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M.D. Lozano



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    MO05 - Prognostic and Predictive Biomarkers II (ID 95)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO05.09 - Activation of the classical complement pathway: a novel biomarker for the early diagnosis and prognosis of lung cancer (ID 964)

      17:05 - 17:10  |  Author(s): M.D. Lozano

      • Abstract
      • Presentation
      • Slides

      Background
      Numerous diagnostic and prognostic molecular markers have been proposed for lung cancer. However, genetic heterogeneity has limited the success of these initiatives. This limitation may be overcome by the use of biomarkers related to the host response to cancer. In this study we tested the capacity of lung cancer cells to activate the complement system and evaluated the diagnostic performance of complement-activation fragments. We demonstrate for the first time that lung cancer cells efficiently activate the classical complement pathway and that fragments of complement activation are of value for detection and prognosis of lung cancer at a very early stage.

      Methods
      We first assessed complement activation in bronchial epithelial and lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; in bronchoalveolar lavage supernatants from 50 patients with lung cancer and 22 non-malignant respiratory diseases; and in plasma samples from different cohorts, including: advanced (n=133) and early (n=84) non-small cell lung cancer patients, subjects with inflammatory lung diseases (n=133) and asymptomatic individuals enrolled in a lung cancer CT-screening program (n=190; 32 of them with lung cancer).

      Results
      Lung cancer cells treated with normal human serum activated complement and deposited C3 more efficiently than non-malignant bronchial epithelial cells. Incubation of cells with different buffer conditions, complement depleted sera and complement inhibitors showed that lung cancer cells bind C1q and activate complement through the classical complement pathway. In a set of lung cancer cell lines, a significant correlation was found between C1q binding and C4 or C3 deposition. The presence of phosphatidylserine inhibited C1q binding and diminished complement activation. Based on these results, C4d, a classical pathway-derived split product, was evaluated as a possible diagnostic or prognostic biomarker in lung cancer. Many lung primary tumors (adenocarcinomas and squamous cell carcinomas) deposited C4d. More importantly, survival was decreased in patients with high C4d deposition in their tumors (HR=3.06; 95% CI=1.18-7.91). Moreover, C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients as compared to patients with non-malignant respiratory diseases (0.61 ± 0.87 vs. 0.16 ± 0.11 µg/ml, respectively; P<0.001). C4d levels in plasma samples from lung cancer patients at both advanced (III and IV) and early (I and II) stages were also increased compared with control subjects (4.13 ± 2.02 vs. 1.86 ± 0.95 µg/ml, P<0.001; and 3.18 ± 3.20 vs. 1.13 ± 0.69 µg/ml, P<0.001, respectively). In addition, C4d plasma levels were associated with shorter survival in patients at advanced (HR=1.59; 95% CI=0.97-2.60) and early stages (HR=5.57; 95% CI=1.60-19.39). Plasma C4d levels were dramatically reduced after surgical removal of lung tumors. Finally, plasma C4d levels were associated with increased lung cancer risk in asymptomatic individuals: OR=4.38; 95% CI=1.61-11.93.

      Conclusion
      Lung tumors activate the classical complement pathway and generate C4d, a stable complement split product. Moreover, C4d is increased in biological samples from lung cancer patients, is associated with poor prognosis, and may be of clinical value for the early detection of lung cancer.

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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): M.D. Lozano

      • 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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      MO26.07 - Usefulness of cytological samples for the assessment of ALK rearrangements in NSCLC patients. (ID 2351)

      11:05 - 11:10  |  Author(s): M.D. Lozano

      • Abstract
      • Presentation
      • Slides

      Background
      ALK gene rearrangement defines a new molecular subtype of NSCLC with response to Crizotinib, (Xalkori®) a dual MET and ALK inhibitor. To date, determination of ALK gene rearrangements has been performed in biopsies and/or surgical specimens. However, advanced lung cancer is often diagnosed by FNA cytology obtained through minimally invasive procedures, and frequently cytological specimens are the only samples available. We assessed the feasibility of determining ALK gene rearrangements in cytological samples.

      Methods
      We studied prospectively 53 cytological samples from 53 NSCLC patients (30 M/23 F) for ALK gene rearrangements by FISH (Abbot dual colour break apart probe). Tumour samples were obtained by bronchoscopy -FNA in 26 cases (49.1%), EBUS-FNA in 7 (13.2%), EUS-FNA in 3 cases (5.7%), CT-FNA in 3 (5.7%), and direct FNA in 6 cases (11.3%). Two cavity fluids (3.8%), 4 imprints from surgical specimens (7.5%), and 2 cellblocks received for consultation (3.8%) were also studied. FISH was performed on Papanicolau stained smears in 15 cases (28.3%), non-stained ThinPrep in 28 cases (52.8%), cell block in 9 cases (17%), and 1 stained ThinPrep. All cases were tested for EGFR and KRAS mutations.

      Results
      Thirty-seven samples (69.8%) were adequate for FISH analysis. Three cases (8.1%) had ALK gene rearrangements: positive cases were non-smoker women with adenocarcinoma, two of them with signet ring cells. One case had a concurrent EGFR mutation in exon 21. FISH study was unsuccessful in 16 cases (30.2%): 10 from Papanicolau stained smears (62.5%), 5 from unstained ThinPrep (31.3%), and 1 from a cell block. Nineteen ThinPrep slides were adequate for FISH analysis (86.4%) as well as 8 out of 9 cell blocks. Correlation cytological / paraffin embedded samples was performed in 4 cases with a concordance rate of 100%.

      Conclusion
      Determination of ALK gene rearrangements in cytological specimens is feasible. It is mandatory an exquisite management and care of the samples to preserve quality. ThinPrep and cell blocks are the most suitable samples for FISH analysis, while Papanicolau stained smears provide poor results. Coexistence of ALK gene rearrangements and EGFR mutations was observed in one case, indicating that such alterations are not necessarily mutually exclusive.

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    P2.18 - Poster Session 2 - Pathology (ID 176)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P2.18-015 - EGFR and KRAS mutational analysis using small amounts of DNA from FNA and small CNB is feasible and reproducible using a commercial real time PCR method. Validation of this PCR method in cytological samples. (ID 2721)

      09:30 - 09:30  |  Author(s): M.D. Lozano

      • Abstract

      Background
      EGFR and KRAS mutations guide treatment selection in NSCLC patients. With 75% of newly diagnosed cases at advanced stages, mutational analysis is performed in small samples: core needle biopsies (CNB) and fine needle aspiration (FNA). Both the cobas EGFR and KRAS test are CE-IVD. No validation studies of the cobas tests have been performed using cytological smears but it is important to extend the benefits of molecular targeted therapy while preserving tissue for additional marker testing.

      Methods
      EGFR and KRAS mutation status were studied in 140 non-selected samples from NSCLC patients: 49 CNB, 91 FNA. DNA was extracted directly from one stained smear in FNA samples and one 5-micron section in CNB using the cobas DNA Sample Preparation Kit. All samples contained ≥ 50% tumor cells. DNA concentration and ratio (A260/280) were recorded. All cases were studied using the cobas EGFR and KRAS mutation tests. Moreover, 123 and 125 cases were analyzed respectively for EGFR and KRAS mutational status using Sanger sequencing.

      Results
      CNB diagnosis was: 29 SqCC, 17 AC, 1 BAC, 1 adenosquamous, 1 NSCLC-NOS. FNA diagnosis was: 64 AC, 13 SqCC, 3BAC, 2 LCC, 2 adenosquamous, 7 NSCLC-NOS. DNA concentrations from CNB were higher and significantly different than DNA from FNA (p<0.001,U Mann Whitney). DNA quality was similar between sample types. Mutational analysis is shown in Table 1. Mutation rate for EGFR was 15.9% and 8.2 % and for KRAS 37% and 16.7% in FNA and CNB, respectively, but should be considered within the context of tumor type. 8.6% and 36.4% of the cases were below the manufacturer’s recommendations of 2ng/µl and 4ng/µl for EGFR and KRAS testing, respectively. Invalid rates were 2.1% (3) for EGFR and 5.5% (9) for KRAS. These results may be due to low DNA concentration (EGFR) or technical performance (KRAS) that was resolved with later samples. Sequencing invalid results were 42.3% for EGFR and 0.8% for KRAS. Table 1.

        FNA CNB
      EGFR WT 74 45
        Exon 19 Del 8 2
        Exon 20 Ins 0 1
        L858R 5 1
        Exon 18 G719X 1 0
        Invalid 3 0
      KRAS WT 51 40
        12/13 Mutation 29 8
        61 Mutation 1 0
        Invalid* 8 1
      * By sequencing 6 WT and 3 mutated

      Conclusion
      In addition to FFPE samples, identification of EGFR and KRAS mutations in FNA and CNB samples using cobas EGFR and KRAS Mutation Tests is faster, easier to use, and reproducible. Although DNA concentrations were lower from FNA, DNA quality was similar to CNB and provided valid results. Sequencing had lower sensitivity and was more time-consuming. Careful sample management, especially for FNA, by the pathologist is critical to ensure quality and to optimize DNA yields.