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M.G. Dal Bello



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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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      MO21.02 - Pretreatment evaluation of the T790M mutation and its correlation with the response to tyrosine kinase inhibitors (TKIs) or chemotherapy in advanced non-small cell lung cancer (NSCLC) patients with activated EGFR mutations (ID 2455)

      10:35 - 10:40  |  Author(s): M.G. Dal Bello

      • Abstract
      • Presentation
      • Slides

      Background
      Preclinical data have shown that the EGFR-T790M mutation confers resistance to reversible EGFR-TKIs (gefitinib, erlotinib) but not to irreversible EGFR-TKIs (afatinib). This study evaluated advanced NSCLC patients (pts) harboring an activated EGFR mutation (exon 18-21) to investigate the incidence of the T790M mutation in pretreatment tumor samples and the correlation between the T790M mutation and the clinical outcome, comparing patients positive for the T790M mutation treated with reversible TKIs, an irreversible TKI or chemotherapy to patients negative for the T790M mutation treated with the same agents.

      Methods
      We screened 317 advanced NSCLC pts for EGFR mutations using the PCR/Sanger sequencing (PSS) method. Tumor tissues from EGFR-mutated pts were analyzed for the EGFR-T790M mutation using a highly sensitive locked nucleic acid-PSS method (LNA-PSS) capable of detecting EGFR-T790M-mutated alleles at extremely low frequencies. The response rate (RR), progression-free survival (PFS) and overall survival (OS) were evaluated retrospectively in these pts.

      Results
      Using PSS, 17.3% (55/317) of pts had an activating mutation in the EGFR-TK domain; 56.3% (31/55) of pts had an in-frame deletion in exon 19, 32.7% (18/55) of pts had point mutation L858R in exon 21, 3.6% (2/55) of pts had an insertion in exon 20, and 7.2% (4/55) of pts had both the T790M mutation and either an exon 19 or 21 mutation. Forty-two pts with EGFR-activating mutations (82.3%) without the T790M mutation (by PSS) were successfully analyzed for the T790M mutation using LNA-PSS. The T790M mutation was detected in 17 (40.5%) pts, with a higher incidence in never smokers (47.7%), adenocarcinoma (76.2%) and females (71.4%). A treatment response evaluation was available in 39 pts, 18 of whom (46.1%) harbored the T790M mutation. Pts with T790M had a lower RR (22.2%) to TKIs than wild-type pts (35.3%); however, mutated pts had better PFS and OS (median PFS 9.2 vs 7 months, respectively; median OS 15.2 vs 11.1 months, respectively). Pts treated with afatinib and positive for T790M obtained longer PFS compared to pts negative for T790M (median PFS 4.7 vs 3.2 months, respectively), but their OS was shorter (median OS, 16.3 vs 18.2 months, respectively). Notably, pts with the T790M mutation had a greater response to chemotherapy (44.4%) compared to pts without the mutation (18.2%) and had a longer PFS (median PFS 8.2 vs 6.1 months, respectively) and OS (median OS 21.8 vs 12.4 months, respectively).

      Conclusion
      In this study, the high proportion of pretreatment tumor samples positive for the EGFR-T790M mutation indicates that its identification at diagnosis is more common than expected using a highly sensitive method. Consequently, in NSCLC pts with EGFR-activating mutations, detection of the T790M mutation at diagnosis can help customize therapy and identify a subset of patients with a relatively more favorable prognosis.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-004 - Molecular mechanism of resistance to afatinib in EGFR-mutated non-small cell lung cancer (NSCLC) cell lines and potential therapeutic implications (ID 1230)

      09:30 - 09:30  |  Author(s): M.G. Dal Bello

      • Abstract

      Background
      Somatic activating mutations in the tyrosine kinase (TK) domain of EGFR are harbored by 10-20% of Caucasian NSCLC patients (pts). Reversible TK inhibitors (TKIs), including erlotinib or gefitinib, have demonstrated significantly longer progression-free survival compared to chemotherapy alone in EGFR-mutated pts. Nevertheless, the majority of these tumors develop drug resistance due to an acquired mutation (T790M) in EGFR that determines disease progression. Recent clinical trials have demonstrated interesting activity of the irreversible EGFR-TKI afatinib (BIBW-2992) in advanced NSCLC carrying EGFR mutations and in unselected pts failing previous treatments with reversible TKIs. The aim of this study was to clarify the mechanisms of acquired resistance to afatinib using in vitro models of resistant cell lines.

      Methods
      A dose-escalation study was performed to establish afatinib-resistant (R) clones in NSCLC cell lines harboring different EGFR mutations: H-1650 (exon 19 delE746-A750) and H-1975 (exon 21 L858R/exon 20 T790M). The entire genomes of parental (P) and R cells were screened by array comparative genomic hybridization (aCGH) using a 105 k oligonucleotide microarray. All EGFR and KRAS exons and 10 known hot spots (5 in genes involved in the EGFR signaling cascade and 5 in genes frequently altered in NSCLC) were deep sequenced using an Ion PGM™ Sequencer in P and R cells. The relative expression of 92 genes belonging to the EGF pathway was studied by quantitative polymerase chain reaction (qPCR). The expression of proteins related to the EGF pathway, including EGFR, AKT and ERK (total and activated forms), was investigated by Western blot.

      Results
      Genomic analysis indicated that both R cell lines had genomic profiles similar to the P cells. However, H-1975-R showed 3p and 12p loss and gains at 4q and 10q compared to the P cell line. Sequence analyses identified a novel frame-shift mutation within exon 14 of MET and confirmed EGFR mutation status in 100% of H1975-R cells. In contrast, H-1650-R cells showed a single-base deletion 12 bp upstream of exon 8 of PIK3CA within a sequence of nine repeated Ts. Furthermore, a novel missense variant (exon 8 K368E) was found in FGFR2 in both R cell lines compared to the P cell line. Gene expression profiles identified an increase in the FGFR2 and PIK3 regulatory subunits and EGFR ligand silencing in H-1975-R. Notably, H-1975-R cells maintained in afatinib-free medium for over 6 months showed higher EGFR and AKT phosphorylation compared to the P cell lines.

      Conclusion
      The lack of novel EGFR mutations suggests the involvement of other mechanisms implicated in afatinib resistance. In particular, the identification of mutations involving MET and FGFR2 in H-1975 and PI3KCA in H-1650 suggests their contribution to resistance against irreversible TKIs, sparing EGFR activation. Furthermore, the different mutation status of the two cell lines indicates that the T790M mutation may be partially responsible for the mechanism of resistance. Validation studies are ongoing to confirm the genomic results. In conclusion, these preliminary data may help identify novel therapeutic strategies to delay or reverse resistance to irreversible TKIs in EGFR-mutated NSCLC patients.

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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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      P3.06-030 - Circulating tumor cells as a novel predictive marker in patients with advanced adenocarcinoma of the lung treated with platinum and pemetrexed (ID 2443)

      09:30 - 09:30  |  Author(s): M.G. Dal Bello

      • Abstract

      Background
      Circulating tumor cells (CTCs) are cells that have spread from the primary tumor into the bloodstream, and they play a crucial role in the development of distant metastases. CTCs have been detected in several cancers and associated with aggressive disease. The aim of this study was to evaluate the correlation between the numeric variation of CTCs in the blood of patients (pts) with advanced adenocarcinoma (ADK) of the lung during chemotherapy (CHT) and the radiological response to explore their potential role as an early predictive indicator of treatment response.

      Methods
      Peripheral blood samples and computed tomography (CT) scans were obtained before any treatment (baseline) from 25 pts with advanced ADK who were candidates for first-line CHT (platinum-based combination with pemetrexed). Blood samples were collected every two CHT cycles, and radiological responses were concomitantly assessed by CT according to the RECIST v. 1.1 criteria. CTCs were isolated from blood and diluted in a buffer containing formaldehyde to lyse red blood cells and fix CTCs using a filtration-based device (ScreenCell[®], France) to sort CTCs by size. CTCs were isolated by size using a microporous membrane filter composed of polycarbonate material containing circular pores that are calibrated (7.5±0.36 μm) and randomly distributed throughout the filter (1×10[5 ]pores/cm[2]). At the end of filtration, hematoxylin and eosin (HE) staining and immunofluorescence (IF) using CK7 were performed to enumerate and characterize the CTCs. Moreover, variations in the CTC count were compared with tumor size variations observed in CT scans.

      Results
      Baseline CTC counts and CT scans were obtained from 25 pts, including 18 males and 7 females, with a median age of 68 (range: 45-81) years. H&E staining revealed that the CTCs were morphologically compatible with tumor cells and were present in all 25 pts at baseline (range: 2-25 CTCs/ml). Furthermore, the epithelial origin of the CTCs was confirmed by CK7 positivity (demonstrated by IF). CTCs and CT images were assessed in 19 pts after at least two CHT cycles; the best response was a partial response (PR) in 2 pts, progressive disease (PD) in 3 pts and stable disease (SD) in 13 pts. The CTC number varied with the tumor size in 77.8% (14/18) of pts; decreased CTC counts were observed in 87.5% (7/8) of pts with reduced tumor size, whereas increased CTC counts were found in 70% (7/10) of pts with increased tumor size. Notably, variations in CTC count and tumor size were concordant in 100% (5/5) of pts achieving a PR or with PD as the best response.

      Conclusion
      This study demonstrates the feasibility of isolating CTCs in all advanced lung ADK pts using a low-cost, size-based technique. Interestingly, the concordance between the CTC analysis and CT suggests that CTCs may represent an early predictive marker of disease outcome. To our knowledge, this is the first study suggesting a relationship between CTC variation and treatment response in lung cancer.

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    P3.12 - Poster Session 3 - NSCLC Early Stage (ID 206)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.12-024 - A multi-center phase II randomized study of customized neoadjuvant therapy vs. standard chemotherapy (CT) in non-small cell lung cancer (NSCLC) patients with resectable stage IIIA (N2) disease (CONTEST trial) (ID 3107)

      09:30 - 09:30  |  Author(s): M.G. Dal Bello

      • Abstract

      Background
      Stage IIIA NSCLC constitutes 30% of all NSCLCs. The most powerful prognostic factors identified in this stage are mediastinal lymph node clearance and a pathologic complete response (pCR). A pCR is obtained in 5-15% of patients (pts) with significant prolongation of survival. The identification of molecular biomarkers, such as excision repair cross-complementation 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1) and thymidylate synthase (TS), may predict the response to CT. Similarly, EGFR mutations may predict the response to EGFR inhibitors.

      Methods
      CONTEST, a multicenter (19 Italian centers), randomized (2:1), 2-arm, phase II study, will recruit pts with resectable stage IIIA (N2) NSCLC. Pts will be randomized to receive before resection either standard CT with cisplatin (CDDP) 75 mg/m2 + docetaxel (Doc) 75 mg/m2 on day (d) 1 q 21 d for 3 cycles (cys) or customized therapy using predetermined values for ERCC1, RRM1, TS and EGFR mutations. Specimens will be sent to Response Genetics (Los Angeles, CA, USA) for the evaluation of ERCC1, RRM1 and TS using RT-PCR and EGFR using Sanger sequencing. The customized arms are as follows: -EGFR+: Gefitinib 250 mg/d for 8 weeks. -EGFR-/non-squamous (NS)/TS-/ERCC1-: CDDP 75 mg/m2 + pemetrexed 500 mg/m2 d 1 q 21 d for 3 cys. -EGFR-/squamous (S) or NS TS+/ERCC1-/RRM1+: CDDP 75 mg/m2 + Doc 75 mg/m2 d 1 q 21 d for 3 cys. -EGFR-/S or NS TS+/ERCC1-/RRM1-: CDDP 75 mg/m2 d 1+ gemcitabine (Gem) 1250 mg/m2 d 1, 8 q 21 d for 3 cys. -EGFR-/S or NS TS+/ERCC1+/RRM1+: Doc 75 mg/m2 d 1 + vinorelbine 20 mg/m2 d 1, 8 q 21 d for 3 cys. -EGFR-/S or NS TS+/ERCC1+/RRM1-: Doc 40 mg/m2 y 1, 8 + Gem 1200 mg/m2 d 1, 8 q 21 d for 3 cys. The primary end point is pCR, and all randomized pts will be compared by treatment arm. Because pCR is a surrogate endpoint and given the expected proportion of pCRs in the control group (pc= 5%), the minimal clinically worthwhile effect of this customized treatment is an increase to 20%. To detect such an effect at the 0.05 (1-sided) significance level with 80% power, a total of 168 pts (112 in the investigational arm and 56 in the standard arm) will be enrolled. The secondary endpoints are overall survival, disease-free survival, overall survival at 1, 2 and 5 years, overall response and safety. The major eligibility criteria are as follows: histologically confirmed NSCLC appropriate for surgery; ≥18 years old; ECOG performance status (PS) 0-1; sufficient tissue to perform marker analyses; medically fit for resection by lobectomy or pneumonectomy; stage IIIA (N2) patients with technically operable disease limited to T1a, b, T2a, b N2 M0; T3 (>7 cm) N2 M0 are eligible; stage IIIA pts limited to T3 N1 M0, T3 (invasion) N2 M0; T4 N0, N1 M0 are not eligible; NSCLC confirmed by mediastinoscopy; informed consent. This study is open for accrual; further details can be found at ClinicalTrials.gov (NCT01784549). Funded by the Italian Ministry of Health – RF 2009-1530324.

      Results
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      Conclusion
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