Alterations of LKB1 and KRAS and risk of brain metastasis: Comprehensive characterization by mutation analysis, copy number, and gene expression in non-small-cell lung carcinoma

doi:10.1016/j.lungcan.2014.08.013

Lung Cancer

Volume 86, Issue 2, November 2014, Pages 255-261

https://doi.org/10.1016/j.lungcan.2014.08.013 Get rights and content

Highlights

•
We analyzed how LKB1 and KRAS alteration, measured by mutation, gene expression and copy number (CN) are associated with brain metastasis in NSCLC.
•
LKB1 CN and KRAS mutation were significantly associated with brain metastasis after adjusting for other variables.
•
We constructed a predictive model based on LKB1 CN and KRAS mutation for brain metastasis in NSCLC with good prediction accuracy.
•
If validated, the prediction model may serve as the basis on which further diagnosis and therapies for the treatment of brain metastasis in NSCLC.

Abstract

Background

Brain metastases are one of the most malignant complications of lung cancer and constitute a significant cause of cancer related morbidity and mortality worldwide. Recent years of investigation suggested a role of LKB1 in NSCLC development and progression, in synergy with KRAS alteration. In this study, we systematically analyzed how LKB1 and KRAS alteration, measured by mutation, gene expression (GE) and copy number (CN), are associated with brain metastasis in NSCLC.

Materials and methods

Patients treated at University of North Carolina Hospital from 1990 to 2009 with NSCLC provided frozen, surgically extracted tumors for analysis. GE was measured using Agilent 44,000 custom-designed arrays, CN was assessed by Affymetrix GeneChip Human Mapping 250K Sty Array or the Genome-Wide Human SNP Array 6.0 and gene mutation was detected using ABI sequencing. Integrated analysis was conducted to assess the relationship between these genetic markers and brain metastasis. A model was proposed for brain metastasis prediction using these genetic measurements.

Results

17 of the 174 patients developed brain metastasis. LKB1 wild type tumors had significantly higher LKB1 CN (p < 0.001) and GE (p = 0.002) than the LKB1 mutant group. KRAS wild type tumors had significantly lower KRAS GE (p < 0.001) and lower CN, although the latter failed to be significant (p = 0.295). Lower LKB1 CN (p = 0.039) and KRAS mutation (p = 0.007) were significantly associated with more brain metastasis. The predictive model based on nodal (N) stage, patient age, LKB1 CN and KRAS mutation had a good prediction accuracy, with area under the ROC curve of 0.832 (p < 0.001).

Conclusion

LKB1 CN in combination with KRAS mutation predicted brain metastasis in NSCLC.

Introduction

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the United States with brain metastasis as one of the most dreaded complications [1]. Historically, the prognosis of NSCLC with brain metastasis has been poor, with a median overall survival of 4.5 months for patients treated with standard whole brain radiation therapy (WBRT) and 4–11 weeks in untreated patients [2], [3]. The prevalence of brain metastasis in NSCLC is reported to be increasing, possibly due to improved diagnosis in brain imaging and prolonged survival with new systemic treatment options [4]. Therefore, identification of biomarkers that have critical roles in cell growth, metabolism, and tumor recurrence would provide valuable information in disease prognosis and better treatment choices.

In the past few years, several lines of evidence implicate the importance of liver kinase B1 (LKB1, aka, serine-threonine kinase or STK11) as a tumor suppressor gene in lung cancer development and progression in both human and model organisms [5], [6]. LKB1 was first identified in 1997 as the causative mutation in the autosomal-dominant inherited Peutz–Jeghers Syndrome (PJS) [7]. LKB1 loss is one of the most frequent genetic alterations in NSCLC [8], the inactivation of which has also been proposed to be associated with tumor metastasis in lung cancer and other tumor types [5], [6], [9]. Specifically, LKB1 mutation or loss of heterozygosity (LOH) of 19p13.2 which harbors the LKB1 gene was observed in a much higher proportion in brain metastases of lung cancer patients than in the primary tumors [5], [10].

As with many tumor suppressor genes, identifying patients with LKB1 inactivation remains a challenge, with potential mechanisms including homozygous deletion, point mutations and epigenetic silencing [5], [6]. The discrepancy between the high frequency of LOH (often over 50%) of 19p13.3 [11] and the reported rate of LKB1 mutation [5], [8] suggests that many “second hits” to the gene may go undetected by current sequencing techniques or that epigenetic silencing or other inactivating events may be more prevalent than previously recognized. In any case, for the purposes of clinical assessment, investigators are challenged to assess the gene through multiple mechanisms to gain confidence in characterizing the gene as intact or altered. In addition, multiple investigators have now reported coordination between losses of LKB1 and the oncogene, KRAS, particularly in smokers suggesting that coordinated assessment may be clinically relevant.

In this study, we seek to identify how LKB1 alteration, assessed by gene mutation, gene expression (GE) and copy number (CN) change, can predict brain metastasis in a group of NSCLC patients in conjunction with KRAS aberration, which has been shown to have a synergistic effect with LKB1 inactivation in lung cancer development and metastasis [6].

Section snippets

Tumor collection and clinical data abstraction

Frozen tumors were collected from patients who received curative surgery at the University of North Carolina (UNC) hospital with NSCLC diagnosis from December 1990 to September 2009. Tissues were flash-frozen and stored at −80 °C until time of analysis. Tumor histology includes adenocarcinoma [12], adenosquamous carcinoma, bronchioloalveolar carcinoma, large cell carcinoma and squamous cell carcinoma [13]. Patient outcomes were assessed by retrospective chart review for vital status and tumor

Patient characteristics with respect to genetic biomarkers

174 of the patients provided sufficient tissue for at least one measurement of LKB1 alteration and were included in subsequent analysis, in which 172 had GE measurement, 162 had CN and 172 had mutation data. Diagnosis age ranges from 39 to 90 with a median of 66 years; approximately half of these patients (88) are males and most of them (161) had smoking history. The majority of these patients (153) were diagnosed when the tumor was still small (T1 or T2). Half of the patients (87) had

Discussion

The poor outcomes of patients with lung cancer have been widely reported, including the frequent occurrence of brain metastases in patients who have otherwise controlled their disease through primary therapy. In small cell lung cancer progress has been made toward preventing brain metastases through prophylactic cranial radiation which has a proven survival benefit [21]. Attempts to extend this benefit to patients with NSCLC have similarly documented a small benefit in terms of prevention of

Funding

This study was supported by the Thomas G. Labreque Foundation, through Joan's Legacy Foundation and by a Clinical/Translational Award from the UNC Lineberger Comprehensive Cancer Center.

Conflict of interest statement

D. Neil Hayes and N. Zhao hold a provisional patent on the predictive model of brain metastasis.

References (35)

L. Gaspar et al.
Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials
Int J Radiat Oncol Biol Phys
(1997)
F. Ampil et al.
The elderly with synchronous non-small cell lung cancer and solitary brain metastasis: does palliative thoracic radiotherapy have a useful role
Lung Cancer
(2007)
S. Aviel-Ronen et al.
K-ras mutations in non-small-cell lung carcinoma: a review
Clin Lung Cancer
(2006)
M. Schemper et al.
A note on quantifying follow-up in studies of failure time
Control Clin Trials
(1996)
W. Liu et al.
LKB1/STK11 inactivation leads to expansion of a prometastatic tumor subpopulation in melanoma
Cancer Cell
(2012)
T.H. Shaikh
Oligonucleotide arrays for high-resolution analysis of copy number alteration in mental retardation/multiple congenital anomalies
Genet Med
(2007)
J.J.S.A. Laskin et al.
Non-small cell lung cancer
(2005)
A.F. Eichler et al.
The biology of brain metastases-translation to new therapies
Nat Rev Clin Oncol
(2011)
S. Matsumoto et al.
Prevalence and specificity of LKB1 genetic alterations in lung cancers
Oncogene
(2007)
H. Ji et al.
LKB1 modulates lung cancer differentiation and metastasis
Nature
(2007)

A. Hemminki et al.

A serine/threonine kinase gene defective in Peutz–Jeghers syndrome

Nature

(1998)

M. Sanchez-Cespedes et al.

Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung

Cancer Res

(2002)

Z.G. Zhuang et al.

Enhanced expression of LKB1 in breast cancer cells attenuates angiogenesis, invasion, and metastatic potential

Mol Cancer Res

(2006)

M. Tiainen et al.

Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest

Proc Natl Acad Sci U S A

(1999)

S.B. Sobottka et al.

Frequent loss of heterozygosity at the 19p13.3 locus without LKB1/STK11 mutations in human carcinoma metastases to the brain

J Neurooncol

(2000)

M.D. Wilkerson et al.

Differential pathogenesis of lung adenocarcinoma subtypes involving sequence mutations, copy number, chromosomal instability, and methylation

PLoS ONE

(2012)

M.D. Wilkerson et al.

Lung squamous cell carcinoma mRNA expression subtypes are reproducible, clinically important, and correspond to normal cell types

Clin Cancer Res

(2010)

Cited by (65)

D-1553 (Garsorasib), a Potent and Selective Inhibitor of KRAS<sup>G12C</sup> in Patients With NSCLC: Phase 1 Study Results
2023, Journal of Thoracic Oncology
D-1553 (garsorasib) is a potent and selective oral KRAS^G12C inhibitor. We report results from a phase I dose-escalation and dose-expansion study of D-1553 in patients with KRAS G12C–mutated NSCLC in multiple sites in the People’s Republic of China.
Patients with KRAS G12C–mutated NSCLC have administrated D-1553 600 mg orally once daily, 800 mg once daily, 1200 mg once daily, 400 mg twice a day, or 600 mg twice a day in dose escalation. In dose-expansion, all patients received 600 mg twice a day. The safety, pharmacokinetics, and efficacy of D-1553 were evaluated.
Among a total of 79 treated patients, 75 patients (94.9%) reported treatment-related adverse events with 30 patients experiencing grade 3 or 4 events (38.0%). Most of the adverse events were manageable and the patients tolerated the study treatment well. Among 74 patients assessable for efficacy analysis, 30 patients had a partial response and 38 had stable disease with a confirmed objective response rate (ORR) and disease control rate (DCR) of 40.5% and 91.9%, respectively. The median progression-free survival was 8.2 months, and the median duration of response was 7.1 months. Among 62 patients assessable for response at the recommended phase 2 dose, partial response occurred in 24 patients (ORR, 38.7%) and stable disease in 32 patients (DCR, 90.3%). The median progression-free survival and duration of response were 7.6 months and 6.9 months, respectively. In patients with brain metastasis, ORR and DCR were 17% and 100%, respectively.
D-1553 represents a promising therapeutic option for patients with KRAS G12C–mutated NSCLC with a well-tolerated safety profile and encouraging antitumor activity.
Risk scoring based on DNA methylation-driven related DEGs for colorectal cancer prognosis with systematic insights
2023, Life Sciences
Colorectal cancer is a common malignant tumor of the digestive tract. Despite advances in diagnostic techniques and medications. Its prognosis remains challenging. DNA methylation-driven related circulating tumor cells have attracted enormous interest in diagnosing owing to their non-invasive nature and early recognition properties. However, the mechanism through which risk biomarkers act remains elusive.
Here, we designed a risk model based on differentially expressed genes, DNA methylation, robust, and survival-related factors in the framework of Cox regression. The model has satisfactory performance and is independently verified by an external and isolated dataset in terms of C-index value, ROC, and tROC. The model was applied to Colorectal cancer patients who were subsequently divided into high- and low-risk groups. Functional annotations, genomic alterations, tumor immune environment, and drug sensitivity were analyzed. We observed that up-regulated genes are associated with epithelial cell differentiation and MAPK signaling pathways. The down-regulated genes are related to IL-7 signaling and apoptosis-induced DNA fragmentation. Interestingly, the immune system was inhibited in high-risk groups. High-frequency mutation genes tend to co-occur. High-risk score patients are related to copy number amplification events. To address the challenges, we suggested eleven and twenty-one drugs that are sensitive to low- and high-risk patients. Finally, an artificial neural network was provided to evaluate the immunotherapeutic efficiency.
Taken together, the findings demonstrated that our risk score model is robust and reliable for evaluating the prognosis with novel diagnostic and treatment targets. It also yields benefits for the treatment and provides unique insights into developing therapeutic strategies.
Therapeutic strategies for non-small cell lung cancer: Experimental models and emerging biomarkers to monitor drug efficacies
2023, Pharmacology and Therapeutics
While new targeted therapies have considerably changed the treatment and prognosis of non-small cell lung cancer (NSCLC), they are frequently unsuccessful due to primary or acquired resistances. Chemoresistance is a complex process that combines cancer cell intrinsic mechanisms including molecular and genetic abnormalities, aberrant interactions within the tumor microenvironment, and the pharmacokinetic characteristics of each molecule. From a pharmacological point of view, two levers could improve the response to treatment: (i) developing tools to predict the response to chemo- and targeted therapies and (ii) gaining a better understanding of the influence of the tumor microenvironment. Both personalized medicine approaches require the identification of relevant experimental models and biomarkers to understand and fight against chemoresistance mechanisms. After describing the main therapies in NSCLC, the scope of this review will be to identify and to discuss relevant in vitro and ex vivo experimental models that are able to mimic tumors. In addition, the interests of these models in the predictive responses to proposed therapies will be discussed. Finally, this review will evaluate the involvement of novel secreted biomarkers such as tumor DNA or micro RNA in predicting responses to anti-tumor therapies.
STK11 Prevents Invasion through Signal Transducer and Activator of Transcription 3/5 and FAK Repression in Cutaneous Melanoma
2022, Journal of Investigative Dermatology
Citation Excerpt :
The evaluation of STK11 expression on melanoma TMAs revealed that STK11 was highly expressed in primary tumors but significantly decreased in metastases, with the lowest levels observed in brain metastases. This finding is consistent with previous observations in nonsmall cell lung carcinoma where an oncogenic KRAS mutation and low STK11 copy number were shown to be associated with brain metastasis (Zhao et al., 2014). In addition, STK11 deletion is frequently shown in brain metastases of different carcinomas (Sobottka et al., 2000).
The STK11/LKB1 is a tumor suppressor involved in metabolism and cell motility. In BRAF^V600E melanoma, STK11 is inactivated by extracellular signal‒regulated kinase and RSK, preventing it from binding and activating adenosine monophosphate-activated protein kinase and promoting melanoma cell proliferation. Although STK11 mutations occur in 5‒10% of cutaneous melanoma, few functional studies have been performed. By knocking out STK11 with CRISPR/Cas9 in two human BRAF-mutant melanoma cell lines, we found that STK11 loss reduced the sensitivity to a BRAF inhibitor. More strikingly, STK11 loss led to an increased invasive phenotype in both three-dimensional spheroids and in vivo zebrafish xenograft models. STK11 overexpression consistently reversed the invasive phenotype. Interestingly, STK11 knockout increased invasion also in an NRAS-mutant melanoma cell line. Furthermore, although STK11 was expressed in primary human melanoma tumors, its expression significantly decreased in melanoma metastases, especially in brain metastases. In the STK11-knockout cells, we observed increased activating phosphorylation of signal transducer and activator of transcription 3/5 and FAK. Using inhibitors of signal transducer and activator of transcription 3/5 and FAK, we reversed the invasive phenotype in both BRAF- and NRAS-mutated cells. Our findings confirm an increased invasive phenotype on STK11 inactivation in BRAF- and NRAS-mutant cutaneous melanoma that can be targeted by signal transducer and activator of transcription 3/5 and FAK inhibition.
Cancer driver gene and non-coding RNA alterations as biomarkers of brain metastasis in lung cancer: A review of the literature
2021, Biomedicine and Pharmacotherapy
Brain metastasis (BM) is the most common event in patients with lung cancer. Despite multimodal treatments and advances in systemic therapies, development of BM remains one of the main factors associated with poor prognosis and mortality in patients with lung cancer. Therefore, better understanding of mechanisms involved in lung cancer brain metastasis (LCBM) is of great importance to suppress cancer cells and to improve the overall survival of patients. Several cancer-related genes such as EGFR and KRAS have been proposed as potential predictors of LCBM. In addition, there is ample evidence supporting crucial roles of non-coding RNAs (ncRNAs) in mediating LCBM. In this review, we provide comprehensive information on risk assessment, predictive, and prognostic panels for early detection of BM in patients with lung cancer. Moreover, we present an overview of LCBM molecular mechanisms, cancer driver genes, and ncRNAs which may predict the risk of BM in lung cancer patients. Recent clinical studies have focused on determining mechanisms involved in LCBM and their association with diagnosis, prognosis, and treatment outcomes. These studies have shown that alterations in EGFR, KRAS, BRAF, and ALK, as the most frequent coding gene alterations, and dysregulation of ncRNAs such as miR-423, miR-330–3p, miR-145, piR-651, and MALAT1 can be considered as potential biomarkers of LCBM.
Driver and novel genes correlated with metastasis of non-small cell lung cancer: A comprehensive analysis
2021, Pathology Research and Practice
Citation Excerpt :
Then 10584 records were found through literature searching, and 171 of full-text articles were assessed for eligibility after duplications deletion and abstracts screening (Fig. 1B). Subsequently, 112 studies were included due to the inclusion criteria [21–132]. Besides, four additional records were identified through Gene Expression Omnibus (GEO) databases [133–136].
Although mutations of genes are crucial events in tumorigenesis and development, the association between gene mutations and lung cancer metastasis is still largely unknown. The goal of this study is to identify driver and novel genes associated with non-small cell lung cancer (NSCLC) metastasis. Candidate genes were identified using a novel comprehensive analysis, which was based on bioinformatics technology and meta-analysis. Firstly, EGFR, KRAS, ALK, TP53, BRAF and PIK3CA were identified as candidate driver genes. Further meta-analysis identified that EGFR (Pooled OR 1.33, 95% CI 1.19, 1.50; P < .001) and ALK (Pooled OR 1.52, 95% CI 1.22, 1.89; P < .001) mutations were associated with distant metastasis of NSCLC. Besides, ALK (Pooled OR 2.40, 95% CI 1.71, 3.38; P < .001) mutation was associated with lymph node metastasis of NSCLC. In addition, thirteen novel gene mutations were identified to be correlated with NSCLC metastasis, including SMARCA1, GGCX, KIF24, LRRK1, LILRA4, OR2T10, EDNRB, NR1H4, ARID4A, PRKCI, PABPC5, ACAN and TLN1. Furthermore, elevated mRNA expression level of SMARCA1 and EDNRB was associated with poor overall survival in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), respectively. Additionally, pathway and protein-protein interactions network analyses found the two genes were correlated with epithelial-mesenchymal transition process. In conclusion, mutations of EGFR and ALK were significantly correlated with NSCLC metastasis. In addition, thirteen novel genes were identified to be associated with NSCLC metastasis, especially SMARCA1 in LUAD and EDNRB in LUSC.

View all citing articles on Scopus

¹: These authors are equally contributed to the manuscript.

View full text

Alterations of LKB1 and KRAS and risk of brain metastasis: Comprehensive characterization by mutation analysis, copy number, and gene expression in non-small-cell lung carcinoma

Highlights

Abstract

Background

Materials and methods

Results

Conclusion

Introduction

Section snippets

Tumor collection and clinical data abstraction

Patient characteristics with respect to genetic biomarkers

Discussion

Funding

Conflict of interest statement

Int J Radiat Oncol Biol Phys

Lung Cancer

Clin Lung Cancer

Control Clin Trials

Cancer Cell

Genet Med

Non-small cell lung cancer

The biology of brain metastases-translation to new therapies

Nat Rev Clin Oncol

Prevalence and specificity of LKB1 genetic alterations in lung cancers

Oncogene

LKB1 modulates lung cancer differentiation and metastasis

Nature

A serine/threonine kinase gene defective in Peutz–Jeghers syndrome

Nature

Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung

Cancer Res

Enhanced expression of LKB1 in breast cancer cells attenuates angiogenesis, invasion, and metastatic potential

Mol Cancer Res

Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest

Proc Natl Acad Sci U S A

Frequent loss of heterozygosity at the 19p13.3 locus without LKB1/STK11 mutations in human carcinoma metastases to the brain

J Neurooncol

Differential pathogenesis of lung adenocarcinoma subtypes involving sequence mutations, copy number, chromosomal instability, and methylation

PLoS ONE

Lung squamous cell carcinoma mRNA expression subtypes are reproducible, clinically important, and correspond to normal cell types

Clin Cancer Res