Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer
Curator, Writer: Stephen J. Williams, Ph.D.
(photo credit: cancer.gov)
A report Lung Cancer Genome Surveys Find Many Potential Drug Targets, in the NCI Bulletin,
http://www.cancer.gov/ncicancerbulletin/091812/page2
summarizes the clinical importance of five new lung cancer genome sequencing projects. These studies have identified genetic and epigenetic alterations in hundreds of lung tumors, of which some alterations could be taken advantage of using currently approved medications.
The reports, all published this month, included genomic information on more than 400 lung tumors. In addition to confirming genetic alterations previously tied to lung cancer, the studies identified other changes that may play a role in the disease.
Collectively, the studies covered the main forms of the disease—lung adenocarcinomas, squamous cell cancers of the lung, and small cell lung cancers.
“All of these studies say that lung cancers are genomically complex and genomically diverse,” said Dr. Matthew Meyerson of Harvard Medical School and the Dana-Farber Cancer Institute, who co-led several of the studies, including a large-scale analysis of squamous cell lung cancer by The Cancer Genome Atlas (TCGA) Research Network.
Some genes, Dr. Meyerson noted, were inactivated through different mechanisms in different tumors. He cautioned that little is known about alterations in DNA sequences that do not encode genes, which is most of the human genome.
Four of the papers are summarized below, with the first described in detail, as the Nature paper used a multi-‘omics strategy to evaluate expression, mutation, and signaling pathway activation in a large cohort of lung tumors. A literature informatics analysis is given for one of the papers. Please note that links on GENE names usually refer to the GeneCard entry.
Paper 1. Comprehensive genomic characterization of squamous cell lung cancers[1]
The Cancer Genome Atlas Research Network Project just reported, in the journal Nature, the results of their comprehensive profiling of 230 resected lung adenocarcinomas. The multi-center teams employed analyses of
- microRNA
- Whole Exome Sequencing including
- Exome mutation analysis
- Gene copy number
- Splicing alteration
- Methylation
- Proteomic analysis
Summary:
Some very interesting overall findings came out of this analysis including:
- High rates of somatic mutations including activating mutations in common oncogenes
- Newly described loss of function MGA mutations
- Sex differences in EGFR and RBM10 mutations
- driver roles for NF1, MET, ERBB2 and RITI identified in certain tumors
- differential mutational pattern based on smoking history
- splicing alterations driven by somatic genomic changes
- MAPK and PI3K pathway activation identified by proteomics not explained by mutational analysis = UNEXPLAINED MECHANISM of PATHWAY ACTIVATION
however, given the plethora of data, and in light of a similar study results recently released, there appears to be a great need for additional mining of this CGAP dataset. Therefore I attempted to curate some of the findings along with some other recent news relevant to the surprising findings with relation to biomarker analysis.
Makeup of tumor samples
230 lung adenocarcinomas specimens were categorized by:
Subtype
33% acinar
25% solid
14% micro-papillary
9% papillary
8% unclassified
5% lepidic
4% invasive mucinous
Gender
Smoking status
81% of patients reported past of present smoking
The authors note that TCGA samples were combined with previous data for analysis purpose.
A detailed description of Methodology and the location of deposited data are given at the following addresses:
Publication TCGA Web Page: https://tcga-data.nci.nih.gov/docs/publications/luad_2014/
Sequence files: https://cghub.ucsc.edu
Results:
Gender and Smoking Habits Show different mutational patterns
WES mutational analysis
- a) smoking status
– there was a strong correlations of cytosine to adenine nucleotide transversions with past or present smoking. In fact smoking history separated into transversion high (past and previous smokers) and transversion low (never smokers) groups, corroborating previous results.
→ mutations in groups Transversion High Transversion Low
TP53, KRAS, STK11, EGFR, RB1, PI3CA
KEAP1, SMARCA4 RBM10
- b) Gender
Although gender differences in mutational profiles have been reported, the study found minimal number of significantly mutated genes correlated with gender. Notably:
- EGFR mutations enriched in female cohort
- RBM10 loss of function mutations enriched in male cohort
Although the study did not analyze the gender differences with smoking patterns, it was noted that RBM10 mutations among males were more prevalent in the transversion high group.
Whole exome Sequencing and copy number analysis reveal Unique, Candidate Driver Genes
Whole exome sequencing revealed that 62% of tumors contained mutations (either point or indel) in known cancer driver genes such as:
KRAS, EGFR, BRMF, ERBB2
However, authors looked at the WES data from the oncogene-negative tumors and found unique mutations not seen in the tumors containing canonical oncogenic mutations.
Unique potential driver mutations were found in
TP53, KEAP1, NF1, and RIT1
The genomics and expression data were backed up by a proteomics analysis of three pathways:
- MAPK pathway
- mTOR
- PI3K pathway
…. showing significant activation of all three pathways HOWEVER the analysis suggested that activation of signaling pathways COULD NOT be deduced from DNA sequencing alone. Phospho-proteomic analysis was required to determine the full extent of pathway modification.
For example, many tumors lacked an obvious mutation which could explain mTOR or MAPK activation.
Altered cell signaling pathways included:
- Increased MAPK signaling due to activating KRAS
- Higher mTOR due to inactivating STK11 leading to increased proliferation, translation
Pathway analysis of mutations revealed alterations in multiple cellular pathways including:
- Reduced oxidative stress response
- Nucleosome remodeling
- RNA splicing
- Cell cycle progression
- Histone methylation
Summary:
Authors noted some interesting conclusions including:
- MET and ERBB2 amplification and mutations in NF1 and RIT1 may be unique driver events in lung adenocarcinoma
- Possible new drug development could be targeted to the RTK/RAS/RAF pathway
- MYC pathway as another important target
- Cluster analysis using multimodal omics approach identifies tumors based on single-gene driver events while other tumor have multiple driver mutational events (TUMOR HETEROGENEITY)
Paper 2. A Genomics-Based Classification of Human Lung Tumors[2]
The paper can be found at
http://stm.sciencemag.org/content/5/209/209ra153
by The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM),*,†
Paper Summary
This sequencing project revealed discrepancies between histologic and genomic classification of lung tumors.
Methodology
– mutational analysis by whole exome sequencing of 1255 lung tumors of histologically
defined subtypes
– immunohistochemistry performed to verify reclassification of subtypes based on sequencing data
Results
- 55% of all cases had at least one oncogenic alteration amenable to current personalized treatment approaches
- Marked differences existed between cluster analysis within and between preclassified histo-subtypes
- Reassignment based on genomic data eliminated large cell carcinomas
- Prospective classification of 5145 lung cancers allowed for genomic classification in 75% of patients
- Identification of EGFR and ALK mutations led to improved outcomes
Conclusions:
It is feasible to successfully classify and diagnose lung tumors based on whole exome sequencing data.
Paper 3. Genomic Landscape of Non-Small Cell Lung Cancer in Smokers and Never-Smokers[3]
A link to the paper can be found here with Graphic Summary: http://www.cell.com/cell/abstract/S0092-8674%2812%2901022-7?cc=y?cc=y
Methodology
- Whole genome sequencing and transcriptome sequencing of cancerous and adjacent normal tissues from 17 patients with NSCLC
- Integrated RNASeq with WES for analysis of
- Variant analysis
- Clonality by variant allele frequency anlaysis
- Fusion genes
- Bioinformatic analysis
Results
- 3,726 point mutations and more than 90 indels in the coding sequence
- Smokers with lung cancer show 10× the number of point mutations than never-smokers
- Novel lung cancer genes, including DACH1, CFTR, RELN, ABCB5, and HGF were identified
- Tumor samples from males showed high frequency of MYCBP2 MYCBP2 involved in transcriptional regulation of MYC.
- Variant allele frequency analysis revealed 10/17 tumors were at least biclonal while 7/17 tumors were monoclonal revealing majority of tumors displayed tumor heterogeneity
- Novel pathway alterations in lung cancer include cell-cycle and JAK-STAT pathways
- 14 fusion proteins found, including ROS1-ALK fusion. ROS1-ALK fusions have been frequently found in lung cancer and is indicative of poor prognosis[4].
- Novel metabolic enzyme fusions
- Alterations were identified in 54 genes for which targeted drugs are available. Drug-gable mutant targets include: AURKC, BRAF, HGF, EGFR, ERBB4, FGFR1, MET, JAK2, JAK3, HDAC2, HDAC6, HDAC9, BIRC6, ITGB1, ITGB3, MMP2, PRKCB, PIK3CG, TERT, KRAS, MMP14
Table. Validated Gene-Fusions Obtained from Ref-Seq Data
Note: Gene columns contain links for GeneCard while Gene function links are to the gene’s GO (Gene Ontology) function.
† Author’s Note:
There has been a recent literature on the importance of the EML4-ALK fusion protein in lung cancer. EML4-ALK positive lung tumors were found to be les chemo sensitive to cytotoxic therapy[5] and these tumor cells may exhibit an epitope rendering these tumors amenable to immunotherapy[6]. In addition, inhibition of the PI3K pathway has sensitized EMl4-ALK fusion positive tumors to ALK-targeted therapy[7]. EML4-ALK fusion positive tumors show dependence on the HSP90 chaperone, suggesting this cohort of patients might benefit from the new HSP90 inhibitors recently being developed[8].
Table. Significantly mutated genes (point mutations, insertions/deletions) with associated function.
| Gene |
Function |
| TP53 |
tumor suppressor |
| KRAS |
oncogene |
| ZFHX4 |
zinc finger DNA binding |
| DACH1 |
transcription factor |
| EGFR |
epidermal growth factor receptor |
| EPHA3 |
receptor tyrosine kinase |
| ENSG00000205044 |
|
| RELN |
cell matrix protein |
| ABCB5 |
ABC Drug Transporter |
Table. Literature Analysis of pathways containing significantly altered genes in NSCLC reveal putative targets and risk factors, linkage between other tumor types, and research areas for further investigation.
Note: Significantly mutated genes, obtained from WES, were subjected to pathway analysis (KEGG Pathway Analysis) in order to see which pathways contained signicantly altered gene networks. This pathway term was then used for PubMed literature search together with terms “lung cancer”, “gene”, and “NOT review” to determine frequency of literature coverage for each pathway in lung cancer. Links are to the PubMEd search results.
| KEGG pathway Name |
# of PUBMed entries containing Pathway Name, Gene ANDLung Cancer |
| Cell cycle |
1237 |
| Cell adhesion molecules (CAMs) |
372 |
| Glioma |
294 |
| Melanoma |
219 |
| Colorectal cancer |
207 |
| Calcium signaling pathway |
175 |
| Prostate cancer |
166 |
| MAPK signaling pathway |
162 |
| Pancreatic cancer |
88 |
| Bladder cancer |
74 |
| Renal cell carcinoma |
68 |
| Focal adhesion |
63 |
| Regulation of actin cytoskeleton |
34 |
| Thyroid cancer |
32 |
| Salivary secretion |
19 |
| Jak-STAT signaling pathway |
16 |
| Natural killer cell mediated cytotoxicity |
11 |
| Gap junction |
11 |
| Endometrial cancer |
11 |
| Long-term depression |
9 |
| Axon guidance |
8 |
| Cytokine-cytokine receptor interaction |
8 |
| Chronic myeloid leukemia |
7 |
| ErbB signaling pathway |
7 |
| Arginine and proline metabolism |
6 |
| Maturity onset diabetes of the young |
6 |
| Neuroactive ligand-receptor interaction |
4 |
| Aldosterone-regulated sodium reabsorption |
2 |
| Systemic lupus erythematosus |
2 |
| Olfactory transduction |
1 |
| Huntington’s disease |
1 |
| Chemokine signaling pathway |
1 |
| Cardiac muscle contraction |
1 |
| Amyotrophic lateral sclerosis (ALS) |
1 |
A few interesting genetic risk factors and possible additional targets for NSCLC were deduced from analysis of the above table of literature including HIF1-α, mIR-31, UBQLN1, ACE, mIR-193a, SRSF1. In addition, glioma, melanoma, colorectal, and prostate and lung cancer share many validated mutations, and possibly similar tumor driver mutations.
please click on graph for larger view
Paper 4. Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing[9]
For full paper and graphical summary please follow the link: http://www.cell.com/cell/abstract/S0092-8674%2812%2901061-6
Highlights
- Exome and genome characterization of somatic alterations in 183 lung adenocarcinomas
- 12 somatic mutations/megabase
- U2AF1, RBM10, and ARID1A are among newly identified recurrently mutated genes
- Structural variants include activating in-frame fusion of EGFR
- Epigenetic and RNA deregulation proposed as a potential lung adenocarcinoma hallmark
Summary
Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.
Paper 5. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer[10]
Highlights
- Whole exome and transcriptome (RNASeq) sequencing 29 small-cell lung carcinomas
- High mutation rate 7.4 protein-changing mutations/million base pairs
- Inactivating mutations in TP53 and RB1
- Functional mutations in CREBBP, EP300, MLL, PTEN, SLIT2, EPHA7, FGFR1 (determined by literature and database mining)
- The mutational spectrum seen in human data also present in a Tp53-/- Rb1-/- mouse lung tumor model
Curator Graphical Summary of Interesting Findings From the Above Studies
The above figure (please click on figure) represents themes and findings resulting from the aforementioned studies including
questions which will be addressed in Future Posts on this site.
References:
- Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012, 489(7417):519-525.
- A genomics-based classification of human lung tumors. Science translational medicine 2013, 5(209):209ra153.
- Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, Maher CA, Fulton R, Fulton L, Wallis J et al: Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012, 150(6):1121-1134.
- Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, Asaka R, Hamanaka W, Ninomiya H, Uehara H et al: RET, ROS1 and ALK fusions in lung cancer. Nature medicine 2012, 18(3):378-381.
- Morodomi Y, Takenoyama M, Inamasu E, Toyozawa R, Kojo M, Toyokawa G, Shiraishi Y, Takenaka T, Hirai F, Yamaguchi M et al: Non-small cell lung cancer patients with EML4-ALK fusion gene are insensitive to cytotoxic chemotherapy. Anticancer research 2014, 34(7):3825-3830.
- Yoshimura M, Tada Y, Ofuzi K, Yamamoto M, Nakatsura T: Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene. Oncology reports 2014, 32(1):33-39.
- Yang L, Li G, Zhao L, Pan F, Qiang J, Han S: Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2014.
- Workman P, van Montfort R: EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence. Cancer discovery 2014, 4(6):642-645.
- Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A et al: Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012, 150(6):1107-1120.
- Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, Plenker D, Leenders F, Sun R, Zander T et al: Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics 2012, 44(10):1104-1110.
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