Annamaria Siggillino

Phosphoinositide-3-Kinase Catalytic Alpha and KRAS Mutations are Important Predictors of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients with Advanced Non-small Cell Lung Cancer

Background: Specific mutations of the epidermal growth factor receptor (EGFR) gene are predictive for favorable response to tyrosine kinase inhibitors (TKIs) and are associated with a good prognosis. In contrast, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation has been shown to predict poor response to such therapy. Nevertheless, tumor that initially responds to EGFR-TKIs almost inevitably becomes resistant later. Other mechanisms of resistance to EGFR inhibitors could involve activating mutations of the other main EGFR effector pathway, i.e., the phosphoinositide-3-kinase/phosphate and tensin homologue deleted from chromosome 10 (PTEN)/alpha serine/threonine protein kinase (AKT) pathway. The aim of this study was to investigate the role of phosphoinositide-3-kinase catalytic alpha (PIK3CA), EGFR, and KRAS gene mutations in predicting response and survival in patients with non-small cell lung cancer (NSCLC) treated with EGFR-TKIs. Patients and Methods: A total of 166 patients with advanced NSCLC treated with EGFR-TKI with available archival tissue specimens were included. PIK3CA, EGFR, and KRAS mutations were analyzed using polymerase chain reaction-based sequencing. Results: EGFR mutation was detected in 25.3% of patients, PIK3CA mutation in 4.1%, and KRAS mutation in 6.7%. PIK3CA mutation correlated with shorter median time to progression (TTP) (p = 0.01) and worse overall survival (OS) (p < 0.001). EGFR mutation (p < 0.0001) correlated with favorable response to TKIs treatment and longer TTP (p < 0.0001). KRAS mutation correlated with progressive disease (p = 0.05) and shorter median TTP (p = 0.003) but not with OS. Cox multivariate analysis including histology and performance status showed that PIK3CA mutation was an independent factor to predict worse OS (p = 0.0001) and shorter TTP (p = 0.03), while KRAS mutation to predict shorter TTP (p = 0.01). Conclusion: PIK3CA and KRAS mutations seem to be indicators of resistance and poor survival in patients with NSCLC treated with EGFR-TKIs.

Impact of specific mutant KRAS on clinical outcome of EGFR-TKI-treated advanced non-small cell lung cancer patients with an EGFR wild type genotype

INTRODUCTION This retrospective study was undertaken to investigate the impact of specific mutant KRAS on clinical outcome to either gefitinib or erlotinib (EGFR tyrosine kinase inhibitor, EGFR-TKI) in patients with EGFR wild type (WT) advanced non-small cell lung cancer (NSCLC). METHODS Patients with an EGFR WT genotype who were treated with an EGFR-TKI for advanced disease at our Institution were identified. Simultaneous availability of KRAS mutation status was required for study inclusion. RESULTS Sixty-seven patients were eligible. Median age was 60 years (39-84), and 10 patients (14.9%) had received an EGFR-TKI as upfront therapy. Overall, the median progression-free survival (PFS) and overall survival (OS) were 2.9 months and 18.0 months, respectively. KRAS mutant patients (n=18) experienced a significantly shorter PFS compared with those carrying a KRAS WT genotype (n=49) (1.6 months vs 3.0 months, respectively, P=0.04; HR=1.92). However, within the KRAS mutant group a great variability in terms of sensitivity to treatment was noted (PFS ranging from 0.7 months to 38.7 months). KRAS codon 13 mutant patients (n=4) experienced the worse outcome when compared with KRAS codon 12 mutants (n=14) and KRAS WT patients (P<0.0001 and P=0.01 for PFS and OS, respectively). CONCLUSIONS Though we found that EGFR WT/KRAS mutant advanced NSCLC patients are associated with an increased resistance to treatment, specific mutant KRAS may account for differential sensitivity to an EGFR-TKI. KRAS codon 13 mutants are those who seem to experience the worse clinical outcome.

Association of Cytidine Deaminase and Xeroderma Pigmentosum Group D Polymorphisms with Response, Toxicity, and Survival in Cisplatin/Gemcitabine-Treated Advanced Non-small Cell Lung Cancer Patients

Background: Selecting patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in non-small cell lung cancer (NSCLC). Genetic variations in drug metabolism may affect the clinical response, toxicity, and prognosis of NSCLC patients treated with cisplatin/gemcitabine-based therapy. Patients and Methods: We evaluated seven single-nucleotide polymorphisms of six genes CDA Lys27Gln (A/C); CDA C435T; ERCC1 C118T; XRCC3 Thr241Met (C/T); XPD Lys751Gln (A/C); P53 Arg72Pro (G/C), and RRM1 C524T in 192 chemotherapy-naive patients with advanced NSCLC treated with cisplatin/gemcitabine-based regimen by TaqMan probe-based assays with 7300 Real-Time PCR System, using genomic DNA extracted from blood samples. Results: The CDA 435 T/T genotype was significantly associated with better response (p = 0.03). The CDA 435 C/T genotype was associated with a significantly increased risk of nonhematological toxicity of grade ≥3 (p = 0.02) after adjusting for performance status, age, and type of treatment regimen. In the multivariate Cox model, the XPD 751 C/C genotype was a significant prognostic factor of longer progression-free survival (p = 0.006). Conclusion: Our data suggest polymorphic variations of drug metabolic gene were associated with response and toxicity of cisplatin/gemcitabine-based therapy and progression-free survival of patients with advanced NSCLC.

Gene identification for risk of relapse in stage I lung adenocarcinoma patients: a combined methodology of gene expression profiling and computational gene network analysis.

Risk assessment and treatment choice remains a challenge in early non-smallcell lung cancer (NSCLC). The aim of this study was to identify novel genes involved in the risk of early relapse (ER) compared to no relapse (NR) in resected lung adenocarcinoma (AD) patients using a combination of high throughput technology and computational analysis. We identified 18 patients (n.13 NR and n.5 ER) with stage I AD. Frozen samples of patients in ER, NR and corresponding normal lung (NL) were subjected to Microarray technology and quantitative-PCR (Q-PCR). A gene network computational analysis was performed to select predictive genes. An independent set of 79 ADs stage I samples was used to validate selected genes by Q-PCR. From microarray analysis we selected 50 genes, using the fold change ratio of ER versus NR. They were validated both in pool and individually in patient samples (ER and NR) by Q-PCR. Fourteen increased and 25 decreased genes showed a concordance between two methods. They were used to perform a computational gene network analysis that identified 4 increased (HOXA10, CLCA2, AKR1B10, FABP3) and 6 decreased (SCGB1A1, PGC, TFF1, PSCA, SPRR1B and PRSS1) genes. Moreover, in an independent dataset of ADs samples, we showed that both high FABP3 expression and low SCGB1A1 expression was associated with a worse disease-free survival (DFS). Our results indicate that it is possible to define, through gene expression and computational analysis, a characteristic gene profiling of patients with an increased risk of relapse that may become a tool for patient selection for adjuvant therapy.

Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors

Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets. Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to laser capture microdissection and reverse phase protein microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC). Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02). This finding was verified in an independent population by IHC (p=0.03). KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications.

Enteric-type adenocarcinoma of the lung harbouring a novel KRAS Q22K mutation with concomitant KRAS polysomy: a case report.

This case describes a novel KRAS Q22K mutation with simultaneous KRAS polysomy in a patient with advanced, enteric-type, adenocarcinoma of the lung. Despite the administration of systemic chemotherapy, the disease underwent rapid progression and led to the patient’s death in a short period of time. Such an aggressive clinical course suggests that, in this specific case, KRAS dependency was the major genetic driver of poor prognosis. Direct deoxy ribonucleic acid (DNA) sequencing of the KRAS gene allows for the detection of novel KRAS mutations, and it might be advocated in patients with advanced non-small cell lung cancer in view of the emerging role of KRAS as a potential therapeutic target.

MYC and human telomerase gene (TERC) copy number gain in early-stage non-small cell lung cancer.

Objectives:We investigated the frequency of MYC and TERC increased gene copy number (GCN) in early-stage non–small cell lung cancer (NSCLC) and evaluated the correlation of these genomic imbalances with clinicopathologic parameters and outcome. Materials and Methods:Tumor tissues were obtained from 113 resected NSCLCs. MYC and TERC GCNs were tested by fluorescence in situ hybridization (FISH) according to the University of Colorado Cancer Center (UCCC) criteria and based on the receiver operating characteristic (ROC) classification. Results:When UCCC criteria were applied, 41 (36%) cases for MYC and 41 (36%) cases for TERC were considered FISH-positive. MYC and TERC concurrent FISH-positive was observed in 12 cases (11%): 2 (17%) cases with gene amplification and 10 (83%) with high polysomy. By using the ROC analysis, high MYC (mean ≥2.83 copies/cell) and TERC (mean ≥2.65 copies/cell) GCNs were observed in 60 (53.1%) cases and 58 (51.3%) cases, respectively. High TERC GCN was associated with squamous cell carcinoma (SCC) histology (P=0.001). In univariate analysis, increased MYC GCN was associated with shorter overall survival (P=0.032 [UCCC criteria] or P=0.02 [ROC classification]), whereas high TERC GCN showed no association. In multivariate analysis including stage and age, high MYC GCN remained significantly associated with worse overall survival using both the UCCC criteria (P=0.02) and the ROC classification (P=0.008). Conclusions:Our results confirm MYC as frequently amplified in early-stage NSCLC and increased MYC GCN as a strong predictor of worse survival. Increased TERC GCN does not have prognostic impact but has strong association with squamous histology.

Variations in gene expression of lung macromolecules after induction chemotherapy for lung cancer

OBJECTIVES Preoperative chemotherapy may play a role in postoperative respiratory complications due to subclinical parenchymal damage. We investigated the gene expression of lung tissue components after neoadjuvant chemotherapy of alveolar-capillary membrane, extracellular matrix and membrane proteins. METHODS The study group included 14 patients submitted to pulmonary resection for lung cancer after 3 cycles of gemcitabine-cisplatin, while the control group included 14 naive-treatment patients. RNA was extracted from frozen tissue obtained by healthy lung specimens using EZ1 RNA Universal Tissue kit and automatically purified by BioRobot EZ1 instrument. Three hundred nanograms of total RNA was reverse transcribed to complementary DNA and used to evaluate the gene expression of type I and III collagen, elastin, syndecan, metalloproteinase 13 and aquaporins (AQPs) in real-time polymerase chain reaction. Results were expressed as the mean ± standard deviation of 3 independent experiments. Analysis of variance followed by Sheffes F-test was performed. RESULTS Among the alveolar-capillary membrane and extracellular matrix genes, type I-III collagens and syndecan were significantly up-regulated (+645%, +327% and +261%, respectively), while elastin and metalloproteinase 13 were down-regulated in the study group versus control group (-46% and -77%, respectively). Furthermore, chemotherapy was associated with a significant up-regulation of AQP expressions (AQP1:+51% and AQP5:+36%). CONCLUSIONS We observed, in the treated group, increases in the mean values of gene expressions for macromolecules involved in the remodelling of both the alveolar septa and parenchyma scaffold, thereby supporting the hypothesis that induction chemotherapy may foster a fibrosing effect on the pulmonary parenchyma and lead to altering the alveolar-capillary membrane.

KRAS mutation and DNA repair and synthesis genes in non‑small‑cell lung cancer

The aim of the present study was to assess the expression of select DNA repair and synthesis genes in non-small-cell lung cancer (NSCLC) according to KRAS mutation status. ERCC1, TS, RRM1, and BRCA1 mRNA expression levels were assessed from either primary or metastatic tumor specimens of patients diagnosed with epidermal growth factor receptor (EGFR) wild-type (WT) advanced NSCLC. Total RNA was isolated from paraffin-embedded tumor specimens using the RNeasy FFPE kit and automatically purified using a QiaCube instrument. Quantification levels were analyzed by real-time one-step RT-PCR using QuantiFast technology, and the results were compared considering β-actin as the internal reference gene. One hundred and eighty-four patients with advanced NSCLC were evaluated for the analysis, of which 92 were KRAS-mutants. Nearly all patients had adenocarcinoma histology (96.7%). Among KRAS-mutants, the majority had a KRAS codon 12 mutation (88%), the most common being G12C (44.4% of cases). Mean ERCC1 levels were indicated to be significantly higher in KRAS-mutants when compared with KRAS WT patients (3,234±6.63 vs. 184±1.24; P=0.05). However, mean TS levels were significantly lower in the KRAS-mutant subgroup compared with the KRAS WT subgroup (4,481±3.756 vs. 5,941±6.4; P=0.039). KRAS-mutant NSCLCs are more likely to express high ERCC1 and low TS levels. This finding may suggest different sensitivity to cytotoxic chemotherapy according to KRAS mutation status.

Long-term survival with erlotinib in advanced lung adenocarcinoma harboring synchronous EGFR G719S and KRAS G12C mutations

Although epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) mutations were thought to be mutually exclusive in patients with non-small cell lung cancer (NSCLC), the development of high sensitive large-scale mutation analysis, has increasingly shown that activating EGFR mutations occasionally coexist with other dominant genetic alterations. Herein, we discuss the case of a patient with advanced NSCLC harboring both the uncommon EGFR G719S and the KRAS G12C mutations, who was treated for 9 years with erlotinib achieving a long-term survival. In light of their rarity, multiple mutations are very challenging for the decision of tyrosine kinase inhibitors (TKIs) treatment, especially when EGFR mutations occur together with mutations known to provide resistance to EGFR TKIs, such as KRAS.