Non-small Cell Lung Cancer Status Research Articles

Relationship between maximum standardized uptake values of 18F-FDG PET-CT and clinicopathological characteristics in non-small cell lung cancer

Objective To analyze the correlation of clinicopathological characteristics and maximum standardized uptake value (SUVmax) detected by 18F-FDG PET-CT in non-small cell lung cancer (NSCLC). Methods 105 patients with NSCLC who underwent 18F-FDG PET-CT scan before surgical resection were reviewed retrospectively. Clinicopathological factors which might affect SUVmax were evaluated, including sex, age, smoking history, CEA level, tumor site, histological type, TNM stage, T factor, N factor, tumor size, lymphovascular invasion and pleural invasion features. Independent factors were identified by multiple regression analysis. The diagnostic efficiency and best cut-off point of SUVmax were calculated by the receiver operating characteristic curve. Results It was identified by the univariate analysis that the CEA level (P=0.002), tumor size (P<0.001), histological type (P<0.001), TNM stage (P<0.001), T factor (P<0.001), N factor (P<0.001), and lymphovascular invasion (P=0.001) were factors affecting SUVmax. While histological type (P=0.004), tumor size (P=0.036), N factor (P=0.043) were found to be significant independent factors according to multivariate regression analysis. The SUVmax of primary tumor was a predictor for lymphatic metastasis with the highest diagnostic accuracy at a cut-off value of 6.75, the sensitivity and specificity were 72.2 % and 81.6 %, respectively. Conclusions The SUVmax is correlated with histological type, tumor diameter, nodal status in NSCLC, and is higher in patients with non-adenocarcinoma, lager tumor and lymphatic metastasis. Furthermore, the probability of lymphatic metastasis could be predicted by SUVmax of the primary tumor. Key words: Carcinoma, non-small-cell lung; PET-CT; Deoxyglucose; Standardized uptake value; Pathology

Intact or broken-apart RNA: an alternative concept for ALK fusion screening in non-small cell lung cancer (NSCLC).

Anaplastic lymphoma kinase (ALK) break-apart fluorescent in situ hybridization (FISH) is currently used in diagnostics for the selection of non-small cell lung cancer (NSCLC) patients to receive crizotinib. We evaluated ALK status in NSCLC with a novel ALK mRNA test based on the break-apart FISH concept, which we called break-apart transcript (BAT) test. ALK5' and ALK3' transcript patterns were established with qPCR for ALK-expressing controls including fusion-negative neuroblastomas, as well as fusion-positive anaplastic large cell lymphomas and NSCLC. The BAT test was evaluated on 271 RNA samples from routinely processed paraffin NSCLC tissues. Test results were compared with ALK FISH (n=121), immunohistochemical (IHC) analysis (n=86), and automated quantitative analysis (AQUA, n=83). On the basis of the nonoverlapping ALK BAT patterns in ALK-expressing controls (P<0.0001), 8/174 adenocarcinomas (4.6%) among 259 informative NSCLC were predicted as fusion positive. Overall concordance for paired method results was high (94.1% to 98.8%) but mainly concerned negative prediction because of the limited availability of positive-matched cases. Tumors with 100% cytoplasmic IHC staining of any intensity (n=3) were positive for AQUA, FISH, and BAT test; tumors with lower IHC positivity and different staining patterns were AQUA-negative. Upon multiple reevaluations, ALK gene status was considered as originally misinterpreted by FISH in 3/121 cases (2.5%). Tumors with >4 ALK gene copies were associated with longer overall survival upon first-line chemotherapy. In conclusion, application of the ALK BAT test on routinely processed NSCLC tissues yields the same fusion partner independent information as ALK break-apart FISH but is more robust and cost-effective. The BAT concept may be considered for the development of further drug-predictive translocation tests.

Circulating tumor DNA is effective for the detection of EGFR mutation in non-small cell lung cancer: a meta-analysis.

Circulating tumor DNA (ctDNA) has offered a minimally invasive and feasible approach for detection of EGFR mutation for non-small cell lung cancer (NSCLC). This meta-analysis was designed to investigate the diagnostic value of ctDNA, compared with current "gold standard," tumor tissues. We searched PubMed, EMBASE, Cochrane Library, and Web of Science to identify eligible studies that reported the sensitivity and specificity of ctDNA for detection of EGFR mutation status in NSCLC. Eligible studies were pooled to calculate the pooled sensitivity, specificity, and diagnostic odds ratio (DOR). The summary ROC curve (SROC) and area under SROC (AUSROC) were used to evaluate the overall diagnostic performance. Twenty-seven eligible studies involving 3,110 participants were included and analyzed in our meta-analysis, and most studies were conducted among Asian population. The pooled sensitivity, specificity, and DOR were 0.620 [95% confidence intervals (CI), 0.513-0.716), 0.959 (95% CI, 0.929-0.977), and 38.270 (95% CI, 21.090-69.444), respectively. The AUSROC was 0.91 (95% CI, 0.89-0.94), indicating the high diagnostic performance of ctDNA. ctDNA is a highly specific and effective biomarker for the detection of EGFR mutation status. ctDNA analysis will be a key part of personalized cancer therapy of NSCLC.

Diagnostic value of circulating free DNA for the detection of EGFR mutation status in NSCLC: a systematic review and meta-analysis

Epidermal growth factor receptor (EGFR) mutation is a reliable and sensitive biomarker for EGFR-TKI therapy in non-small-cell lung cancer (NSCLC). However, detection of EGFR mutation in tissues has obvious limitations. Circulating free DNA (cfDNA) has been reported as an alternative approach for the detection of EGFR mutations. This systematic review and meta-analysis was designed to assess the diagnostic performance of cfDNA, compared with tissues. True-positive (TP), false-positive (FP), false-negative (FN), and true-negative (TN) values were extracted or calculated for each study. Pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated. A summary receiver operating characteristic curve (SROC) and area under curve (AUC) were used to evaluate the overall diagnostic performance. 20 eligible studies involving 2012 cases were included in this meta-analysis. The pooled sensitivity, specificity, PLR, NLR, andDORwere 0.674 (95%CI: 0.517–0.800), 0.935 (95%CI: 0.888–0.963), 10.307 (95%CI: 6.167–17.227), 0.348 (95%CI: 0.226–0.537), and 29.582 (95%CI: 4.582–60.012), respectively. The AUC was 0.93 (95% CI: 0.90–0.95). The meta-analysis suggests that detection of EGFR mutation by cfDNA is of adequate diagnostic accuracy and cfDNA analysis could be a promising screening test for NSCLC.

Role of [¹⁸F]FDG PET in prediction of KRAS and EGFR mutation status in patients with advanced non-small-cell lung cancer.

The tumour molecular profile predicts the activity of epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). However, tissue availability and tumour heterogeneity limit its assessment. We evaluated whether [(18)F]FDG PET might help predict KRAS and EFGR mutation status in NSCLC. Between January 2005 and October 2011, 340 NSCLC patients were tested for KRAS and EGFR mutation status. We identified patients with stage III and IV disease who had undergone [(18)F]FDG PET/CT scanning for initial staging. SUVpeak, SUVmax and SUVmean of the single hottest tumour lesions were calculated, and their association with KRAS and EGFR mutation status was assessed. A receiver operator characteristic (ROC) curve analysis and a multivariate analysis (including SUVmean, gender, age and AJCC stage) were performed to identify the potential value of [(18)F]FDG PET/CT for predicting KRAS mutation. From 102 patients staged using [(18)F]FDG PET/CT, 28 (27%) had KRAS mutation (KRAS+), 22 (22%) had EGFR mutation (EGFR+) and 52 (51%) had wild-type KRAS and EGFR profiles (WT). KRAS+ patients showed significantly higher [(18)F]FDG uptake than EGFR+ and WT patients (SUVmean 9.5, 5.7 and 6.6, respectively; p < 0.001). No significant differences were observed in [(18)F]FDG uptake between EGFR+ patients and WT patients. ROC curve analysis for KRAS mutation status discrimination yielded an area under the curve of 0.740 for SUVmean (p < 0.001). The multivariate analysis showed a sensitivity and specificity of 78.6% and 62.2%, respectively, and the AUC was 0.773. NSCLC patients with tumours harbouring KRAS mutations showed significantly higher [(18)F]FDG uptake than WT patients, as assessed in terms of SUVpeak, SUVmax and SUVmean. A multivariate model based on age, gender, AJCC stage and SUVmean might be used as a predictive marker of KRAS mutation status in patients with stage III or IV NSCLC.

EGFR mutational status in a large series of Caucasian European NSCLC patients: data from daily practice

Background:The prognosis of metastatic non-small cell lung cancer (NSCLC) is still poor. Activating epithelial growth factor receptor (EGFR) mutations are important genetic alterations with dramatic therapeutical implications. Up to now, in contrast to Asian populations only limited data on the prevalence of those mutations are available from patients with Caucasian and especially European ethnicity.Methods:In this multicentre study, 1201 unselected NSCLC patients from Southern Germany were tested in the daily clinical routine for EGFR mutation status.Results:Activating EGFR mutations were found in 9.8% of all tumours. Mutations in exons 18, 19 and 21 accounted for 4.2%, 61.9% and 33.1% of all mutations, respectively. Non-smokers had a significantly higher rate of EGFR mutations than smokers or ex-smokers (24.4% vs 4.2% P<0.001). Non-lepidic-non-mucinous adenocarcinomas (G2) accounted for 45.5% of all activating EGFR mutations and 3.5% of all squamous cell carcinomas were tested positive. Thyroid transcription factor 1 protein expression was significantly associated with EGFR mutational status.Conclusion:These comprehensive data from clinical routine in Germany add to the knowledge of clinical and histopathological factors associated with EGFR mutational status in NSCLC.

Abstract 62: Detection and comparison of EGFR mutations in matched tumor tissue, cell block, pleural effusion and serum with PNA clamping and direct sequencing in NSCLC with malignant pleural effusion.

Abstract Background: Peptide nucleic acid (PNA)-mediated real-time PCR clamping was recently used to detect mutations because it has higher sensitivity than conventional direct sequencing. Pleural effusion and serum samples could be good sources of detecting the epidermal growth factor receptor (EGFR) mutational status of non-small cell lung cancer (NSCLC) patients. Methods: We studied 37 NSCLC patients with malignant pleural effusion. In each patient, EGFR mutational status was measured from the samples of tumor tissue, cell block, pleural effusion and serum using both PNA clamping and direct sequencing. The concordance between PNA clamping and direct sequencing, and the diagnostic performance of pleural effusion was investigated. Results: The detection rate of EGFR mutation in the pleural effusion and the serum was 27% and 2.8%, respectively by both PNA clamping and direct sequencing. The κ coefficient between the two methods were 0.68 (p-value = 0.0007), 0.91 (p-value &amp;lt; 0.0001), 0.75 (p-value &amp;lt; 0.0001) and -0.01 (p-value = 0.8639) in tissue, cell block, pleural effusion and serum, respectively. The diagnostic performance of pleural effusion compared with combined tumor tissue plus cell block showed sensitivity 89%, specificity 100%, positive predictive value 100% and negative predictive value 95% with PNA clamping, and sensitivity 67%, specificity 90%, positive predictive value 75% and negative predictive value 86% with directing sequencing. Interestingly, a patient who was confirmed EGFR mutation only with the PNA clamping showed significant response to EGFR TKI (tyrosine kinase inhibitor). Conclusion: Despite the limited role of serum samples, pleural effusion had a diagnostic performance comparable with tumor tissue and cell block to detect EGFR mutational status in NSCLC. Although the diagnostic performance of PNA clamping was similar to that of direct sequencing, additional benefit is expected considering the response to the EGFR TKI. Citation Format: Chan Kwon Park, Chang Dong, Jin Woo Jin Woo, Kwan Hyoung Kim, Jick Hwan Ha, Chin Kook Rhee, Young Kyoon Kim, Sang Haak Lee, Mi Sun Park, Hyeon Woo Yim, Seung Joon Kim. Detection and comparison of EGFR mutations in matched tumor tissue, cell block, pleural effusion and serum with PNA clamping and direct sequencing in NSCLC with malignant pleural effusion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2013-62

Epidermal growth factor receptor and Kras gene expression: Reliability of mutational analysis on cytological samples

Epidermal growth factor receptor (EGFR) and Kras gene mutations are crucial for discriminating patients responsive to anti-EGFR drugs in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), respectively. The majority of NSCLCs come to clinical attention at an advanced stage when surgery is no longer recommended and a considerable number of them are diagnosed by cytology only. A large number of metastatic CRCs are also diagnosed by imaging and minimally invasive techniques such as fine-needle aspiration biopsy. Here, we report our experience in the mutation analysis of EGFR and Kras on cytological material obtained from superficial and deep lesions of NSCLC and CRC. Our series included 63 cytological specimens from primary or metastatic lesions of 42 NSCLCs and 21 CRCs. The cytological material was adequate for the mutation analysis in 39/42 (93%) NSCLCs and in 20/21(95%) CRCs. EGFR and Kras mutations were found in 9 (23%) and 9 (23%) NSCLC cases, respectively. Kras mutations were found in 9/20 (45%) CRC specimens. Histological samples from the primary tumors were available in 9/42 NSCLCs and in 17/21 CRCs. The agreement of EGFR and Kras mutational status in cytological vs. histological samples was 100% for NSCLC and 88% for CRC. Our results suggest that standard cytology provides adequate material for the assessment of EGFR and Kras mutational status in NSCLC and CRC patients and could be specifically indicated in patients not eligible for surgery but candidate to anti-EGFR therapy.

Influence of Chemotherapy on EGFR Mutation Status Among Patients With Non–Small-Cell Lung Cancer

EGFR mutation is a predictor of epidermal growth factor receptor-tyrosine kinase inhibitor treatment response in patients with non-small-cell lung cancer (NSCLC). However, it remains unclear whether chemotherapy affects EGFR mutation status in NSCLC. We investigated the influence of chemotherapy on EGFR mutations in plasma and tumor tissues from patients with NSCLC. Samples were derived from three cohorts: one, 264 patients with advanced NSCLC who received first-line chemotherapy with matched pre- and postchemotherapy blood samples; two, 63 patients with stages IIb to IIIb disease with pre- and post-neoadjuvant chemotherapy tumor tissues; and three, 79 patients with advanced NSCLC who underwent palliative surgery. EGFR mutation status was determined and analyzed to reveal potential impact of chemotherapy. In the first cohort, EGFR mutations were detected in 34.5% of the prechemotherapy plasma samples (91 of 264) but in only 23.1% of the postchemotherapy plasma samples (61 of 264). The decrease in EGFR mutation rate was statistically significant (P < .001). Patients whose EGFR mutations switched from positive to negative after chemotherapy had a better partial response (PR) than patients with a reverse change (P = .037). A similar decrease in EGFR mutation rate was observed in tissues after neoadjuvant chemotherapy in the second cohort (34.9% [22 of 63] v 19.0% [12 of 63]; P = .013). In the third cohort, 38.0% of the tumors (30 of 79) showed an intratumor heterogeneity of EGFR mutation, whereas 62.0% (49 of 79) were homogeneous, either with EGFR mutation or no mutation. Our results suggest that chemotherapy may reduce EGFR mutation frequency in patients with NSCLC, likely the result of a preferential response of subclones with EGFR mutations in tumors with heterogeneous tumor cell populations.

Abstract LB-449: KRAS mutation and amplification status predicts sensitivity to antifolate therapies in non-small-cell lung cancer

Abstract Somatic genetic mutation in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been linked to poor prognosis and resistance to various targeted therapeutics in Non Small Cell Lung Cancer (NSCLC). Therapeutic strategies that target tumors harboring these mutations represent an unmet medical need. In this study, we investigated the relationship between antifolate sensitivity and KRAS mutation/amplification status in NSCLC. Human NSCLC cell lines (KRAS wild type, KRAS mutant non-amplified and KRAS mutant amplified) were treated with Methotrexate (MTX) or Pemetrexed (PEM) and assayed for proliferation. In these studies, KRASwt (wildtype) and KRASmut (mutant) amplified cells showed resistance to MTX treatment (IC50 &amp;gt;10μM). In contrast, growth of all KRASmut non-amplified cell lines studied was inhibited with MTX treatment (IC50 &amp;lt;100nM). Similar effects were observed for PEM in this study. Interrogation of the NCI Developmental Therapeutics Program drug screen database for the relationship between KRAS mutation status and drug efficacy also revealed a similar trend in other NSCLC cell lines for MTX and other anti-folates. qPCR analysis demonstrated a dramatic downregulation of KRAS gene expression in KRASwt and KRASmut cells with antifolate treatment. However, KRAS gene expression was less affected in antifolate treated KRASmut amplified cells. Co-treatment of KRASmut cells with antifolates and hypoxanthine/thymidine (which compensate for folate pathway inhibition) prevented downregulation of KRAS gene expression and rescued KRASmut cells. qPCR array analysis of miRNA expression in antifolate treated cells revealed increased expression of specific miRNAs, including miR-181c, with treatment compared to untreated controls. Transfection of a miR-181c mimic led to downregulation of KRAS gene expression in cells. Furthermore, antagomirs targeting miR-181c partially inhibited the downregulation of KRAS by antifolates. Importantly, we present clinical data describing rapid and durable radiographic responses in KRAS mutant NSCLC cancer patients. Collectively, these studies identify higher sensitivity to antifolates in KRASmut non-amp NSCLC cell lines. Antifolate therapies decrease KRAS gene expression in KRASwt and KRASmut but do not do so in KRASmut amplified cells. We propose that decreased KRAS gene expression is detrimental to KRASmut cells due to their dependency on this survival pathway. We also propose that decreases in KRAS gene expression are mechanistically linked to stress (folate inhibition) induced miRNA expression which target KRAS gene expression. Overall, antifolates represent a novel method to target KRAS and as such should be investigated further for use in this subtype of NSCLC. As clinical evidence emerges, both KRAS mutation and amplification status should be incorporated for patient stratification prior to antifolate treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-449. doi:1538-7445.AM2012-LB-449

Intratumor heterogeneity and chemotherapy-induced changes in EGFR status in non-small cell lung cancer

Biomarker expression is increasingly being used to customize treatment in non-small cell lung cancer (NSCLC). The choice of systemic treatment usually depends on biomarker expression in the initial diagnostic biopsy taken before initiation of first-line treatment. Chemotherapy induces DNA damages in the tumor cells, and thus, biomarker expression in the tumor after systemic treatment might not be identical to biomarker expression in the diagnostic biopsy. NSCLC is highly heterogeneous and biomarker expression may vary in different areas within the same tumor. This review explores the tumor heterogeneity and chemotherapy-induced changes in EGFR biomarker status in NSCLC. A literature search was performed in August 2011 using pubmed. Fifteen trials explored EGFR status in primary tumor and subsequent resected primary tumor, lymph node metastases, or organ metastases. Four papers compared EGFR status in primary tumor or metastases before and after systemic treatment. All trials included relatively few patients and used different chemotherapy regimes, biopsy locations, or time intervals between biopsies. Tumor heterogeneity and probably also previous systemic treatment may be an obstacle for correct interpretation of EGFR status in NSCLC. Heterogeneity regarding EGFR mutations is probably rare and previously reported intra and intertumor heterogeneity may be due to methodological issues. In the current and future clinical scenario with many different options for systemic treatment both as 2nd line and beyond, it is increasingly important to further elucidate the role extent of chemotherapy-induced changes in biomarker expression for proper use of biomarkers in order to customize treatment and thus improve prognosis.

Abstract PR-2: KRAS mutation and amplification status predicts sensitivity to antifolate therapies in non-small cell lung cancer.

Abstract Somatic genetic mutation in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been linked to poor prognosis and resistance to various targeted therapeutics in Non Small Cell Lung Cancer (NSCLC). Therapeutic strategies that target tumors harboring these mutations represent an unmet medical need. In this study, we investigated the relationship between antifolate sensitivity and KRAS mutation/amplification status in NSCLC. Human NSCLC cell lines (KRAS wild type, KRAS mutant non-amplified and KRAS mutant amplified) were treated with Methotrexate (MTX) or Pemetrexed (PEM) and assayed for proliferation after 72h. In these studies, 5 out of 7 KRASwt (wildtype) cells and all KRASmut (mutant) amplified cells showed resistance to MTX treatment (IC50 &amp;gt;10μM). In contrast, growth of all KRASmut non-amplified cell lines studied was inhibited with MTX treatment (IC50 &amp;lt;100nM). Similar effects were observed for PEM in this study. Interrogation of the NCI Developmental Therapeutics Program cancer drug screen database for the relationship between KRAS mutation status and drug efficacy also revealed a similar trend in other NSCLC cell lines for MTX and other anti-folates. Cell cycle analysis of MTX treated cells demonstrated induction of S phase growth arrest in MTX treated KRASmut non-amplified NSCLC cells, an effect that is not observed in KRASwt or KRASmut amplified NSCLC cell lines. qRT-PCR analysis demonstrated a dramatic downregulation of KRAS gene expression in KRASwt and KRASmut cells with antifolate treatment. However, KRAS gene expression was less affected in antifolate treated KRASmut amplified cells. Co-treatment of KRASmut cells with antifolates and hypoxanthine/thymidine (which compensate for folate pathway inhibition) prevented downregulation of KRAS gene expression and rescued KRASmut cells. qPCR array analysis of miRNA expression in antifolate treated cells revealed increased expression of KRAS specific miRNAs including miR-181 and miR-143 with treatment compared to untreated controls. Finally, we present patient data describing unprecedented rapid and durable radiographic responses in KRAS mutant NSCLC cancer patients. Collectively, these studies identify increased sensitivity to antifolates in KRASmut non-amp NSCLC cell lines. Anti-folate therapies decrease KRAS gene expression in KRASwt and KRASmut cells but do not do so in KRASmut amplified cells. We propose that decreased KRAS gene expression is detrimental to KRASmut cells due to their dependency on this pathway for survival. We also propose that decreases in KRAS gene expression are mechanistically linked to stress (folate inhibition) induced miRNA expression which target KRAS gene expression. Overall, antifolates represent a novel method to target KRAS and as such should be investigated further for use in this subtype of NSCLC. As clinical evidence emerges, it is apparent that both KRAS mutation and amplification status should be considered for patient stratification prior to antifolate treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-2.

Detection of Brk expression in non-small cell lung cancer: clinicopathological relevance

Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk upregulation and oncogenic properties have been found in several malignant tumors, including breast, colon carcinomas, and melanomas, but the expression of Brk and its clinical significance in non-small cell lung cancer (NSCLC) remains unclear. In the current study, we examined the expression of Brk and its correlation with clinicopathological features involving p53, ki67, and E-cadherin status in NSCLC tissue using immunohistochemistry. We also used immunocytochemistry and immunofluorescent staining to examine the Brk expression and its subcellular localization in NSCLC cell lines, including LTE and H460. We further confirmed cytoplasmic and nucleus expression of Brk in LTE and H460 cells using Western blotting. The Brk expression in NSCLC cells was mainly found in cytoplasm (59/122, 48.4%) with some nucleus staining (17/122, 13.9%) with a total positive rate of 53.3% (65/122). Cytoplasmic Brk expression in NSCLC was higher than that in normal lung tissues (24/122, 19.7%) (P < 0.05). Increased cytoplasmic Brk expression in NSCLC was associated with large tumor size (≥ 3 cm), lymph node metastasis, and advanced tumor-node-metastasis (TNM) stages (III and IV) (P < 0.05). Moreover, increased cytoplasmic Brk expression was positively associated with Ki67 status in NSCLC (P < 0.05). Reduced E-cadherin expression was also found to be associated with lymph node metastasis and advanced TNM stages (III and IV) in NSCLC (P < 0.05). Brk expression was not associated with E-cadherin expression and P53 status in NSCLC (P > 0.05). The present findings indicate an increase of cytoplasmic Brk expression in NSCLC which may play a role in tumor development, including tumor expansion and lymph node metastasis in which Ki67, but not E-cadherin, and P53 status may be involved.

Prognostic Impact of MiR-155 in Non-Small Cell Lung Cancer Evaluated by in Situ Hybridization

BackgroundIn recent years, microRNAs (miRNAs) have been found to play an essential role in tumor development. In lung tumorigenesis, targets and pathways of miRNAs are being revealed, and further translational research in this field is warranted. MiR-155 is one of the miRNAs most consistently involved in various neoplastic diseases. We aimed to investigate the prognostic impact of the multifunctional miR-155 in non-small cell lung cancer (NSCLC) patients.MethodsTumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with four cores from each tumor specimen. In situ hybridization (ISH) was used to evaluate the expression of miR-155.ResultsThere were 191 squamous cell carcinomas (SCCs), 95 adenocarcinomas (ACs), 31 large cell carcinomas and 18 bronchioalveolar carcinomas. MiR-155 expression did not have a significant prognostic impact in the total cohort (P = 0.43). In ACs, high miR-155 expression tended to a significant negative prognostic effect on survival in univariate analysis (P = 0.086) and was an independent prognostic factor in multivariate analysis (HR 1.87, CI 95% 1.01 - 3.48, P = 0.047). In SCC patients with lymph node metastasis, however, miR-155 had a positive prognostic impact on survival in univariate (P = 0.034) as well as in multivariate (HR 0.45, CI 95% 0.21-0.96, P = 0.039) analysis.ConclusionsThe prognostic impact of miR-155 depends on histological subtype and nodal status in NSCLC.

Telomere length maintenance: a factor to be considered in personalized medicine?

telomerase is required to sustain their growth. The fact that cancer cells have shorter telomeres than normal cells, together with the fact that cancer growth appears to depend on telomerase reactivation, indicates that therapeutic strategies that are aimed at inhibiting telomerase will preferentially kill the tumor cells and have no toxicity on normal cells [3]. Since telomerase and telomere maintenance play a key role in the development of malignancies, several approaches targeting either the telomerase enzyme or the telomeres have been developed. However, while many efforts have been carried out to discover new anti-telomere/telomerase drugs, to date, very few compounds are exploitable for clinical use owing to low effectiveness or toxicity in vivo and also because of our partial knowledge on telomere and telomerase regulation and its functions [4]. There is growing evidence that telomere length carries information that may be of clinical importance for cancer patients. It is well established that telomere length is important for senescence in normal cells and there appears to be a connection between telomere shortening, genetic aberrations and the risk of cell transformation. Interests in investigating telomere length as a potential clinical biomarker in cancer has grown considerably in recent years. For hematogical malignancies, published data demon strate that short telomeres indicate progressive disease and a poor outcome [5]. For solid tumors the data are more heterogeneous. Thus, in colorectal cancer (CRC) telomere reduction appears to occur in parallel in noncancerous mucosa and colon cancer, with a correlation between both. Functional studies have implicated human TRF1 in telomere length Telomeres form specialized structures at the ends of eukaryotic chromosomes, preventing them from being wrongly recognized as damaged DNA. In normal cells, the DNA replication machinery is unable to completely duplicate telomeric DNA; thus, telomeres are shortened after every cell division. When telomeres become critically shortened and compromise genomic stability, chromosome ends activate DNA damage response pathways that can induce apoptosis. In cancer, these mechanisms are often inactivated, and telomeres can become dysfunctional via several mechanisms, such as loss or alteration of telomere-binding proteins involved in telomere maintenance. These proteins are critical for the functions of telomeres as they prevent unwanted DNA repair activity [1]. Tumor cells acquire the hallmark properties of cancer during the carcinogenic selection process. Cell immortality is one of the principal features acquired, which involves the stabilization of telomere length. It is achieved mainly by telomerase activation. Thus, the discovery of telo meres and telomerase allowed an understanding of the mechanisms by which cells can become immortalized [2]. Generally tumors have shorter telomeres than the surrounding normal tissue, owing to the fact that they have had a longer proliferative history in the absence of telomerase activity. Telomere shortening can eventually lead to increased cell death and loss of cell viability within the tumor. However, telomerase is reactivated in more than 90% of all types of human tumor, thereby rescuing and perpetuating cells with short telomeres and high chromosomal instability. Similarly, most metastases also contain telomerase-positive cells, which indicate that

KRAS mutations (MTs) in non-small cell lung cancer (NSCLC) versus colorectal cancer (CRC): Implications for cetuximab therapy.

10529 Background: KRAS MTs have been associated with a diversity of biologic functions, and have both prognostic and predictive consequences in NSCLC and CRC. Little data exist comparing the specifics of KRAS MTs in the two cancers. In CRC, benefit from the cetuximab is mainly limited to patients whose tumors are KRAS wild-type (WT). However, recent studies show no such predictive value in NSCLC. We hypothesized that the unique molecular biology and tobacco carcinogen-related etiology of NSCLC contribute to a divergence in KRAS MT type and EGFR-related dependency. Here, we compare KRAS and EGFR status in NSCLC versus (vs) CRC in the large Response Genetics (RGI) database. Methods: KRAS MTs were assessed in 2,693 specimens (838 CRC and 1165 NSCLC) using DNA from microdissected formalin fixed paraffin embedded tumor specimens. EGFR MTs were determined in 649 NSCLCs. A PCR approach with direct sequencing was used to detect 7 different base substitutions in KRAS and 28 EGFR MTs. Statistics: Fisher's Exact Test. Results: KRAS MTs were found in 39% of CRC and 23% of NSCLC specimens (p < 0.001). EGFR MTs were identified in 16% of NSCLCs. In 447 specimens with both assays, EGFR and KRAS MTs were largely exclusive; 4 specimens harbored both (p < 0.001). The ratio of base transversions to transitions was 3.27 in NSCLC versus 0.77 in CRC (p < 0.001). With regard to histology, in NSCLC KRAS MTs were observed in 28.7% (186/649) of adenocarcinomas vs 3.5% (7/198) of squamous cell carcinomas (p < 0.001). Tobacco carcinogenesis-associated G>T transversions (codon 12 GGT>TGT plus GGT>GTT) comprised 61% of KRAS MTs in NSCLC vs 39% in CRC (p < 0.001). The GGT>GAT transition was most prevalent in CRC at 33.3%. Conclusions: This analysis of the RGI database demonstrates differences between NSCLC vs CRC in both incidence and type of KRAS MTs. Increased frequencies of DNA KRAS transversions were seen in NSCLC, likely linked to tobacco exposure. Distinct MT patterns and biologic function may contribute to differences in predictive value for cetuximab therapy between NSCLC and CRC. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics Amgen, AstraZeneca, Biodesix, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genoptix, GlaxoSmithKline, Lilly, Merck, Novartis, Pfizer, Response Genetics, sanofi-aventis Response Genetics DxS, Response Genetics Abbott Laboratories, Bristol-Myers Squibb, Genentech, Lilly, Merck, Novartis, OSI, Pfizer

ACR Appropriateness Criteria ® Nonsurgical Treatment for Non-Small-Cell Lung Cancer: Good Performance Status/Definitive Intent

The optimal strategy for the non-surgical definitive treatment of patients with good performance status non-small cell lung cancer (mostly with locally advanced disease) has dramatically evolved over time. This article presents evidence-based data to review this literature. Several decades ago, the standard treatment for most stage III inoperable NSCLC was definitive radiation therapy alone. Randomized trials have since shown superior results with sequential chemotherapy and radiation, and more recently with concurrent chemoradiation, the current standard of care. Studies suggest a limited role for induction or adjuvant systemic therapy in addition to concurrent chemoradiation. The role of altered radiation fractionation techniques, such as hyperfractionation for locally advanced disease or hypofractionation for early stage disease is also discussed. More recently, the application of more advanced radiation techniques has been explored, including intensity modulated radiation therapy (IMRT) and proton beam radiation. Finally, various case variants are presented as examples of state-of-the-art treatment approaches.

Abstract 3015: The miR-21:miR-221 ratio as a serum-based diagnostic for non-small cell lung cancer

Abstract MicroRNAs (miRNA) are small (21-25 nucleotides) non-coding regulatory RNAs that control protein expression at the transcriptional level through various mechanisms. Previous studies have shown that circulating miRNA can be utilized as a tool to gain a better understanding of both benign and malignant tumor conditions. Their altered expression has been linked with several cancers including lung cancer. Lung cancer is the leading cause of death worldwide with approximately 85% of diagnoses being attributed to non-small cell lung cancer (NSCLC). The goal of this study was to determine if the presence of circulating miRNAs from NSCLC patients can serve as non-invasive diagnostic biomarkers of disease and therapy monitoring. We initially evaluated the expression of 16 NSCLC-specific miRNAs (miR-146b, miR-155, miR-205, miR-21, miR-221, miR-199a-5p, miR-222, miR-320, miR-223, miR-25, miR-16, miR-191, miR-20a, miR-24, miR-145, and miR-152) in 46 NSCLC serum samples ranging from stage I to IV, including 20 adenocarcinoma and 26 squamous cell carcinoma samples. Eighteen healthy controls were used for comparison. RNA was extracted from serum using the single-step Trizol method and real-time PCR (Taqman). Of the 16 miRNAs tested, six miRNAs were significantly upregulated in the NSCLC when compared to the normals (p&amp;lt;0.0001 with AUROCs of 0.80 to 0.93). Top Scoring Pair analyses identified the ratio of miR-21 to miR-221 as the most robust predictor of NSCLC status compared to healthy subjects with an AUROC of 0.95 (95% CI; 0.91-0.99, p &amp;lt; 0.001) and 17% error rate (leave-one-out cross validation). This miRNA pair was then validated in an independent set of 19 NSCLC and 26 normal serum samples with overall accuracy of 80% (89% and 73% for NSCLC and normal, respectively) and AUC of 0.88 (95%CI: 0.78-0.98). These findings demonstrate the potential of using miR-21 and miR-221 in detecting NSCLC from the serum. Furthermore, these miRNAs could lead to the development of non-invasive biomarkers for early diagnosis, prognosis, and therapy monitoring in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3015.

Abstract B180: A novel mutant-enriched liquidchip technology (MEL) for detecting circulating EGFR somatic mutations in non small cell lung cancer

Abstract Epidermal growth factor receptor (EGFR) is a key target for developing targeted therapeutics to treat cancer. The US FDA has approved some EGFR targeted drugs that include monoclonal antibody drugs Erbitux, Panitumumab, as well as the EGFR tyrosine kinase inhibitors Iressa and Tarceva. However, clinical data indicated that only a small percentage of patients were responsive to these targeted drugs. Further studies have revealed that up to 20% of non small cell lung cancer (NSCLC) tumors that harbor the EGFR Exon 19 or 21 somatic mutations are more likely responsive, while patients without these mutations are not responsive. Therefore, physicians have a responsibility to test whether or not the patient's EGFR is mutated before prescribing these targeted drugs. In general, a very small amount of free DNA fragments can be found in the blood, especially in patients with malignant tumors. However, it is technically very difficult to detect a very small amount of DNA somatic mutations that are usually masked under a huge amount of the wild-type (WT) sequence. To cater to the clinical need, we have successfully developed a novel technology that creatively combines a mutant-enriched method to a liquidchip platform. The principle of MEL is to create a unique restriction enzyme site at the WT gene sequence in order to remove the WT gene by the restriction enzyme digestion before the mutated gene sequence could be selectively amplified by the PCR. The mutated PCR product will then be hybridized to a specific probe on polystyrene microspheres and analyzed by the Luminex. This technology is extremely sensitive and therefore allows us to use the serum or plasma sample to detect somatic gene mutations. It is also high-throughput; which allows us to assay up to 96 samples at once and read up to 100 different mutations from one sample. MEL technology has been successfully used in the clinic for detecting other somatic mutations from serum and plasma samples as well, such as KRAS, BRAF, PIK3CA, EGFR exon 20, etc. The detection sensitivity is one mutant DNA in the presence of 1×103 wild-type copies. As low as 10 copies of mutant DNA in a sample could be detected. To evaluate the MEL, the EGFR mutation status of NSCLC was tested on tissue samples using both sequencing and MEL methods. The positive samples of EGFR mutation by sequencing method were in agreement with the results by the MEL. However, 31.8% (7/22) of the negative samples by sequencing were positive by MEL. To date, our results have shown that 32.1% (146/455) serum samples of NSCLC patients showed EGFR mutant positive, from which 9.2% (42/455) were exon 19 deletion mutations; 19.8% (90/455), exon 21 single nucleotide polymorphism mutation; and 3.1% (14/455), both. Our study also showed that EGFR mutations were detected more frequently in patients with partial response following the targeted therapy than those with stable disease or progressive disease (p=0.027). In conclusion, MEL is a valuable method for detecting circulating EGFR mutations in serum or plasma of NSCLC patients for pharmacogenomic diagnostics. This method could be used on tissue samples as well. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B180.

Morphometric analysis of regional lymph nodes in surgically resected non-small cell lung cancer

Nodal staging is a crucial factor in choosing the treatment option for non-small cell lung cancer (NSCLC). However, so far as we know, a computer-based histomorphometric analysis of lymph nodes in NSCLC has never been developed. We studied 299 surgically resected lymph nodes from 108 patients with NSCLC. Microscopic digital images were analyzed with Scion Image software. Seventy lymph nodes had at least one metastatic focus. The metastasis occupancy area per node ranged from 0.01 to 209.58 mm(2) (mean, 29.58 +/- 5.87 mm(2)). The metastasis occupancy ratio ranged from 0.01% to 100% (mean, 48.70% +/- 42.10%). The short-axis diameter of malignant lymph nodes was significantly longer than that of benign lymph nodes (P = 0.0002). The average metastasis occupancy area in the regional lymph nodes of NSCLC is quite small. Various inflammatory conditions can result in a false-positive diagnosis when these techniques are used. Studies that combine analysis of primary tumor size and serum carcinoembryonic antigen (CEA) levels with imaging methods should be considered. Finally, the use of mediastinoscopy or video-assisted thoracoscopic surgery is encouraged in determining the exact nodal status in NSCLC.