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M. Milella



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    MO20 - Preclinical Therapeutic Models II (ID 93)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO20.06 - Histone deacetylase inhibition downregulates thymidylate synthase (TS) expression and enhances pemetrexed-induced cytotoxicity in NSCLC models (ID 2010)

      10:30 - 12:00  |  Author(s): M. Milella

      • Abstract
      • Presentation
      • Slides

      Background
      Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM’s molecular targets) expression. Recently, TS expression has been found to be regulated by histone acetylation status, thus raising the interesting hypothesis that histone deacetylase inhibitors (HDACi) may sensitize NSCLC cells to PEM cytotoxicity.

      Methods
      Molecular and functional effects of single and combined HDAC inhibition and PEM exposure were assessed in NSCLC cell lines (A549, H1299, H1650, Calu-1) and patient-derived lung cancer stem cells (L-CSC). Pharmacologic interactions were assessed by conservative isobologram analysis using the Chou-Talalay method and the Calcusyn software. TS expression was studied by WB analysis and real-time PCR. Apoposis induction was assessed by flow cytometry and WB. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells, detection of acidic vesicle organelles (AVO) formation and WB. In vivo experiments were conducted in xenograft models established by i.m. injection of NSCLC cells into 6-8 week-old male athymic mice (nu/nu).

      Results
      In NSCLC cell lines and L-CSC, the HDACi ITF2357 dose-dependently inhibited cell growth (IC~50~: <1-20 mM), induced histone H3 acetylation, and downregulated TS expression at the mRNA and protein levels. Combined HDAC inhibition and PEM exposure was then tested using three different administration schedules: simultaneous exposure to both drugs, ITF2357 followed by PEM, and the reverse sequence. Simultaneous PEM/ITF2357 treatment resulted in antagonistic growth inhibitory interactions (combination index – CI >1) in all cell lines tested, while ITF2357 followed by PEM had additive effects in A549 cells and slightly synergistic effects in H1299 and Calu-1 cells; conversely, PEM followed by ITF2357 had strikingly synergistic effects (CI <<1) in all NSCLC cell lines, as well as in the L-CSC143. Most notably, only the ITF2357 followed by PEM sequence synergistically induced apoptosis, resulting in approximately 50% Annexin V-positive cells; apoptosis was only partially rescued by caspase inhibition by z-VAD-fmk, which led us to investigate autophagy as an alternative mechanism of combination-induced cell death. Indeed, ITF2357, and to a significantly greater extent PEM followed by ITF2357, induced autophagy as evidenced by AVO formation, LC3BII processing, p62 downregulation, and Beclin1 induction. Most importantly, autophagy induction was instrumental to the cytotoxic interaction between PEM and ITF2357, as Beclin1 silencing by shRNA completely reversed their growth inhibitory synergism and prevented both autophagy and apoptosis induction. The synergistic cytotoxic interaction between PEM and ITF2357 was at least partly due to ITF2357 ability to prevent PEM-induced TS upregulation, as TS silencing by siRNA further enhanced apoptosis induction by single and combined PEM/ITF2357 exposure. Finally, both H1650 and H1299 xenografts had a robust response to sequential PEM/ITF2357 administration in vivo, resulting in an approximately doubled mice survival in the H1650 model.

      Conclusion
      Overall, our data indicate that HDAC inhibition by ITF2357 downregulates TS expression and synergistically potentiates apoptosis and autophagy induction following PEM exposure, supporting the clinical investigation of sequential PEM/ITF2357 schedules for the treatment of advanced NSCLC.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-041 - Prognostic impact of cytoskeleton regulatory protein human Mena (hMena) isoforms in resected, node-negative, non-small-cell lung cancer: validation of a clinic-molecular prognostic model. (ID 2609)

      09:30 - 16:30  |  Author(s): M. Milella

      • Abstract

      Background
      Human Mena and the isoform hMena[+11a] are cytoskeleton regulatory proteins involved in adhesion, motility, regulated in the epithelio-mesenchimal transition. Here, we investigated their potential prognostic value in node-negative non-small-cell lung cancer (NSCLC) patients.

      Methods
      Pan-hMena, hMena[+11a], E-cadherin, vimentin, ER-beta, EGFR, HER-2, pAKT, detected immunohystochemically on duplicate TMA and clinical factors (sex, age, histology, grading, T-size, number of resected nodes, RN) were correlated to 3-yr disease-free (DFS), cancer-specific (CSS), and overall survival (OS) using a Cox model. ROC analysis provided optimal cut-off values and model validation. A logistic equation including regression analysis coefficients was constructed to estimate individual patients’ probability (IPP) of relapse. Internal cross-validation (100 simulations with 80% of the dataset) and external validation was accomplished.

      Results
      In a training set of 248 patients (median follow-up: 36 months, range 1-96), Pan-hMmena and hMena+11a were the only biological variables displaying significant correlation with outcome(s), confirmed by the cross-validation (replication rate: 78%, 83%), with a prognostic model accuracy of 61% (standard error 0.04, p=0.0001). Patients with high pan-hMENA expression had a non-significant trend towards a worse outcome, while patients with high hMena+11a expression had a significant and borderline significant advantage in DFS (p=0.03) and OS (p=0.056), respectively, and a non-significant trend towards a better CSS. Univariate and multivariate 3-yr median individual patient probabilities of recurrence were 70.9 (range 40.3-94.4) and 41.2 (range 13.6-86.5), respectively (data not shown). The subgroup of patients with High Pan-hMena/Low hMena11a relative expression fared significantly better than any of the other 3 groups (p≤0.002 for all outcomes). On the basis of the combination between this molecular hybrid variable and T-size and RN, a 3-class risk stratification model was generated; the derived 3-risk class survival model strikingly discriminated between patients at different risk of relapse, cancer-related death, and death for any cause, with a prognostic accuracy of 61% (standard error 0.03, p=0.01), according to ROC analysis. The 3-risk class survival model was externally validated in an independent dataset of 133 patients, and significantly discriminated between patients at Intermediate- and High-Risk of relapse and cancer-related death.

      Conclusion
      The expression of the hMena and its isoform may represent a powerful prognostic factor in early NSCLC and usefully complements clinical parameters to accurately predict individual patient risk..

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    P1.11 - Poster Session 1 - NSCLC Novel Therapies (ID 208)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P1.11-033 - Afatinib in EGFR mutant non-small-cell lung cancer patients with acquired resistance to reversible EGFR-TKIs (ID 2285)

      09:30 - 16:30  |  Author(s): M. Milella

      • Abstract

      Background
      Afatinib, an irreversible EGFR-HER2 dual inhibitor, demonstrated superiority versus standard platinum-based chemotherapy as front-line therapy in non-small-cell lung cancer patients (NSCLC) with activating Epidermal Growth Factor Receptor (EGFR) mutations. In pretreated NSCLC afatinib failed to improve survival when compared to placebo in patients refractory to gefitinib or erlotinib and not selected for EGFR status. Aim of the present study was to evaluate clinical efficacy of afatinib in EGFR mutant NSCLC patients (pts) with secondary resistance to reversible EGFR-TKIs.

      Methods
      We retrospectively analyzed a cohort of 97 EGFR mutant lung cancer pts resistant to EGFR-TKI according to criteria used in the LUX-Lung 1 trial (Miller VA, Lancet Oncol 2012) and treated with Afatinib at the daily dose of 40-50 mg. The drug was given as compassionate use.

      Results
      The study included individuals with a median age of 62,5 year. The majority were females (N=63/64.9%), never/former smokers (N=94/96,9%), with good performance status (ECOG PS 0-1; N=90/90.2%) and pretreated with > 3 therapy lines (N=68/70.0%). EGFR status was assessed in tumor tissue obtained at the time of original diagnosis. The majority of pts (N=64, 66%) harbored a deletion in exon 19, while T790M mutation was detected in two cases including one case with double exon 19 and T790M mutation. Among the 95 pts evaluable for toxicity, 54.7% had any grade skin rash, including 11.6% with grade 3, and 50,5% had any grade of diarrhea, with grade 3 recorded in 10,5%. Among the 87 pts evaluable for efficacy, response rate (RR) was 11.5%, with a median progression free-survival and overall survival of 3.9 months and 7.3 months respectively. In 25 pts a tumor biopsy was repeated immediately before starting Afatinib therapy and 1 patient out of 5 individuals harboring T790M mutation showed a short extracerebral partial response, with following brain progression.

      Conclusion
      Our findings suggest that afatinib is modestly effective in EGFR mutant NSCLC with acquired resistance to reversible EGFR-TKIs.

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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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      P3.06-026 - Thymidylate synthase (TS) mRNA and protein expression in advanced non-small cell lung cancer (NSCLC) patients treated with pemetrexed-based therapy. (ID 2355)

      09:30 - 16:30  |  Author(s): M. Milella

      • Abstract

      Background
      In NSCLC, higher Thymidylate Synthase (TS) levels have been reported in both squamous and large cell carcinomas compared to adenocarcinoma. In clinical practice, Pemetrexed, a potent antifolate inhibitor of TS, showed a selective benefit in patients with "non-squamous" NSCLC. Two retrospective studies have shown that low TS protein levels are associated with better clinical outcome in NSCLC patients treated with pemetrexed. Aim of this study was to explore, in a series of advanced stage IV patients receiving pemetrexed-based regimens in first line of treatment, the association between TS mRNA and protein expression with overall survival (OS) and therapeutic response.

      Methods
      Two series of histologically confirmed non squamous-NSCLC, assessed in formalin-fixed and paraffin embedded specimens from patients treated with pemetrexed-based regimens were collected: the first series at San Luigi Hospital (n=64), the second series at Regina Elena National Cancer Institute (n=8). Due to the limited amounts of tissue specimens available, total RNA extraction was possible in 52 out of 72 cases. TS protein expression was performed using immunohistochemistry (mouse monoclonal TS106 antibody) and scored through H-SCORE method, considering both staining intensity (0 no staining; +1 weak; +2 moderate; or +3 strong) and percentage of tumor cells stained, resulting in semiquantitative H-scores ranging from 0 to 300. TS nuclear and cytoplasmic staining, respectively, were separately scored. Statistical analyses were performed using the STATISTICA10 software.

      Results
      The differential H-SCORE assessment showed a strong importance of TS localisation for clinical outcome prediction: in Cox regression analysis, a statistically significant association was observed between nuclear TS expression and OS (p < 0.009) indicating that lower nuclear TS expression levels were associated with longer OS. In addition, lower nuclear TS levels were significantly associated with a better response to therapy (p<0.001). On the contrary, TS cytoplasmic staining did not affect patients’ survival or clinical response (p>0.05). Four subgroups of patients, based on the dichotomized low/high TS expression in both nucleus and cytoplasm, were obtained: both high, both low, nucleus high/cytoplasm low and nucleus low/cytoplasm high. Significant differences in overall survival among these four groups were detected (p=0.017), confirming the strong and selective influence of nuclear TS, as compared to cytoplasmic TS, expression in clinical outcome. Moreover, Chi[2] test revealed a significant association between low nuclear TS and partial response to pemetrexed treatment, independently of cytoplasmic TS expression (p<0.001). No correlation between TS protein expression data and clinico-pathological data (age, gender) were identified. TS gene expression analyses are ongoing.

      Conclusion
      This retrospective study suggests that TS protein expression, selectively assessed at nuclear level, has a potential predictive role in advanced stage IV patients, receiving pemetrexed in first line of treatment. Patients with low nuclear TS expression showed prolonged overall survival and better response to therapy. Such preliminary results define TS assessment as a potential tool which may select the most appropriate group of patients to be treated with pemetrexed.

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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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      P3.12-023 - International Tailored Chemotherapy Adjuvant Trial: ITACA Trial (ID 1452)

      09:30 - 16:30  |  Author(s): M. Milella

      • Abstract

      Background
      This is an ongoing phase III multicenter randomized trial comparing adjuvant pharmacogenomic-driven chemotherapy, based on thymidilate synthase (TS) and excision-repair cross-complementing-1 (ERCC1) gene expression versus standard adjuvant chemotherapy in completely resected Stage II-IIIA non-small cell lung cancer (EudraCT #: 2008-001764-36).

      Methods
      For all the registered patients (pts) the expression of ERCC1 and TS is assessed by qRT-PCR on paraffin-embedded tumor specimens in a central laboratory. Randomization is stratified by stage and smoking status. Trial was emended on Feb, 2011 with the 7th staging system. Primary end point is overall survival; secondary end points include recurrence-free survival, therapeutic compliance, toxicity profile and comparative evaluation of ERCC1 and TS mRNA versus protein expression. It is assumed that the 5-year survival rate in the control arm is 45% and the hazard reduction associated to the experimental treatment is 30%. With a power of 90% to detect the estimated effect with log-rank test, a significant level of 5% (2 tails), 336 events have to be observed; the expected total number of pts is 700. The final statistical analysis will compare all pts in the control arms versus all those treated in the tailored chemotherapies groups. Efficacy analysis will be done on an intent-to-treat basis. Cox proportional hazard model will be used for estimating hazard ratios after adjusting for relevant variables. Within 45 days post-surgery, pts in each genetic profile are randomized to receive either a standard chemotherapy selected by the investigator (cisplatin/vinorelbine, cisplatin/docetaxel or cisplatin/gemcitabine) or an experimental treatment (tailored arms) selected as follows: 1) high ERCC1 and high TS 4 cycles of single agent paclitaxel 2) high ERCC1 and low TS 4 cycles of single agent pemetrexed 3) low ERCC1 and high TS 4 cycles of cisplatin/gemcitabine 4) low ERCC1 and low TS 4 cycles of cisplatin/pemetrexed. All chemotherapy regimens are administered for a total of 4 cycles on a 3-weekly basis.

      Results
      Not applicable

      Conclusion
      Currently, 558 pts have been randomized from 26 institutions mainly located in Italy and Germany (average enrolment: 19 patients/month). This is one of the pharmacogenomic-driven trial in the adjuvant setting in the European scenario.