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N. Karachaliou



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    MO21 - Prognostic and Predictive Biomarkers V - EGFR (ID 98)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO21.05 - Integrated genomic analysis by whole exome and transcriptome sequencing of tumor samples from EGFR-mutant non-small-cell lung cancer (NSCLC) patients with acquired resistance to erlotinib. (ID 1426)

      10:50 - 10:55  |  Author(s): N. Karachaliou

      • Abstract
      • Presentation
      • Slides

      Background
      NSCLC patients with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs) but ultimately relapse. Sub-genomic molecular studies indicate that the EGFR T790M mutation and the activation of MET, PI3K, AXL, HER2 and MAPK can lead to acquired resistance to EGFR TKIs. To date, no integrated comprehensive genomic investigation of EGFR TKI resistance has been reported.

      Methods
      FFPE biopsies of erlotinib-sensitive and erlotinib-resistant tumors were obtained from 13 EGFR mutant NSCLC patients. The samples were analyzed by whole exome sequencing and whole transcriptome sequencing utilizing the Illumina HiSeq2500 platform. In addition, targeted gene sequencing was performed with the Illumina TruSeq Amplicon-Cancer Panel and run on the MiSeq system.

      Results
      Erlotinib resistant NSCLC specimens harbored known resistance drivers, including EGFR T790M mutations (9/13; 69%), MET amplification (3/13; 23%), HER2 amplification (3/13; 23%), and AXL upregulation (3/13; 23%). Differential expression analysis between resistant and pre-treatment states revealed enrichment in the pre-treatment tumors of immune signaling pathways, and in the resistant tumors upregulation of ERBB2, mTOR, PI3 kinase and ribosomal signaling pathways. PI3K/AKT pathway upregulation also occurred through somatic mutations in AKT and LKB1 in the resistant tumors. Copy number analysis demonstrated both large scale and focal amplifications and deletions in the resistant tumors, including the focal loss of EGFR and gain of c-Myc and NKX2-1. There was strong correlation between the copy number changes observed and the expression mRNA levels of the involved cancer-associated genes. Of note, each resistant tumor exhibited greater copy number similarity to the corresponding matched pre-treatment sample compared to other tumors within the resistance cohort.

      Conclusion
      We conducted the first ever comprehensive integrated genomic analysis of EGFR TKI resistant NSCLC patients, and identified both known and potentially novel drivers of EGFR TKI resistance. This study demonstrated the feasibility and utility of comprehensive genomic analysis in the clinical management of NSCLC receiving targeted therapy. Together, our data provide unprecedented insight into the molecular pathogenesis of escape from EGFR oncogene inhibition in NSCLC. We are now conducting a prospective observational study in additional NSCLC patients on targeted therapy.

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    MO26 - Anatomical Pathology II (ID 129)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      MO26.08 - The concomitant presence of echinoderm microtubule associated protein like 4 - anaplastic lymphoma kinase (EML4-ALK) EML4-ALK fusion gene in EGFR-mutant non-small-cell lung cancer (NSCLC) patients treated with erlotinib or chemotherapy in the EURTAC trial</b> (ID 1109)

      11:10 - 11:15  |  Author(s): N. Karachaliou

      • Abstract
      • Presentation
      • Slides

      Background
      Activating mutations in the epidermal growth factor receptor (EGFR) confer sensitivity to gefitinib and erlotinib in patients with NSCLC. However, response is often short-lived, and patients ultimately relapse, indicating that other concomitant actionable mutations could influence outcome in these patients. The EML4-ALK fusion gene has recently been identified in a subset of NSCLCs, but its specific role remains unclear. We have evaluated the frequency and impact of the concomitant presence of EML4-ALK in patients included in the randomized phase III EURTAC trial.

      Methods
      The EURTAC study enrolled 173 EGFR-mutant NSCLC patients who were randomized to receive erlotinib or standard chemotherapy with cisplatin or carboplatin plus docetaxel or gemcitabine. Tumor specimens were available from 95 of these patients for the analysis of EML4-ALK. EML4-ALK variants 1 and 3 (v1, v3) were analyzed by an independent single round of PCR followed by sequencing, using cDNA as a sample.

      Results
      EML4-ALK was detected in 15 samples (15.79%). Nine tumors contained v1 (E13;A20) and six v3 (E6;A20). No significant differences were found in baseline characteristics between patients with and without EML4-ALK. Progression-free survival was 10.4 months (m) for patients harboring the EML4-ALK fusion gene compared to 7.1 m for those without EML4-ALK. Overall survival (OS) was not reached in patients with EML4-ALK, compared to 22.9 m in those without. Complete data on outcome according to treatment arm will be presented.

      Conclusion
      Our findings indicate that the EML4-ALK rearrangement is concomitant with EGFR mutations in a considerable number of NSCLC patients and may affect outcome.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-001 - MED12, a component of the transcriptional MEDIATOR complex, and STAT3 influence outcome to platinum-based chemotherapy in patients (p) with advanced non-small-cell lung cancer (NSCLC) (ID 323)

      09:30 - 09:47  |  Author(s): N. Karachaliou

      • Abstract

      Background
      MED12 negatively regulates TGF-b receptor signaling. Loss of MED12 induces an EMT-like phenotype associated with chemotherapy resistance. IDO and IL6 activate the JAK2/STAT3 signaling pathway, which – together with NFkB (RelA) signaling – is often altered in lung cancer. BIM could influence response to chemotherapy. We have examined these components and KRAS mutations in NSCLC tumor samples and correlated results with progression-free survival (PFS).

      Methods
      The mRNA expression of MED12, IDO, JAK2, STAT3, RelA and BIM was examined in microdissected tumor samples from p with stage IV NSCLC. mRNA levels were assessed by RT-PCR and categorized by terciles (high vs low/intermediate). KRAS mutations were assessed by high resolution melting.

      Results
      A total of 55 p with performance status (PS) 0-1, treated with platinum plus either gemcitabine or pemetrexed: median age, 62 years; 27.6% females; 84.2% smokers; 66% adenocarcinoma; 16% with KRAS mutations. There was no correlation between gene expression levels and KRAS mutation status. In the multivariate analysis, including gene expression levels, histology and PS, only MED12 and STAT3 were associated with PFS (low MED12: HR=11.6, P=0.005; high STAT3: HR=6.5, P=0.01). HR for low BIM expression was 2.4 (P=0.16). None of the markers were associated with overall survival.

      Conclusion
      To the best of our knowledge, this is the first time that low expression of MED12 with significantly shorter PFS in NSCLC p receiving platinum-based chemotherapy. MED12 could be a new biomarker of chemoresistance and inhibition of TGF-bR signaling can restore chemotherapy response in patients with low MED12.

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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P2.06-012 - Impact of EGFR T790M mutations and BIM mRNA expression on progression-free survival (PFS) and overall survival (OS) in patients with EGFR-mutant non-small-cell lung cancer (NSCLC) treated with erlotinib or chemotherapy in the randomized phase III EURTAC trial (ID 1167)

      09:30 - 09:30  |  Author(s): N. Karachaliou

      • Abstract

      Background
      Activating EGFR mutations confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in patients with NSCLC, but responses are transient, with delay in disease progression but no impact on survival. Concomitant genetic alterations could account for these incomplete clinical responses. Erlotinib-treated EGFR-mutant NSCLC patients harboring the EGFR T790M mutation had shorter PFS than those without the mutation (12 vs 18 months [m]). Low BIM levels were associated with gefitinib resistance in EGFR-mutant NSCLC.

      Methods
      The efficacy results of the EURTAC trial were updated at January 24, 2013. We have evaluated the frequency and potential impact of pretreatment EGFR T790M mutations and BIM mRNA expression in 95 patients with EGFR-mutant NSCLC included in the EURTAC trial.

      Results
      T790M mutations were detected in 65.26% of patients. PFS to erlotinib was 9.7 m for those with T790M mutations and 15.8 m for those without, while among patients receiving chemotherapy, it was 6 and 5.1 m, respectively (P<0.0001). BIM expression was successfully analyzed in 83 patients. PFS to erlotinib was 12.9 m for those with high BIM levels and 7.2 m for those with low/intermediate BIM levels, while among chemotherapy-treated patients, it was 5.8 and 5.5 m, respectively (P=0.0003). OS was 28.6 m for patients with high BIM expression and 22.1 m for those with low/intermediate BIM expression (P=0.0364). The multivariate analyses showed that erlotinib was a marker of longer PFS (HR, 0.35; P=0.0003), while high BIM expression was a marker of longer PFS (HR, 0.49; P=0.0122) and OS (HR, 0.53; P=0.0323).

      Conclusion
      BIM mRNA expression is a biomarker of PFS and OS in EGFR-mutant NSCLC. T790M mutations and BIM mRNA expression can potentially be used for designing combination therapeutic strategies for use in lieu of EGFR TKI monotherapy.

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      P2.06-046 - EML4-ALK monitoring of crizotinib response in blood platelets and plasma of NSCLC patients (ID 3267)

      09:30 - 09:30  |  Author(s): N. Karachaliou

      • Abstract

      Background
      The discovery of various genetic alterations that underlie lung cancer has opened-up a new era in the development of specifically targeted therapies employing specific alteration-dependent inhibitors. In the vast majority of NSCLC patients genetic alterations occur in EGFR (20-25%), BRAF (3%), and ALK (3-10%), seemingly in a predominant mutual exclusive manner. Hence, patient stratification for EGFR, KRAS, BRAF, and ALK alterations is becoming common practice among oncologists. The mutational status of NSCLC patients is considered dynamic and can change in due course of the disease and selection in response to therapy. Longitudinal monitoring of the mutational status of NSCLC patients is of crucial importance to tailor targeted therapy by adapting treatment regimens according to mutational status. However, technical challenges associated with serial tumor biopsy constitute a major challenge in longitudinal molecular monitoring and targeted treatment of NSCLC patients. Blood-based assays may overcome such limitations and allow for frequent assessment of the mutational status.

      Methods
      Here, tumor tissue, platelets, and/or plasma of NSCLC patients were collected and analyzed for the presence of translocated ALK using FISH, IHC, and/or RT-PCR. Monitoring of EML4-ALK in platelets and plasma was performed by RT-PCR on EML4-ALK positive patients treated with crizotinib and correlated to the clinical response as measured by serial radiographic CT.

      Results
      From a multicenter cohort of NSCLC patients we identified EML4-ALK positive patients by FISH, IHC, and RT-PCR, for blood platelet and/or plasma isolation. In blood platelets of ALK positive NSCLC patients (n=24) and ALK negative control subjects (n=54) we demonstrated an EML4-ALK test sensitivity of 70-80% and specificity of 100%. We detected EML4-ALK in plasma of ALK positive NSCLC patients (n=22), however with inferior sensitivity of 20-30%. In addition, longitudinal EML4-ALK monitoring in blood platelets of an ALK positive NSCLC patient correlated with the crizotinib response.

      Conclusion
      We demonstrate here that blood platelets of NSCLC patients are a biosource for the detection of the EML4-ALK translocation and may proof useful for longitudinal monitoring of ALK inhibitors in NSCLC patients, with superior outcome over plasma.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-009 - <b>ROR1 as a novel therapeutic target for EGFR-mutant non-small-cell lung cancer (NSCLC) patients with the EGFR T790M mutation</b> (ID 1395)

      09:30 - 09:30  |  Author(s): N. Karachaliou

      • Abstract

      Background
      Molecular cross-talk between EGFR and other signaling pathways creates alternative means of tumor cell proliferation and promotes resistance to single-agent erlotinib therapy in NSCLC driven by EGFR mutations. ROR1 knockdown inhibited the growth of NCI-H1975 cells (harboring EGFR L858R and T790M mutations). A pro-survival function for ROR1/MEK/ERK signaling in cooperation with AKT has been demonstrated. We have assessed ROR1 expression in 45 patients from the EURTAC trial (clinicaltrials.gov NCT00446225), 27 of whom harbored pretreatment concomitant EGFR T790M mutations, and correlated results with outcome.

      Methods
      ROR1 mRNA expression was examined by quantitative RT-PCR and categorized by terciles; patients were classified as having low/intermediate or high ROR1 expression. The T790M mutation was determined by Taqman with a PNA to inhibit amplification of the wild-type (wt) allele. Tumor samples were run in octuplicates; this method can detect 1 mutated allele among 10,000 wt alleles.

      Results
      Median age 65; 68.9% female; 57.8% never-smokers; 95.6% ECOG PS <2; 91.1% adenocarcinoma; 68.9% exon 19 deletion. No differences in baseline characteristics were observed according to ROR1 expression levels. 24 patients (53.3%) were treated with erlotinib and 21 (46.7%) with chemotherapy. 10 (41.7%) erlotinib-treated patients and 6 (28.6%) chemotherapy-treated patients had high ROR1 mRNA levels. Among erlotinib-treated patients, response rate (RR) was 40% for the 10 patients with high ROR1 levels vs 71.4% for the 14 with low/intermediate levels (P=0.058). Among chemotherapy-treated patients, RR for the 15 patients with low/intermediate ROR1 levels was 6.7%; the 6 patients with high ROR1 levels did not respond. Progression-free survival (PFS) was 11.8 months (m) for erlotinib-treated patients with low/intermediate ROR1 levels vs 5.8 m for those with high levels. PFS for chemotherapy-treated patients was 5.6 and 9 m, respectively (P=0.0165). 15 erlotinib-treated patients harbored concomitant T790M mutations; for these patients, PFS was10.8 m for those with low/intermediate ROR1 levels vs 2.7 m for those with high levels (P=0.0138).

      Conclusion
      ROR1 expression has a differential effect on outcome to erlotinib and chemotherapy in EGFR-mutant NSCLC patients. High ROR1 expression significantly limits PFS in erlotinib-treated patients with T790M mutations and ROR1-directed therapies can enhance the efficacy of treatment. In contrast, high ROR1 expression confers longer PFS to chemotherapy in the same group of patients. The role of chemotherapy and erlotinib in EGFR-mutant NSCLC patients with high ROR1 expression warrants further investigation.

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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-006 - Integrated genomic analysis of EGFR-mutant non-small cell lung cancer immediately following erlotinib initiation in patients (ID 1003)

      09:30 - 09:30  |  Author(s): N. Karachaliou

      • Abstract

      Background
      Major obstacles limiting the clinical success of EGFR TKI therapy in EGFR mutant non-small cell lung cancer (NSCLC) patients are heterogeneity and variability in the initial anti-tumor response to treatment. The underlying molecular basis for this heterogeneity has not been explored in patients immediately after initiation of therapy.

      Methods
      We conducted CT-guided core needle biopsies immediately prior to erlotinib treatment initiation and at 6 days and 60 days post erlotinib initiation in a patient with histologically confirmed NSCLC harboring an established activating mutation in EGFR. DNA and RNA from each of the frozen biopsies were analyzed by whole exome sequencing and whole transcriptome sequencing, respectively. High-resolution CT images were also obtained at the time of each biopsy to assess the degree of molecular and radiographic responses observed.

      Results
      Two established activating somatic mutations were identified in EGFR (p.G719A and p. R776H). Gene expression analysis revealed that several proapoptotic genes including BID, CASP3 and several growth inhibitory genes including GADD45B, GADD45G were upregulated at 6 days post erlotinib treatment, while at 60 days their expression levels decreased to below pretreatment levels. Other proapoptotic genes such as BAD and BAX and were noted to be upregulated most significantly 60 days, as was growth inhibitory gene CDKN1A. In contrast, the growth-promoting genes CCNB1 and CCND3 exhibited a progressive decrease in expression across time points. Whole exome sequencing demonstrated the progressive evolution of global copy number abnormalities. High-resolution CT scans revealed no interval radiographic change in tumor size after 6 days of erlotinib treatment, and a decrease in tumor size 60 days into therapy. No clinical complications were encountered.

      Conclusion
      This study is the first reported longitudinal integrated genomic analysis of EGFR-mutant NSCLC in a patient treated with an EGFR TKI. We documented the feasibility, safety and utility of this strategy to establish initial drug efficacy at the molecular level prior to any radiographic evidence of response (6 days), as well as evidence that acquired resistance can emerge early in the course of TKI therapy. Serial integrated genomic analysis is ongoing in other TKI treated patients to enhance the management of NSCLC patients on targeted therapy.