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R. Bruno
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-077 - Whole-Transcriptome Gene Expression Analysis of Pulmonary Sarcomatoid Carcinomas (ID 5477)
14:30 - 14:30 | Author(s): R. Bruno
- Abstract
Background:
Pulmonary sarcomatoid carcinomas (PSCs) are rare, poorly differentiated non-small cell lung cancers (NSCLCs) containing sarcoma or sarcoma-like features, with a worse prognosis than other NSCLCs. The World Health Organization (WHO) classifies three histopathologic subtypes: subtype-1 includes pleomorphic, spindle-cell, and giant-cell carcinomas (PSCGC), subtype-2 carcinosarcoma and subtype-3 blastoma. The value of different subtypes for clinical management and their molecular characteristics are unclear. The aim of this study is to get new insights into PSCs carcinogenesis by a high-throughput sequencing of RNA (RNA-seq).
Methods:
A whole-transcriptome targeted-gene quantification analysis was retrospectively performed on RNA from formalin-fixed paraffin-embedded tissues of 13 PSCs (5 pleomorphic, 2 spindle-cell, 2 giant-cell carcinomas, 4 carcinosarcomas). RNA-seq reads were mapped to the amplicon sequences of the panel and quantified by tools based on alignment algorithms. Differentially expressed genes between subtype-1and -2 were determined using a non-parametric Mann-Whitney U-test (p-value < 0.01) with linearity correction. Moreover, within subtype-1 we compared gene expression levels between monophasic (spindle- and giant-cell) and biphasic (pleomorphic) carcinomas.
Results:
216 genes resulted down-regulated and 15 up-regulated in PSCGC compared to carcinosarcomas (Table 1). There were not significant differences between monophasic and biphasic PSCGC. Figure 1
Conclusion:
PSCs are heterogeneous tumours, barely characterized from a molecular point of view. WHO has recently classified PSCGC and carcinosarcoma as two distinct entities, and our results demonstrated that they effectively have different gene expression profiles. Deregulated genes mostly belong to pathways crucial for cancer, like p53-, MAPK- and Wnt-signaling, and future investigation should clarify their specific role in PSCs. Interestingly, we did not find statistically deregulated genes among monophasic and biphasic carcinomas of subtype-1, thus indicating their molecular similarity. Although this is a preliminary and explorative study, needing further validation, it constitutes a starting point to increase our knowledge of these rare tumours.
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P3.02a - Poster Session with Presenters Present (ID 470)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.02a-010 - Evaluation of Aberrant ALK Expression in Lung Cancer by RT-PCRÂ and Comparison with FISH and Immunohistochemistry (ID 5490)
14:30 - 14:30 | Author(s): R. Bruno
- Abstract
Background:
In advanced lung cancer patients the gold standard for detecting ALK gene rearrangements is fluorescence in situ hybridization (FISH), and ALK protein expression can be also evaluated by immunohistochemistry (IHC). A single analysis performed alone may not detect all the ALK-positive cases and some patients with discordant FISH and IHC respond to tyrosine kinase inhibitors (TKIs). In this study we evaluated ALK aberrant expression in lung cancer patients by a reverse-transcription (RT)-PCR, to investigate its clinical utility and its concordance with FISH and IHC.
Methods:
ALK aberrant expression was retrospectively investigated on RNA from formalin-fixed paraffin-embedded tissue (FFPEt) of 24 advanced lung adenocarcinoma patients, previously evaluated by FISH and IHC. We used a one-step Scorpion RT-PCR that allowed in a single reaction either the mRNA reverse transcription and the cDNA amplification for ALK kinase-domain, normally not expressed, and a control gene, to assess RNA quality.
Results:
Results are reported in Table 1.Figure 1
Conclusion:
Despite the instability of mRNA from FFPEt, only 2 samples resulted inadequate for RT-PCR. RT-PCR was in disagreement with both FISH and IHC in one case, which is likely to be a RT-PCR false positive. RT-PCR did not detect ALK aberrant expression in a FISH positive case, which was negative also by IHC; unfortunately, this patient died after a cycle of pemetrexed therapy, before undergoing a second line TKI treatment. The presence of ALK rearrangements does not necessarily imply increased protein levels, because of the complex transcriptional and post-transcriptional regulations, so further analysis at RNA levels may clarify discrepancy between FISH and IHC allowing a better stratification of patients who could benefit from TKIs. Therefore, according to our results the RT-PCR evaluating ALK aberrant expression regardless of the fusion partners should be considered for introduction into routine ALK testing in lung cancer.
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P3.02b - Poster Session with Presenters Present (ID 494)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 2
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.02b-027 - Detection of EGFR Mutations in Plasma of Lung Adenocarcinoma Patients Using Real-Time PCR and Mass Spectrometry (ID 5475)
14:30 - 14:30 | Author(s): R. Bruno
- Abstract
Background:
Lung adenocarcinoma patients harbouring sensitizing EGFR mutations can benefit from treatment with tyrosine kinase inhibitors (TKI). Whenever tumour tissue is inadequate or unavailable, detection of EGFR mutations in circulating cell-free tumour (ct) DNA from plasma is crucial to predict and monitor response to therapy. In this study we compared EGFR status between tumour tissue and plasma, using real-time PCR. Moreover, we evaluated the adequacy of ctDNA for a multi-target mass spectrometry (MS) analysis.
Methods:
EGFR mutations were investigated in paired plasma and tumour tissues from a prospective series of 105 lung adenocarcinoma patients: 79 had no prior TKI treatment and 26 underwent re-biopsy for TKI-acquired resistance. Molecular analyses were performed on tissue by a routine MS test (evaluation of 307 hot-spots in 10 genes including EGFR) and on ctDNA by a validated Scorpion/LNA real-time PCR (evaluation of 30 EGFR mutations). In the 26 postTKI patients ctDNA analysis was performed also by MS.
Results:
1-Plasma versus tissue by real-time PCR: overall sensitivity, specificity and concordance were 64.8%, 95.4% and 82%. In preTKI patients, 17 harboured EGFR sensitizing mutations on tissue,10 detected also in plasma (sensitivity 58.8 %, specificity 100%, concordance 91%). All 26 postTKI patients preserved EGFR sensitizing mutations. Regarding the detection of EGFR T790M resistance mutation, sensitivity, specificity and concordance were 63.6%, 80% and 73%. Specifically, 11 patients were T790M-positive (42%): 7 on both specimens, 4 only on tissue and 3 only on ctDNA. 2-Plasma versus tissue by MS: sensitivity, specificity and concordance for T790M were 50%, 93.3% and 76%. Particularly, 6 patients had a T790M-positive ctDNA: 5 concordant and 1 discordant with tissue; 4 T790M-positive cases on tissue were undetected in plasma; 1 sample was not evaluable.
Conclusion:
The real-time PCR on ctDNA showed a sensitivity consistent with literature and a high specificity, mostly in preTKI group. Concordance rates, influenced by biological and methodological factors, were lower in postTKI group. Indeed, some T790M mutations were detected only on ctDNA, which is expected to effectively mirror tumour heterogeneity better than bioptic samples, thus giving a global view of tumour. Finally, we demonstrated the adequacy of ctDNA for MS, in terms of quantity and quality. The use of a multi-target analysis on ctDNA might improve tumour characterization and response monitoring, evaluating important oncogenes other than EGFR, like PIK3CA, KRAS and BRAF. However, further studies are needed to better explore MS applicability on ctDNA.
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P3.02b-034 - Clinical Impact of Pretreatment EGFR T790M Mutation in Lung Adenocarcinoma Patients (ID 5463)
14:30 - 14:30 | Author(s): R. Bruno
- Abstract
Background:
About a half of lung adenocarcinoma patients with an activating EGFR mutation undergoing tyrosine kinase inhibitor (TKI) therapy develop the EGFR T790M resistance mutation. Previous studies have reported that T790M positive clones are already present before the TKI treatment in the 0.32-80% of cases, depending on methodology and population. Whereas a statistical association between the presence of pretreatment T790M and the L858R activating mutation has been demonstrated, little is known about the influence of preexisting T790M clones on the clinical outcome. The aim of our study is to investigate whether pretreatment T790M affect the response to TKIs.
Methods:
We selected 18 patients who developed a T790M-related resistance to TKI therapy. Their sensitizing mutations were L858R or exon 19 deletion in 8 and 10 cases respectively. For all patients pre- and post-treatment tumor tissues were available to detect and quantify T790M mutant alleles with a highly sensitive digital PCR analysis (Raindance Technologies).
Results:
Pretreatment T790M was found in 5 out of 18 patients (28%), with a mutation frequency ranging from 0.03% and 0.14% (mean frequency 0.08%). The mean T790M allele frequency in posttreatment tumors was 12%. The presence of T790M before TKIs was not associated with a specific activating mutation (L858R or exon 19 deletion), nor with the disease stage and worse response to treatment, independently from the type of TKI drug.
Conclusion:
Our results confirm that T790M-positive clones can coexist with the activating mutation clones in lung adenocarcinoma even before TKI treatment. A previously reported analysis, performed with a methodology as much sensitive as ours by Watanabe et al., showed a pretreatment T790M mutation frequency raising the 80% in a series of lung adenocarcinoma patients harboring an EGFR activating mutation, with no regard to TKI treatment or resistance. In contrast, we found 28% of cases having T790M before treatment. This discrepancy can be due to the different criteria adopted for samples selection, since our cohort included only patients found positive for T790M after TKI therapy. In our series of cases, the presence of T790M before TKIs did not correlate with significant clinical parameters. In conclusion, in this preliminary study we did not identify a direct association between the presence of small amounts of pre-TKI T790M mutant alleles and patients’ clinical outcome. However, in order to better assess the impact of T790M in predicting the response to therapy, further studies on larger series of patients are needed.
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P3.03 - Poster Session with Presenters Present (ID 473)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.03-024 - Malignant Pleural Mesothelioma: Gene Expression Profiling of the Main Histological Subtypes (ID 5465)
14:30 - 14:30 | Author(s): R. Bruno
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is a low-incidence, aggressive, asbestos-related tumor, whose treatment options are currently limited. MPM is a heterogeneous tumor with three main histological subtypes: epithelioid (E), sarcomatoid (S) and biphasic (B). S- and B- MPMs are rarer and have a poorer prognosis than the E-subtype. In the present study we compared the expression profile of 117 genes with a crucial role in cancer between the E- and S/B- subtypes, in order to identify histology-specific molecular markers.
Methods:
Gene expression analysis was performed by Nanostring system directly on RNA from 38 formalin-fixed and paraffin-embedded tissues of MPM patients (25 E-subtype, 13 S/B-subtypes). After data normalization, differences of gene expression levels between the two groups were evaluated by a non-parametric Mann-Whitney U-test (p-value < 0.05).
Results:
39 genes were differentially expressed. In particular, 21 genes were statistically up-regulated and 18 down-regulated in E- compared to S/B-subtypes (Table 1). Figure 1
Conclusion:
The identification of gene expression profiles specific for each histological subtype could improve the clinical approach to MPM. In this study we found genes differentially expressed between E- and S/B-subtypes. In detail, up-regulated genes in E-MPM encode for proteins involved in epithelial cell differentiation and regulation of apoptosis, whereas down-regulated genes belong to pathways related to extracellular matrix, cell adhesion and angiogenesis. Moreover, some of the deregulated genes have been already described to influence the sensitivity to chemotherapy, such as ASS1, to play an important role in the mesenchymal transition, like MMP9, and others, among which ESR2, have been proposed as potential therapeutic targets. Our results reveal genes activated or inactivated in a histotype-dependent manner as new potential biomarkers for MPM, however, further studies are needed to better understand their clinical value.
