18%of multiple myelomas (n = 133). Bcl-B immunostaining was also present in normal germinal center centroblasts and centrocytes and in approximately half of diffuse large B-cell lymphoma (n =48) specimens, whereas follicular lymphomas (n = 57) did not contain Bcl-B. In breast (n = 119), prostate (n = 66), gastric (n = 180), and colorectal (n = 106) adenocarcinomas, as well as in non – small cell lung cancers (n = 82), tumor-specific overexpression of Bcl-B was observed. Bcl-B expression was associated with variables of poor prognosis, such as high tumor grade in breast cancer (P = 0.009), microsatellite stability (P = 0.0002), and left-sided anatomic location (P = 0.02) of colorectal cancers, as well as with greater incidence of death from prostate cancer (P = 0.005) and shorter survival of patients with small cell lung cancer (P = 0.009). Conversely, although overexpressed in many gastric cancers, Bcl-B tended to correlate with better outcome (P = 0.01) and more differentiated tumor histology (P < 0.0001).
Tumor-specific alterations in Bcl-B expressionmay define subsets of nonepithelial and epithelial neoplasms with distinct clinical behaviors.
Survival of patients with completely resected non–small-cell lung cancer (NSCLC) is unsatisfactory, and in 2002, the benefit of adjuvant chemotherapy was not established. This phase III study assessed the impact of postoperative adjuvant gefitinib on overall survival (OS).
Patients and Methods
Patients with completely resected (stage IB, II, or IIIA) NSCLC stratified by stage, histology, sex, postoperative radiotherapy, and chemotherapy were randomly assigned (1:1) to receive gefitinib 250 mg per day or placebo for 2 years. Study end points were OS, disease-free survival (DFS), and toxicity.
As a result of early closure, 503 of 1,242 planned patients were randomly assigned (251 to gefitinib and 252 to placebo). Baseline factors were balanced between the arms. With a median of 4.7 years of follow-up (range, 0.1 to 6.3 years), there was no difference in OS (hazard ratio [HR], 1.24; 95% CI, 0.94 to 1.64; P = .14) or DFS (HR, 1.22; 95% CI, 0.93 to 1.61; P = .15) between the arms. Exploratory analyses demonstrated no DFS (HR, 1.28; 95% CI, 0.92 to 1.76; P = .14) or OS benefit (HR, 1.24; 95% CI, 0.90 to 1.71; P = .18) from gefitinib for 344 patients with epidermal growth factor receptor (EGFR) wild-type tumors. Similarly, there was no DFS (HR, 1.84; 95% CI, 0.44 to 7.73; P = .395) or OS benefit (HR, 3.16; 95% CI, 0.61 to 16.45; P = .15) from gefitinib for the 15 patients with EGFR mutation-positive tumors. Adverse events were those expected with an EGFR inhibitor. Serious adverse events occurred in ≤ 5% of patients, except infection, fatigue, and pain. One patient in each arm had fatal pneumonitis.
Although the trial closed prematurely and definitive statements regarding the efficacy of adjuvant gefitinib cannot be made, these results indicate that it is unlikely to be of benefit.
Polarization second harmonic microscopy was used for collagen imaging in human non-small cell lung carcinoma and normal lung tissues ex vivo and revealed significant differences in the nonlinear susceptibility component ratio, demonstrating potential use in cancer diagnosis.
(180.0180) Microscopy; (180.4315) Nonlinear microscopy; (190.4160) Multiharmonic generation
K-ras mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in the molecular pathogenesis. Expression of K-ras oncogene in an immortalized human pancreatic ductal epithelial cell line, originally derived from normal pancreas (H6c7), induced the formation of carcinoma in mice. We hypothesized that K-ras oncogene correlates with increased non-mitochondrial-generated superoxide (O2·−), which could be involved in regulating cell growth contributing to tumor progression. In the H6c7 cell line and its derivatives, H6c7er-Kras+ (H6c7 cells expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene), there was an increase in hydroethidine fluorescence in cell lines that express K-ras. Western blots and activity assays for the antioxidant enzymes that detoxify O2·− were similar in these cell lines suggesting that the increase in hydroethidine fluorescence was not due to decreased antioxidant capacity. To determine a possible non-mitochondrial source of the increased levels of O2·−, Western analysis demonstrated the absence of NADPH oxidase-2 (NOX2) in H6c7 cells but present in the H6c7 cell lines expressing K-ras and other pancreatic cancer cell lines. Inhibition of NOX2 decreased hydroethidine fluorescence and clonogenic survival. Furthermore, in the cell lines with the K-ras oncogene, overexpression of superoxide dismutases, that detoxify non-mitochondrial sources of O2·−, and treatment with the small molecule O2·− scavenger Tempol, also decreased hydroethidine fluorescence, inhibited clonogenic survival and inhibited growth of tumor xenografts. Thus, O2·− produced by NOX2 in pancreatic cancer cells with K-ras, may regulate pancreatic cancer cell growth.
The excision repair cross-complementation group 1 (ERCC1) protein is a potential prognostic biomarker of the efficacy of cisplatin-based chemotherapy in non–small-cell lung cancer (NSCLC). Although several ongoing trials are evaluating the level of expression of ERCC1, no consensus has been reached regarding a method for evaluation.
We used the 8F1 antibody to measure the level of expression of ERCC1 protein by means of immunohistochemical analysis in a validation set of samples obtained from 494 patients in two independent phase 3 trials (the National Cancer Institute of Canada Clinical Trials Group JBR.10 and the Cancer and Leukemia Group B 9633 trial from the Lung Adjuvant Cisplatin Evaluation Biology project). We compared the results of repeated staining of the entire original set of samples obtained from 589 patients in the International Adjuvant Lung Cancer Trial Biology study, which had led to the initial correlation between the absence of ERCC1 expression and platinum response, with our previous results in the same tumors. We mapped the epitope recognized by 16 commercially available ERCC1 antibodies and investigated the capacity of the different ERCC1 isoforms to repair platinum-induced DNA damage.
We were unable to validate the predictive effect of immunostaining for ERCC1 protein. The discordance in the results of staining for ERCC1 suggested a change in the performance of the 8F1 antibody since 2006. We found that none of the 16 antibodies could distinguish among the four ERCC1 protein isoforms, whereas only one isoform produced a protein that had full capacities for nucleotide excision repair and cisplatin resistance.
Immunohistochemical analysis with the use of currently available ERCC1 antibodies did not specifically detect the unique functional ERCC1 isoform. As a result, its usefulness in guiding therapeutic decision making is limited. (Funded by Eli Lilly and others.)
SOX genes are transcription factors with important roles in embryonic development and carcinogenesis. The SOX family of 20 genes is responsible for regulating lineage and tissue specific gene expression patterns, controlling numerous developmental processes including cell differentiation, sex determination, and organogenesis. As is the case with many genes involved in regulating development, SOX genes are frequently deregulated in cancer. In this perspective we provide a brief overview of how SOX proteins can promote or suppress cancer growth. We also present a pan-cancer analysis of aberrant SOX gene expression and highlight potential molecular mechanisms responsible for their disruption in cancer. Our analyses indicate the prominence of SOX deregulation in different cancer types and reveal potential roles for SOX genes not previously described in cancer. Finally, we summarize our recent identification of SOX15 as a candidate tumor suppressor in pancreatic cancer and propose several research avenues to pursue to further delineate the emerging role of SOX15 in development and carcinogenesis.
SOX; SOX15; oncogene; tumor suppressor; development; cancer
Brahma (BRM) has a key function in chromatin remodeling. Two germline BRM promoter insertion–deletion polymorphisms, BRM-741 and BRM-1321, have been previously associated with an increased risk of lung cancer in smokers and head and neck cancer. To further evaluate their role in cancer susceptibility particularly in early disease, we conducted a preplanned case–control study to investigate the association between the BRM promoter variants and stage I/II upper aerodigestive tract (UADT) cancers (i.e., lung, esophageal, head and neck), a group of early-stage malignancies in which molecular and genetic etiologic factors are poorly understood. The effects of various clinical factors on this association were also studied. We analyzed 562 cases of early-stage UADT cancers and 993 matched healthy controls. The double homozygous BRM promoter variants were associated with a significantly increased risk of early stage UADT cancers (adjusted odds ratio [aOR], 2.46; 95% confidence interval [CI], 1.7–3.8). This association was observed in lung (aOR, 2.61; 95% CI, 1.5–4.9) and head and neck (aOR, 2.75; 95% CI, 1.4–5.6) cancers, but not significantly in esophageal cancer (aOR, 1.66; 95% CI, 0.7–5.8). There was a nonsignificant trend for increased risk in the heterozygotes or single homozygotes. The relationship between the BRM polymorphisms and early-stage UADT cancers was independent of age, sex, smoking status, histology, and clinical stage. These findings suggest that the BRM promoter double insertion homozygotes may be associated with an increased risk of early-stage UADT cancers independent of smoking status and histology, which must be further validated in other populations.
BRM; cancer risk; case–control study; esophageal cancer; genetic polymorphisms; head and neck cancer; lung cancer; upper aerodigestive tract cancers
Major issues in the implementation of screening for lung cancer by means of low-dose computed tomography (CT) are the definition of a positive result and the management of lung nodules detected on the scans. We conducted a population-based prospective study to determine factors predicting the probability that lung nodules detected on the first screening low-dose CT scans are malignant or will be found to be malignant on follow-up.
We analyzed data from two cohorts of participants undergoing low-dose CT screening. The development data set included participants in the Pan-Canadian Early Detection of Lung Cancer Study (PanCan). The validation data set included participants involved in chemoprevention trials at the British Columbia Cancer Agency (BCCA), sponsored by the U.S. National Cancer Institute. The final outcomes of all nodules of any size that were detected on baseline low-dose CT scans were tracked. Parsimonious and fuller multivariable logistic-regression models were prepared to estimate the probability of lung cancer.
In the PanCan data set, 1871 persons had 7008 nodules, of which 102 were malignant, and in the BCCA data set, 1090 persons had 5021 nodules, of which 42 were malignant. Among persons with nodules, the rates of cancer in the two data sets were 5.5% and 3.7%, respectively. Predictors of cancer in the model included older age, female sex, family history of lung cancer, emphysema, larger nodule size, location of the nodule in the upper lobe, part-solid nodule type, lower nodule count, and spiculation. Our final parsimonious and full models showed excellent discrimination and calibration, with areas under the receiver-operating-characteristic curve of more than 0.90, even for nodules that were 10 mm or smaller in the validation set.
Predictive tools based on patient and nodule characteristics can be used to accurately estimate the probability that lung nodules detected on baseline screening low-dose CT scans are malignant. (Funded by the Terry Fox Research Institute and others; ClinicalTrials.gov number, NCT00751660.)
The unusually dense stroma of pancreatic cancers is thought to play an important role in their biological aggression. The presence of hypoxia is also considered an adverse prognostic factor. Although it is usually assumed that this is the result of effects of hypoxia on the epithelial component, it is possible that hypoxia exerts indirect effects via the tumor stroma. We therefore measured hypoxia in the stroma of a series of primary pancreatic cancer xenografts. Nine patient-derived pancreatic xenografts representing a range of oxygenation levels were labeled by immunohistochemistry for EF5 and analyzed using semi-automated pattern recognition software. Hypoxia in the tumor and stroma was correlated with tumor growth and metastatic potential. The extent of hypoxia varied from 1%–39% between the different models. EF5 labeling in the stroma ranged from 0–20% between models, and was correlated with the level of hypoxia in the tumor cell area, but not microvessel density. Tumor hypoxia correlated with spontaneous metastasis formation with the exception of one hypoxic model that showed disproportionately low levels of hypoxia in the stroma and was non-metastatic. Our results demonstrate that hypoxia exists in the stroma of primary pancreatic cancer xenografts and suggest that stromal hypoxia impacts the metastatic potential.
pancreatic cancer; tumor hypoxia; tumor-associated stroma; patient-derived pancreatic xenograft models; pattern recognition software
KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRASG12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.
New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker.
We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay.
Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). Fibulin-3 preferentially stained tumor cells in 26 of 26 samples. In an overall comparison of patients with and those without mesothelioma, the receiver-operating-characteristic curve for plasma fibulin-3 levels had a sensitivity of 96.7% and a specificity of 95.5% at a cutoff value of 52.8 ng of fibulin-3 per milliliter. In a comparison of patients with early-stage mesothelioma with asbestos-exposed persons, the sensitivity was 100% and the specificity was 94.1% at a cutoff value of 46.0 ng of fibulin-3 per milliliter. Blinded validation revealed an area under the curve of 0.87 for plasma specimens from 96 asbestos-exposed persons as compared with 48 patients with mesothelioma.
Plasma fibulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with effusion fibulin-3 levels, plasma fibulin-3 levels can further differentiate mesothelioma effusions from other malignant and benign effusions. (Funded by the Early Detection Research Network, National Institutes of Health, and others.)
Small cell lung cancer (SCLC) is a highly aggressive lung neoplasm with extremely poor clinical outcomes and no approved targeted treatments. To elucidate the mechanisms responsible for driving the SCLC phenotype in hopes of revealing novel therapeutic targets, we studied copy number and methylation profiles of SCLC. We found disruption of the E2F/Rb pathway was a prominent feature deregulated in 96% of the SCLC samples investigated and was strongly associated with increased expression of EZH2, an oncogene and core member of the polycomb repressive complex 2 (PRC2). Through its catalytic role in the PRC2 complex, EZH2 normally functions to epigenetically silence genes during development, however, it aberrantly silences genes in human cancers. We provide evidence to support that EZH2 is functionally active in SCLC tumours, exerts pro-tumourigenic functions in vitro, and is associated with aberrant methylation profiles of PRC2 target genes indicative of a “stem-cell like” hypermethylator profile in SCLC tumours. Furthermore, lentiviral-mediated knockdown of EZH2 demonstrated a significant reduction in the growth of SCLC cell lines, suggesting EZH2 has a key role in driving SCLC biology. In conclusion, our data confirm the role of EZH2 as a critical oncogene in SCLC, and lend support to the prioritization of EZH2 as a potential therapeutic target in clinical disease.
We investigated the predictive and prognostic effects of VeriStrat®, a serum or plasma based assay, on response and survival in a subset of patients enrolled on the NCIC Clinical Trials Group (CTG) BR.21 phase III trial of erlotinib versus placebo in previously treated advanced non-small cell lung cancer (NSCLC) patients.
Pretreatment plasma samples were available for 441 of 731 enrolled patients and were provided as anonymized aliquots to Biodesix. The VeriStrat test was performed in a CLIA-accredited laboratory at Biodesix, Inc. Results (Good, Poor) were returned to NCIC CTG, who performed all statistical analyses.
VeriStrat testing was successful in 436 samples (98.9%), with 61% classified as Good. VeriStrat was prognostic for overall survival in both erlotinib-treated patients and those on placebo, independent of clinical covariates. For VeriStrat Good patients, the median survival was 10.5 months on erlotinib vs. 6.6 months for placebo (HR 0.63, 95% C.I. 0.47–0.85, P=0.002). For VeriStrat Poor patients, the median survival was 4 months for patients receiving erlotinib, and 3.1 months for placebo (HR: 0.77, 95% C.I. 0.55–1.06, P=0.11). VeriStrat was predictive for objective response (P =0.002), but was not able to predict for differential survival benefit from erlotinib (interaction p-value 0.48). Similar results were found for progression-free survival (PFS).
We were able to confirm that VeriStrat is predictive of objective response to erlotinib. VeriStrat is prognostic for both OS and PFS, independent of clinical features, but is not predictive of differential survival benefit vs. placebo.
erlotinib; proteomics; metastatic non-small cell lung cancer; biomarkers
Cancer-Testis Antigens (CTAs) are immunogenic proteins that are poor prognostic markers in non-small cell lung cancer (NSCLC). We investigated expression of CTAs in NSCLC and their association with response to chemotherapy, genetic mutations and survival.
We studied 199 patients with pathological N2 NSCLC treated with neoadjuvant chemotherapy (NAC; n = 94), post-operative observation (n = 49), adjuvant chemotherapy (n = 47) or unknown (n = 9). Immunohistochemistry for NY-ESO-1, MAGE-A and MAGE-C1 was performed. Clinicopathological features, response to neoadjuvant treatment and overall survival were correlated. DNA mutations were characterized using the Sequenom Oncocarta panel v1.0. Affymetrix data from the JBR.10 adjuvant chemotherapy study were obtained from a public repository, normalised and mapped for CTAs.
NY-ESO-1 was expressed in 50/199 (25%) samples. Expression of NY-ESO-1 in the NAC cohort was associated with significantly increased response rates (P = 0.03), but not overall survival. In the post-operative cohort, multivariate analyses identified NY-ESO-1 as an independent poor prognostic marker for those not treated with chemotherapy (HR 2.61, 95% CI 1.28–5.33; P = 0.008), whereas treatment with chemotherapy and expression of NY-ESO-1 was an independent predictor of improved survival (HR 0.267, 95% CI 0.07–0.980; P = 0.046). Similar findings for MAGE-A were seen, but did not meet statistical significance. Independent gene expression data from the JBR.10 dataset support these findings but were underpowered to demonstrate significant differences. There was no association between oncogenic mutations and CTA expression.
NY-ESO-1 was predictive of increased response to neoadjuvant chemotherapy and benefit from adjuvant chemotherapy. Further studies investigating the relationship between these findings and immune mechanisms are warranted.
The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable.
Staging of node negative (N0) non-small cell lung cancer is modified in the 7th edition TNM classification. Here, we pool data from JBR.10 and CALGB-9633 to explore the prognostic and predictive effects of the new T-size descriptors and KRAS mutation status.
Node negative patients were reclassified as T2a (>3-≤5cm), T2b (>5-≤7cm), T3 (>7cm) or T≤3 cm (≤3cm but other T2 characteristics).
Of 538 eligible patients, 288 (53.5%) were T2a, 111 (21%) T2b, 62 (11.5%) T3, while 77 (14%) T≤3cm were excluded to avoid confounding. KRAS mutations were detected in 104/390 (27%) patients. T-size was prognostic for disease-free survival (DFS; p=0.03), but borderline for overall survival (OS; p=0.10), on multivariable analysis. Significant interaction between the prognostic value of KRAS and tumor size was observed for OS (p=0.01), but not DFS (p=0.10). There was a non-significant trend (p=0.24) for increased chemotherapy effect on OS with advancing T-size (HR T2a 0.90, [0.63-1.30]; T2b 0.69, [0.38-1.24]; and T3 0.57, [0.28-1.17]). The HR for chemotherapy effect on OS in T2a patients with KRAS wild-type tumors was 0.81 (p=0.36), while a trend for detrimental effect was observed in those with mutant tumors (HR 2.11; p=0.09; interaction p=0.05). Similar trends were observed in T2b-T3 patients with wild-type (HR 0.86; p=0.62), and KRAS mutant tumors (HR 1.16; p=0.74; interaction p=0.58).
Chemotherapy effect appears to increase with tumor size. However, this small study could not identify subgroups of patients who did or did not derive significant benefit from adjuvant chemotherapy based on T-size or KRAS status.
Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
The advent of personalized medicine requires robust, reproducible biomarkers that indicate which treatment will maximize therapeutic benefit while minimizing side effects and costs. Numerous molecular signatures have been developed over the past decade to fill this need, but their validation and up-take into clinical settings has been poor. Here, we investigate the technical reasons underlying reported failures in biomarker validation for non-small cell lung cancer (NSCLC).
We evaluated two published prognostic multi-gene biomarkers for NSCLC in an independent 442-patient dataset. We then systematically assessed how technical factors influenced validation success.
Both biomarkers validated successfully (biomarker #1: hazard ratio (HR) 1.63, 95% confidence interval (CI) 1.21 to 2.19, P = 0.001; biomarker #2: HR 1.42, 95% CI 1.03 to 1.96, P = 0.030). Further, despite being underpowered for stage-specific analyses, both biomarkers successfully stratified stage II patients and biomarker #1 also stratified stage IB patients. We then systematically evaluated reasons for reported validation failures and find they can be directly attributed to technical challenges in data analysis. By examining 24 separate pre-processing techniques we show that minor alterations in pre-processing can change a successful prognostic biomarker (HR 1.85, 95% CI 1.37 to 2.50, P < 0.001) into one indistinguishable from random chance (HR 1.15, 95% CI 0.86 to 1.54, P = 0.348). Finally, we develop a new method, based on ensembles of analysis methodologies, to exploit this technical variability to improve biomarker robustness and to provide an independent confidence metric.
Biomarkers comprise a fundamental component of personalized medicine. We first validated two NSCLC prognostic biomarkers in an independent patient cohort. Power analyses demonstrate that even this large, 442-patient cohort is under-powered for stage-specific analyses. We then use these results to discover an unexpected sensitivity of validation to subtle data analysis decisions. Finally, we develop a novel algorithmic approach to exploit this sensitivity to improve biomarker robustness.
Aldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), a n d metabolizing these lipid substrates plays a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohistochemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 over-expressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared to a normal pancreatic ductal epithelial cell (HPDE6E7), all of six cultured pancreatic adenocarcinoma cell lines had a over-expression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in 1) increased cell apoptosis, 2) increased non-farnesyled HDJ2 protein, and 3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence of that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation.
pancreatic adenocarcinoma; AKR1B10; prenylation; smoking; immunohistochemistry
Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.
IKBKB (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in regulation of NF-κB signaling. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer (NSCLC).
644 tumor- and 90 cell line-genomes were analyzed for gene-dosage status of the individual complex components and IKBKB. Gene expression of these genes, and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition.
We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. While both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity.
Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Here we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in NSCLC.
KEAP1; CUL3; RBX1; IKBKB; NF-κB signaling; genetic disruption
For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.