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G. Fontanini
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MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 1
- Moderators:E. Brambilla, M. Noguchi
- Coordinates: 12/05/2016, 14:20 - 15:50, Stolz 2
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MA02.05 - Distinct Angiogenic microRNA-mRNA Expression Profiles among Subtypes of Lung Adenocarcinoma (ID 5464)
14:50 - 14:56 | Author(s): G. Fontanini
- Abstract
- Presentation
Background:
Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers and adenocarcinoma (ADC) represents the most common histological type with a heterogeneous pattern of growth classified as lepidic, acinar, papillary, solid, and micropapillary. For ADC there are restricted available therapeutic options except for patients that could benefit from target therapy. A valuable therapeutic strategy is represented by angiogenesis inhibitors such as bevacizumab that has been approved for the treatment of NSCLC patients. However, there are concerns about its treatment-related toxicity and the identification of new reliable biomarkers to stratify patients who can really benefit from antiangiogenic drugs is urgently needed. Using miRNA target prediction tools, we selected and investigated the expression level of a panel of miRNAs togheter with their mRNA target involved in the angiogenesis pathway.
Methods:
We designed a custom codeset including probes for six genes (VEGF-A, FLT1, KDR, FLT4, PDGFRa and PDGFRb) and sixteen miRNAs. The expression analysis was performed by the nCounter System® (NanoString Technologies) directly on RNA, enriched of small RNA, purified from the formalin-fixed and paraffin-embedded tumor tissues of 80 ADC patients. Of these 25 were predominatly lepidic (31.25%), 24 were predominatly solid (30%), 20 were predominatly acinar (16%), 11 were predominatly papillary (13.75%).
Results:
Comparing the expression levels of mRNAs with the different histological ADC subtypes we found a significant higher expression of VEGF-A in papillary than in other subtypes (p=0.02). In contrast PDGFRa and PDGFRb were upregulated in lepidic and downregulated in papillary subtypes (both p=0.03). Among 16 miRNAs that target the angiogenic mRNA, 6 were significantly downregulated in papillary compared to other groups.
Conclusion:
Our data suggest a distinct angiogenic miRNA-mRNA expression profile among the subtypes of ADC. The higher level of VEGF-A in papillary than in lepidic subtypes could represent a useful biomarker to stratify patients who can effectively treated with bevacizumab, which is directed against VEGF. Moreover, the regulation of angiogenic mRNA factors by miRNAs could provide a novel therapeutic approach based on their expression pattern specific for distinct ADC subtypes. Further studies are nedeed in a larger cohort of patients to confirm our results and to investigate whether different rates of response to treatment are observed among patients stratified according to the proposed biomarkers.
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- 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): G. Fontanini
- 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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P1.02-078 - Expression Profiling of LKB1 Pathway in Young and Old Lung Adenocarcinoma Patients (ID 5484)
14:30 - 14:30 | Author(s): G. Fontanini
- Abstract
Background:
The youthful lung cancer may constitute an entity with distinct clinicopathologic characteristics. The serine/threonine kinase LKB1, also known as Serine/Threonine Kinase 11-STK11, is a known tumor suppressor gene involved in cellular responses such as energy metabolism, cell polarity and cell growth, but the role of LKB1 pathway in lung adenocarcinoma (ADC) is barely studied, especially in young patients
Methods:
Fifty lung ADC patients were retrospectively analysed. After RNA purification from formalin fixed and paraffin embedded tumor tissues, we analysed the mRNA expression levels of LKB1 and of genes involved in its pathway, such as cyclin D1 (CCND1), beta catenin (CCNNB1), lysyl oxidase (LOX), yes-associated protein-1 (YAP-1), and survivin, with NanoString technology, a new tool for a more accurate expression profiling. KRAS mutations were investigated by pyrosequencing analysis. Clinicopathologic characteristics and survival analysis were available for all patients.
Results:
Patients under 50 years old (including 50) were defined as the younger group and patients above 50 years old were defined as the older group. Among all the clinicopathologic characteristics, in the younger group there were more women, less solid and more acinar adenocarcinoma prevalent pattern in comparison to the older group. Younger and older groups showed similar survival rates, as well as KRAS mutations frequencies. Also, in the comparison between the gene expression level of the analyzed genes and the two different age subgroups,no statistical difference was found. We then focused on the LKB1 pathway in all series, independently from the age stratification, founding LKB1 low expression associated with low cyclin D1 (CCND1) (p<0.0001), beta catenin (CCNNB1) (p<0.0001), and YAP1 (p=0.0024) levels, suggesting a target regulation by LKB1. We next tested the expression level of LOX, one of its downstream target, and we found that lung ADC with a high LOX mRNA expression showed a significantly worse prognosis, either in terms of disease-free interval or overall post-operative survival.
Conclusion:
Based on our preliminary results young patients seem to show similar LKB1 pathway expression levels to older group, even if further investigations will be necessary. Moreover, our data suggest LKB1 as a key pathway in lung ADC regardless of age, with a relevant sfavorable role of LOX. A robust assessment of LKB1 and of its downstream gene, LOX in particular, may elucidate the role of this pathway deregulation in lung adenocarcinoma in order to identify potential target for lung cancer therapy.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-018 - Differential microRNA Expression Profile between Young and Old Lung Adenocarcinoma Patients (ID 5478)
14:30 - 14:30 | Author(s): G. Fontanini
- Abstract
Background:
Lung cancer is the leading cause of cancer related mortality and approximately 80% is represented by non-small cell lung cancer (NSCLC). In the last decade, age of patients at diagnosis has decreased, with an incidence of approximately 13.4% in patients under 50 years. Previous studies have hypotesized that lung cancer in young patients could represent a separated clinicalpathological entity, however it is still controversial whether younger patients have a different outcome compared with their older counterparts. MicroRNAs (miRNAs) have recently been defined to play a key role in cancer pathogenesis and their aberrant expression has been suggested as a potential biomarker of prognosis in lung adenocarcinoma. To understand the molecular features of young and old adenocarcinoma patients, we investigated the expression levels of a panel of miRNAs selected from recent literature.
Methods:
Thirty-five lung ADC from patients under 50 years old were enrolled as the younger group and thirty-five ADC older than 50 years were collected as the older group. After miRNA isolation from formalin-fixed and paraffin-embedded tumor tissues, the expression levels of 30 miRNAs were analyzed by NanoString technology and compared between the two groups. Survival data were used to assess the prognostic impact of miRNAs. The software miRgator v3.0 was used to predict the putative pathways targeted by miRNAs.
Results:
The analysis revealed that 7 miRNAs (miR-25-3p, miR-29c-3p, miR-33a-5p, miR-144-3p, miR-153-3p, miR-342-5p and miR-485-3p) were differently expressed in the two groups (Mann-Whitney U test, p<0.05). All these miRNAs showed higher expression levels in young compared to old patients, and their predicted targets included EGFR, MET, VEGF-A, TP53 and PDGFRa. miR-144-3p had an opposite influence on overall survival, since its upregulation was associated with a better outcome in young patients (p= 0.01) and a worse prognosis in the old group (p= 0.03).
Conclusion:
Our study provides new insights about the role of miRNAs in lung adenocarcinoma occurring in young patients. We observed that lung cancer in young and old patients may be influenced by different regulatory mechanisms since we found 7 miRNAs as downregulated in the older group, probably due to distinct age-related genetic and epigenetic alterations. Moreover, one of the deregulated miRNAs showed a different prognostic impact in the two groups thus confirming that young and old patients deserve a specific clinical approach. Further validations are needed to better define if an age-based genomic signature could be used as prognostic marker in lung cancer.
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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-PCR and Comparison with FISH and Immunohistochemistry (ID 5490)
14:30 - 14:30 | Author(s): G. Fontanini
- 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): G. Fontanini
- 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): G. Fontanini
- 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): G. Fontanini
- 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.
