Quantification and Dynamic Monitoring of EGFR T790M in Plasma Cell-Free DNA by Digital PCR for Prognosis of EGFR-TKI Treatment in Advanced NSCLC

Background

Among advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy.

Materials and Methods

We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification.

Results

Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007) and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001) compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively).

Conclusion

Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.     

Distinct Clinical Outcomes of Non-Small Cell Lung Cancer Patients with Epidermal Growth Factor Receptor (EGFR) Mutations Treated with EGFR Tyrosine Kinase Inhibitors: Non-Responders versus Responders

Introduction

Treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been associated with favorable progression free survival (PFS) in patients with non-small cell lung cancers (NSCLC) harboring EGFR mutations. However, a subset of this population doesn''t respond to EGFR-TKI treatment. Therefore, the present study aimed to elucidate survival outcome in NSCLC EGFR-mutant patients who were treated with EGFR TKIs.

Methods

Among the 580 consecutive NSCLC patients who were treated at our facility between 2008 and 2012, a total of 124 treatment-naïve, advanced NSCLC, EGFR-mutant patients treated with EGFR TKIs were identified and grouped into non-responders and responders for analyses.

Results

Of 124 patients, 104 (84%) responded to treatment, and 20 (16%) did not; and the overall median PFS was 9.0 months. Notably, the PFS, overall survival (OS) and survival rates were significantly unfavorable in non-responders (1.8 vs. 10.3 months, hazard ratio (HR) = 29.2, 95% confidence interval (CI), 13.48–63.26, P<0.0001; 9.4 vs. 17.3 months, HR = 2.74, 95% CI, 1.52–4.94, P = 0.0008; and 58% vs. 82% in 6, 37% vs. 60% in 12, and 19 vs. 40% at 24 months, respectively). In multivariate analysis, treatment efficacy strongly affected PFS and OS, independent of covariates (HR = 47.22, 95% CI, 17.88–124.73, P<0.001 and HR = 2.74, 95% CI, 1.43–5.24, P = 0.002, respectively). However, none of the covariates except of the presence of EGFR exon 19 deletion in the tumors was significantly associated with better treatment efficacy.

Conclusions

A subset of NSCLC EGFR-mutant patients displayed unfavorable survival despite EGFR TKI administration. This observation reinforces the urgent need for biomarkers effectively predicting the non-responders and for drug development overcoming primary resistance to EGFR TKIs. In addition, optimal therapeutic strategies to prolong the survival of non-responders need to be investigated.     

Frequency of EGFR Mutations in 907 Lung Adenocarcioma Patients of Indian Ethnicity

Background

During the past decade, the incidence of EGFR mutation has been shown to vary across different ethnicities. It occurs at the rate of 10–15% in North Americans and Europeans, 19% in African-Americans, 20–30% in various East Asian series including Chinese, Koreans, and Japanese. Frequency of EGFR mutations in India however remains sparsely explored.

Methodology/Principal Findings

We report 23% incidence of Epidermal growth factor receptor (EGFR) mutations in 907 Non small cell lung cancer (NSCLC) patients of Indian ethnicity, in contrast to 10–15% known in Caucasians and 27–62% among East Asians. In this study, EGFR mutations were found to be more common in never-smokers 29.4% as compared to smokers 15.3%. Consistent with other populations, mutation rates among adenocarcinoma-males were predominantly lower than females with 32% incidence. However unlike Caucasians, EGFR mutation rate among adenocarcinoma-never-smoker females were comparable to males suggesting lack of gender bias among never smokers likely to benefit from EGFR targeted therapy.

Conclusions/Significance

This study has an overall implication for establishing relevance for routine EGFR mutation diagnostics for NSCLC patients in clinics and emphasizes effectiveness for adoption of EGFR inhibitors as the first line treatment among Indian population. The intermediate frequency of EGFR mutation among Indian population compared to Caucasians and East Asians is reminiscent of an ancestral admixture of genetic influence from Middle Easterners, Central Asians, and Europeans on modern- Indian population that may confer differential susceptibility to somatic mutations in EGFR.     

Impact of EGFR Mutation Detection Methods on the Efficacy of Erlotinib in Patients with Advanced EGFR-Wild Type Lung Adenocarcinoma

Introduction

Methods used for epidermal growth factor receptor (EGFR) mutation testing vary widely. The impact of detection methods on the rates of response to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-wild type (wt) lung adenocarcinoma patients is unknown.

Methods

We recruited the Group-I patients to evaluate the efficacy of erlotinib in patients with EGFR-wt lung adenocarcinoma by either direct sequencing (DS) or mutant type-specific sensitive (MtS) methods in six medical centers in Taiwan. Cross recheck of EGFR mutations was performed in patients who achieved objective response to erlotinib and had adequate specimens. The independent Group-II lung adenocarcinoma patients whose EGFR mutation status determined by DS were recruited to evaluate the potential limitations of three MtS methods.

Results

In Group-I analysis, 38 of 261 EGFR-wt patients (14.6%) achieved partial response to erlotinib treatment. Nineteen patients (50.0%) had adequate specimens for cross recheck of EGFR mutations and 10 of them (52.6%) had changes in EGFR mutation status, 5 in 10 by DS and 5 in 9 by MtS methods originally. In Group-II analysis, 598 of 996 lung adenocarcinoma patients (60.0%) had detectable EGFR mutations. The accuracy rates of the three MtS methods, MALDI-TOF MS, Scorpions ARMS and Cobas, were 87.8%, 86.8% and 85.8%, respectively.

Conclusions

A significant portion of the erlotinib responses in EGFR-wt lung adenocarcinoma patients were related to the limitations of detection methods, not only DS but also MtS methods with similar percentages. Prospective studies are needed to define the proper strategy for EGFR mutation testing.     

Prognostic Value of Epidermal Growth Factor Receptor Mutations in Resected Non-Small Cell Lung Cancer: A Systematic Review with Meta-Analysis

Background

The prognostic value of epidermal growth factor receptor (EGFR) mutations in resected non-small cell lung cancer (NSCLC) remains controversial. We performed a systematic review with meta-analysis to assess its role.

Methods

Studies were identified via an electronic search on PubMed, Embase and Cochrane Library databases. Pooled hazard ratio (HR) for disease-free survival (DFS) and overall survival (OS) were calculated for meta-analysis.

Results

There were 16 evaluated studies (n = 3337) in the meta-analysis. The combined HR evaluating EGFR mutations on disease free survival was 0.96 (95% CI [0.79–1.16] P = 0.65). The combined HR evaluating EGFR mutations on overall survival was 0.86 (95% CI [0.72–1.04] P = 0.12). The subgroup analysis based on univariate and multivariate analyses in DFS and OS showed no statistically significant difference. There was also no statistically significant difference in DFS and OS of stage I NSCLC patients.

Conclusion

The systematic review with meta-analysis showed that EGFR mutations were not a prognostic factor in patients with surgically resected non-small cell lung cancer. Well designed prospective study is needed to confirm the result.     

Assessment of EGFR Mutations in Circulating Tumor Cell Preparations from NSCLC Patients by Next Generation Sequencing: Toward a Real-Time Liquid Biopsy for Treatment

Introduction

Assessment of EGFR mutation in non-small cell lung cancer (NSCLC) patients is mandatory for optimization of pharmacologic treatment. In this respect, mutation analysis of circulating tumor cells (CTCs) may be desirable since they may provide real-time information on patient''s disease status.

Experimental Design

Blood samples were collected from 37 patients enrolled in the TRIGGER study, a prospective phase II multi-center trial of erlotinib treatment in advanced NSCLC patients with activating EGFR mutations in tumor tissue. 10 CTC preparations from breast cancer patients without EGFR mutations in their primary tumors and 12 blood samples from healthy subjects were analyzed as negative controls. CTC preparations, obtained by the Veridex CellSearch System, were subjected to ultra-deep next generation sequencing (NGS) on the Roche 454 GS junior platform.

Results

CTCs fulfilling all Veridex criteria were present in 41% of the patients examined, ranging in number between 1 and 29. In addition to validated CTCs, potential neoplastic elements were seen in 33 cases. These included cells not fulfilling all Veridex criteria (also known as “suspicious objects”) found in 5 (13%) of 37 cases, and isolated or clustered large naked nuclei with irregular shape observed in 33 (89%) cases. EGFR mutations were identified by NGS in CTC preparations of 31 (84%) patients, corresponding to those present in matching tumor tissue. Twenty-five (96%) of 26 deletions at exon 19 and 6 (55%) of 11 mutations at exon 21 were detectable (P = 0.005). In 4 (13%) cases, multiple EGFR mutations, suggesting CTC heterogeneity, were documented. No mutations were found in control samples.

Conclusions

We report for the first time that the CellSearch System coupled with NGS is a very sensitive and specific diagnostic tool for EGFR mutation analysis in CTC preparations with potential clinical impact.     

Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients

Background

Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods.

Results

All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations.

Conclusions

Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS.     

Prognostic Value Analysis of Mutational and Clinicopathological Factors in Non-Small Cell Lung Cancer

Introduction

Targeting activating oncogenic driver mutations in lung adenocarcinoma has led to prolonged survival in patients harboring these specific genetic alterations. The prognostic value of these mutations has not yet been elucidated. The prevalence of recently uncovered non-coding somatic mutation in promoter region of TERT gene is also to be validated in lung cancer. The purpose of this study is to show the prevalence, association with clinicalpathological features and prognostic value of these factors.

Methods

In a cohort of patients with non-small cell lung cancer (NSCLC) (n = 174, including 107 lung adenocarcinoma and 67 lung squamous cell carcinoma), EGFR, KRAS, HER2 and BRAF were directly sequenced in lung adeoncarcinoma, ALK fusions were screened using FISH (Fluorescence in situ Hybridization).TERT promoter region was sequenced in all of the 174 NSCLC samples. Associations of these somatic mutations and clinicopathological features, as well as prognostic factors were evaluated.

Results

EGFR, KRAS, HER2, BRAF mutation and ALK fusion were mutated in 25.2%, 6.5%, 1.9%, 0.9% and 3.7% of lung adenocarcinomas. No TERT promoter mutation was validated by reverse-sided sequencing. Lung adenocarcinoma with EGFR and KRAS mutations showed no significant difference in Disease-free Survival (DFS) and Overall Survival (OS). Cox Multi-variate analysis revealed that only N stage and HER2 mutation were independent predictors of worse overall survival (HR = 1.653, 95% CI 1.219–2.241, P = 0.001; HR = 12.344, 95% CI 2.615–58.275, P = 0.002).

Conclusions

We have further confirmed that TERT promoter mutation may only exist in a very small fraction of NSCLCs. These results indicate that dividing lung adenocarcinoma into molecular subtypes according to oncogenic driver mutations doesn''t predict survival difference of the disease.     

Icotinib Is an Active Treatment of Non-Small-Cell Lung Cancer: A Retrospective Study

Background

Icotinib hydrochloride is a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with preclinical and clinical activity in non-small cell lung cancer (NSCLC). This retrospective analysis was performed to assess the efficacy of icotinib on patients with non-small-cell lung cancer (NSCLC).

Methods

82 consecutive patients treated with icotinib as first (n = 24) or second/third line (n = 58) treatment at three hospitals in Nanjing were enrolled into our retrospective research. The Response Evaluation Criteria in Solid Tumors (RECIST) was used to evaluate the tumor responses and the progression-free survival (PFS) and overall survival (OS) was evaluated by the Kaplan-Meier method.

Results

Median PFS was 4.0 months (95% CI 2.311–5.689). Median OS was 11.0 months (95% CI 8.537–13.463) in this cohort. Median PFS for first and second/third line were 7.0 months (95% CI 2.151–11.8) and 3.0 months (95% CI 1.042–4.958), respectively. Median OS for first and second/third line were 13.0 months (95% CI 10.305–15.695) and 10.0 months (95% CI 7.295–12.70), respectively. In patients with EGFR mutation (n = 19), icotinib significantly reduced the risk of progression (HR 0.36, 95% CI 0.18–0.70, p = 0.003) and death (HR 0.10, 95% CI 0.02–0.42, p = 0.002) compared with those EGFR status unknown (n = 63). The most common adverse events were acne-like rash (39.0%) and diarrhea (20.7%).

Conclusions

Icotinib is active in the treatment of patients with NSCLC both in first or second/third line, especially in those patients harbouring EGFR mutations, with an acceptable adverse event profile.     

Concomitant Tumor Expression of EGFR and TATI/SPINK1 Associates with Better Prognosis in Colorectal Cancer

Background

Epidermal growth factor receptor (EGFR) activation plays a role in colorectal cancer (CRC) carcinogenesis, and anti-EGFR drugs are used in treatment of advanced CRC. One of the EGFR ligands is tumor-associated trypsinogen inhibitor TATI, also called serine protease inhibitor Kazal type1 (SPINK 1), which we recently showed to be an independent prognostic marker in CRC.

Methods

We studied the prognostic value of immunohistochemical expression of EGFR and concomitant expression of EGFR and TATI/SPINK1 in a series of 619 colorectal cancer patients.

Results

Of the samples, 92% were positive for EGFR. EGFR+/TATI+ was seen in 62.8%, EGFR+/TATI− in 29.5%, EGFR−/TATI+ in 4.9%, and EGFR−/TATI− in 2.7% of patients. EGFR expression correlated with WHO grade (p = 0.040). In univariate analysis, EGFR expression correlated with favourable survival (p = 0.006). EGFR+/TATI+ patients showed better survival than did those with other combinations (p<0.001). In multivariate analysis, EGFR+/TATI+ was an independent prognostic factor of favourable prognosis (p<0.001).

Conclusion

Concomitant positivity of EGFR and TATI/SPINK1 predicts favourable prognosis in CRC.     

The Molecular Detection and Clinical Significance of ALK Rearrangement in Selected Advanced Non-Small Cell Lung Cancer: ALK Expression Provides Insights into ALK Targeted Therapy

Background

This study aimed to elucidate clinical significance of anaplastic lymphoma kinase (ALK) rearrangement in selected advanced non-small cell lung cancer (NSCLC), to compare the application of different ALK detection methods, and especially evaluate a possible association between ALK expression and clinical outcomes in crizotinib-treated patients.

Methods

ALK status was assessed by fluorescent in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) in 173 selected advanced NSCLC patients. Clinicopathologic data, genotype status and survival outcomes were analyzed. Moreover, the association of ALK expression with clinical outcomes was evaluated in ALK FISH-positive crizotinib-treated patients including two patients with concurrent epidermal growth factor receptor (EGFR) mutation.

Results

The positivity detection rate of ALK rearrangement by FISH, IHC and qRT-PCR was 35.5% (59/166), 35.7% (61/171), and 27.9% (34/122), respectively. ALK rearrangement was observed predominantly in young patients, never or light smokers, and adenocarcinomas, especially with signet ring cell features and poor differentiation. Median progression-free survival (PFS) of crizotinib-treated patients was 7.6 months. The overall survival (OS) of these patients was longer compared with that of crizotinib-naive or wild-type cohorts, but there was no significant difference in OS compared with patients with EGFR mutation. ALK expression did not associate with PFS; but, when ALK expression was analyzed as a dichotomous variable, moderate and strong ALK expression had a decreased risk of death (P = 0.026). The two patients with concomitant EGFR and ALK alterations showed difference in ALK expression, response to EGFR and ALK inhibitors, and overall survival.

Conclusions

Selective enrichment according to clinicopathologic features in NSCLC patients could highly improve the positivity detection rate of ALK rearrangement for ALK-targeted therapy. IHC could provide more clues for clinical trial design and therapeutic strategies for ALK-positive NSCLC patients including patients with double genetic aberration of ALK and EGFR.     

Clinicopathologic Features of Patients with Non-Small Cell Lung Cancer Harboring the EML4-ALK Fusion Gene: A Meta-Analysis

Background

The frequencies of EML4-ALK fusion gene in non-small cell lung cancer (NSCLC) with different clinicopathologic features described by previous studies are inconsistent. The key demographic and pathologic features associated with EML4-ALK fusion gene have not been definitively established. This meta-analysis was conducted to compare the frequency of the EML4-ALK fusion gene in patients with different clinicopathologic features and to identify an enriched population of patients with NSCLC harboring EML4-ALK fusion gene.

Methods

The Pubmed and Embase databases for all studies on EML4-ALK fusion gene in NSCLC patients were searched up to July 2014. A criteria list and exclusion criteria were established to screen the studies. The frequency of the EML4-ALK fusion gene and the clinicopathologic features, including smoking status, pathologic type, gender, and EGFR status were abstracted.

Results

Seventeen articles consisting of 4511 NSCLC cases were included in this meta-analysis. A significant lower EML4-ALK fusion gene positive rate was associated with smokers (pooled OR = 0.40, 95% CI = 0.30–0.54, P<0.00001). A significantly higher EML4-ALK fusion gene positivity rate was associated with adenocarcinomas (pooled OR = 2.53, 95% CI = 1.66–3.86, P<0.0001) and female (pooled OR = 0.61, 95% CI = 0.41–0.90, P = 0.01). We found that a significantly lower EML4-ALK fusion gene positivity rate was associated with EGFR mutation (pooled OR = 0.07, 95% CI = 0.03–0.19, P<0.00001). No publication bias was observed in any meta-analysis (all P value of Egger''s test >0.05); however, because of the small sample size, no results were in the meta-analysis regarding EGFR gene status.

Conclusion

This meta-analysis revealed that the EML4-ALK fusion gene is highly correlated with a never/light smoking history, female and the pathologic type of adenocarcinoma, and is largely mutually exclusive of EGFR.     

TP53 Mutational Status Is a Potential Marker for Risk Stratification in Wilms Tumour with Diffuse Anaplasia

Purpose

The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information.

Patients and Methods

We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling.

Results

From the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes.

Conclusion

This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.     

EGFR Mutation Testing in Patients with Advanced Non-Small Cell Lung Cancer: A Comprehensive Evaluation of Real-World Practice in an East Asian Tertiary Hospital

Introduction

Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea.

Methods

Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records.

Results

This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001). The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%), pulmonologists (31.9%), and thoracic surgeons (6.6%). EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma.

Conclusions

In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants the need for generalized testing in Asian NSCLC patients.     

Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers

Purpose

We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.

Experimental Design

We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.

Results

152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.

Conclusion

The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.     

The Prognostic Value of BRAF Mutation in Colorectal Cancer and Melanoma: A Systematic Review and Meta-Analysis

Background

Mutation of BRAF is a predominant event in cancers with poor prognosis such as melanoma and colorectal cancer. BRAF mutation leads to a constitutive activation of mitogen activated protein kinase pathway which is essential for cell proliferation and tumor progression. Despite tremendous efforts made to target BRAF for cancer treatment, the correlation between BRAF mutation and patient survival is still a matter of controversy.

Methods/Principal Findings

Clinical studies on the correlation between BRAF mutation and patient survival were retrieved from MEDLINE and EMBASE databases between June 2002 and December 2011. One hundred twenty relevant full text studies were categorized based on study design and cancer type. Publication bias was evaluated for each category and pooled hazard ratio (HR) with 95% confidence interval (CI) was calculated using random or fixed effect meta-analysis based on the percentage of heterogeneity. Twenty six studies on colorectal cancer (11,773 patients) and four studies on melanoma (674 patients) were included in our final meta-analysis. The average prevalence of BRAF mutation was 9.6% in colorectal cancer, and 47.8% in melanoma reports. We found that BRAF mutation increases the risk of mortality in colorectal cancer patients for more than two times; HR = 2.25 (95% CI, 1.82–2.83). In addition, we revealed that BRAF mutation also increases the risk of mortality in melanoma patients by 1.7 times (95% CI, 1.37–2.12).

Conclusions

We revealed that BRAF mutation is an absolute risk factor for patient survival in colorectal cancer and melanoma.     

KRAS Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in Prediction of Lung Metastasis

Background

KRAS mutations in colorectal cancer primary tumors predict resistance to anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody therapy in patients with metastatic colorectal cancer, and thus represent a true indicator of EGFR pathway activation status.

Methodology/Principal Findings

KRAS mutations were retrospectively studied using polymerase chain reactions and subsequent sequencing of codons 12 and 13 (exon 2) in 110 patients with metastatic colorectal tumors. These studies were performed using tissue samples from both the primary tumor and their related metastases (93 liver, 84%; 17 lung, 16%). All patients received adjuvant 5-Fluorouracil-based polychemotherapy after resection of metastases. None received anti-EGFR therapy. Mutations in KRAS were observed in 37 (34%) of primary tumors and in 40 (36%) of related metastases, yielding a 94% level of concordance (kappa index 0.86). Patients with primary tumors possessing KRAS mutations had a shorter disease-free survival period after metastasis resection (12.0 vs 18.0 months; P = 0.035) than those who did not. A higher percentage of KRAS mutations was detected in primary tumors of patiens with lung metastases than in patients with liver metastases (59% vs 32%; p = 0.054). To further evaluate this finding we analyzed 120 additional patients with unresectable metastatic colorectal cancer who previously had their primary tumors evaluated for KRAS mutational status for clinical purposes. Separately, the analysis of these 120 patients showed a tendency towards a higher degree of KRAS mutations in primary tumors of patients with lung metastases, although it did not reach statistical significance. Taken together the group of 230 patients showed that KRAS was mutated significantly more often in the primary tumors of patients with lung metastases (57% vs 35%; P = 0.006).

Conclusions/Significance

Our results suggest a role for KRAS mutations in the propensity of primary colorectal tumors to metastasize to the lung.     

Alternative HER/PTEN/Akt pathway activation in HPV positive and negative penile carcinomas

Background

The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH).

Methodology/Principal Findings

148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs.

Conclusions/Significance

EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection. Our results suggest that PSCC patients could benefit from therapies developed to target HER receptors.     

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

Introduction

In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI.

Patient and Methods

Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis.

Results

Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively.

Conclusion

One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival.