Combined clinical and genomic signatures for the prognosis of early stage non-small cell lung cancer based on gene copy number alterations.

BACKGROUND: The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. RESULTS: A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). CONCLUSION: The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.

New syngeneic inflammatory-related lung cancer metastatic model harboring double KRAS/WWOX alterations.

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition), accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.

Phenotypic and genetic characterization of circulating tumor cells by combining immunomagnetic selection and FICTION techniques.

The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies.

Glucose availability controls ATF4-mediated MITF suppression to drive melanoma cell growth.

It is well know that cancer cells have adopted an altered metabolism and that glucose is a major source of energy for these cells. In melanoma, enhanced glucose usage is favoured through the hyper-activated MAPK pathway, which suppresses OXPHOS and stimulates glycolysis. However, it has not been addressed how glucose availability impacts on melanoma specific signaling pathways that drive melanoma cell proliferation. Here we show that melanoma cells are dependent on high glucose levels for efficient growth. Thereby, glucose metabolism controls the expression of the melanoma fate transcription factor MITF, a master regulator of melanoma cell survival and proliferation, invasion and therapy resistance. Restriction of glucose availability to physiological concentrations induces the production of reactive oxygen species (ROS). Increased ROS levels lead to the up-regulation of AFT4, which in turn suppresses MITF expression by competing with CREB, an otherwise potent inducer of the MITF promoter. Our data give new insight into the complex regulation of MITF, a key regulator of melanoma biology, and support previous findings that link metabolic disorders such as hyperglycemia and diabetes with increased melanoma risk.

Molecular profiling of computed tomography screen-detected lung nodules shows multiple malignant features.

RATIONALE AND PURPOSE: Low-dose spiral computerized axial tomography (spiral CT) is effective for the detection of small early lung cancers. Although published data seem promising, there has been a significant degree of discussion concerning the potential of overdiagnosis in the context of spiral CT-based screening. The objective of the current study was to analyze the phenotypic and genetic alterations in the small pulmonary malignancies resected after detection in the University of Navarra/International Early Lung Cancer Action Project spiral CT screening trial and to determine whether their malignant molecular features are similar to those of resected lung tumors diagnosed conventionally. EXPERIMENTAL DESIGN: We analyzed 17 biomarkers of lung epithelial malignancy in a series of 11 tumors resected at our institution during the last 4 years (1,004 high-risk individuals screened), using immunohistochemistry and fluorescence in situ hybridization (FISH). A parallel series of 11 gender-, stage-, and histology-matched lung cancers diagnosed by other means except screening was used as control. RESULTS: The molecular alterations and the frequency of phenotypic or genetic aberrations were very similar when screen-detected and nonscreen-detected lung cancers were compared. Furthermore, most of the alterations found in the screen-detected cancers from this study were concordant with what has been described previously for stage I-II lung cancer. CONCLUSIONS: Small early-stage lung cancers resected after detection in a spiral CT-based screening trial reveal malignant molecular features similar to those found in conventionally diagnosed lung cancers, suggesting that the screen-detected cancers are not overdiagnosed.

Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer.

alpha CP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of alpha CP-4 and alpha CP-4a, an alternatively spliced variant of alpha CP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of alpha CP-4 and alpha CP-4a in normal lung bronchiolar epithelium. In contrast, alpha CP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed alpha CP-4a. Besides, lack of alpha CP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of alpha CP-4 and alpha CP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of alpha CP-4 in lung cancer, possibly associated with an altered processing of the alpha CP-4 mRNA leading to a predominant expression of alpha CP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of alpha CP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.

NUP98 is fused to HOXA9 in a variant complex t(7;11;13;17) in a patient with AML-M2.

The t(7;11)(p15;p15.4) has been reported to fuse the NUP98 gene (11p15), a component of the nuclear pore complex, with the class-1 homeobox gene HOXA9 at 7p15. This translocation has been associated with myeloid leukemias, predominantly acute myeloid leukemia (AML) M2 subtype with trilineage myelodysplastic features, and with a poor prognosis. The derived fusion protein retains the FG repeat motif of NUP98 N-terminus and the homeodomain shared by the HOX genes, acting as an oncogenic transcription factor critical for leukemogenesis. We report here a new complex t(7;11)-variant, i.e., t(7;11;13;17)(p15;p15;p?;p1?2) in a patient with AML-M2 and poor prognosis. The NUP98-HOXA9 fusion transcript was detected by RT-PCR, suggesting its role in the malignant transformation as it has been postulated for other t(7;11)-associated leukemias. No other fusion transcripts involving the NUP98 or HOXA9 genes were present, although other mechanisms involving several genes on chromosomes 13 and 17 may also be involved. To our knowledge, this is the first t(7;11) variant involving NUP98 described in hematological malignancies.

Variations in molecular profile in NSCLC can be analyzed using cytological samples: development of EGFR resistance mutations and coexistence of ALK-EML4 translocation in an EGFR-sensitive patient.

As a result of therapeutic advances, a revolution is taking place in the lung cancer field with major implications for pathologic diagnosis and tissue management. We report a case of a non-small cell lung carcinoma patient with coexistence of EGFR mutations and ALK-EML4 rearrangements that responded to EGFR inhibitors and in which the development of a new resistance mutation in exon 20 of EGFR-determined treatment resistance. All the molecular determinations were performed in cytological samples. To our knowledge, this is the first case reported with these characteristics, and the 11th case described with coexistence of EGFR mutations and ALK-EML4 rearrangements. The EGFR L858R mutation in exon 21 was found at diagnosis, and the patient presented a 4-year response to erlotinib. On progression, the T790M resistance mutation in the EGFR exon 20 was also confirmed in cytological samples. At this point, fluorescence in situ hybridization also detected ALK-EML4 translocation. This case emphasizes the usefulness of cytological samples for molecular analysis in lung adenocarcinoma, as well as the relevance of repeating biopsies/fine-needle aspirations in tumor recurrences to assess the mutation profile of the disease.

A core of kinase-regulated interactomes defines the neoplastic MDSC lineage.

Myeloid-derived suppressor cells (MDSCs) differentiate from bone marrow precursors, expand in cancer-bearing hosts and accelerate tumor progression. MDSCs have become attractive therapeutic targets, as their elimination strongly enhances anti-neoplastic treatments. Here, immature myeloid dendritic cells (DCs), MDSCs modeling tumor-infiltrating subsets or modeling non-cancerous (NC)-MDSCs were compared by in-depth quantitative proteomics. We found that neoplastic MDSCs differentially expressed a core of kinases which controlled lineage-specific (PI3K-AKT and SRC kinases) and cancer-induced (ERK and PKC kinases) protein interaction networks (interactomes). These kinases contributed to some extent to myeloid differentiation. However, only AKT and ERK specifically drove MDSC differentiation from myeloid precursors. Interfering with AKT and ERK with selective small molecule inhibitors or shRNAs selectively hampered MDSC differentiation and viability. Thus, we provide compelling evidence that MDSCs constitute a distinct myeloid lineage distinguished by a "kinase signature" and well-defined interactomes. Our results define new opportunities for the development of anti-cancer treatments targeting these tumor-promoting immune cells.

Contrasting responses of non-small cell lung cancer to antiangiogenic therapies depend on histological subtype.

The vascular endothelial growth factor (VEGF) pathway is a clinically validated antiangiogenic target for non-small cell lung cancer (NSCLC). However, some contradictory results have been reported on the biological effects of antiangiogenic drugs. In order to evaluate the efficacy of these drugs in NSCLC histological subtypes, we analyzed the anticancer effect of two anti-VEGFR2 therapies (sunitinib and DC101) in chemically induced mouse models and tumorgrafts of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Antiangiogenic treatments induced vascular trimming in both histological subtypes. In ADC tumors, vascular trimming was accompanied by tumor stabilization. In contrast, in SCC tumors, antiangiogenic therapy was associated with disease progression and induction of tumor proliferation. Moreover, in SCC, anti-VEGFR2 therapies increased the expression of stem cell markers such as aldehyde dehydrogenase 1A1, CD133, and CD15, independently of intratumoral hypoxia. In vitro studies with ADC cell lines revealed that antiangiogenic treatments reduced pAKT and pERK signaling and inhibited proliferation, while in SCC-derived cell lines the same treatments increased pAKT and pERK, and induced survival. In conclusion, this study evaluates for the first time the effect of antiangiogenic drugs in lung SCC murine models in vivo and sheds light on the contradictory results of antiangiogenic therapies in NSCLC.