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Marek Sawicki
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EP1.03 - Biology (ID 193)
- Event: WCLC 2019
- Type: E-Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:
- Coordinates: 9/08/2019, 08:00 - 18:00, Exhibit Hall
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EP1.03-15 - Comparison of Plasma Expression of Drosha and Dicer mRNAs in Early and Advanced Stages of NSCLC Patients (Now Available) (ID 628)
08:00 - 18:00 | Author(s): Marek Sawicki
- Abstract
Background
Drosha and Dicer are the enzymes necessary during the miRNA biogenesis. They have the same effect on all microRNAs. Many studies have focused on profiling the expression of selected miRNAs or whole miRNom’s analysis in serum or plasma, as potential tool for early detection of diseases. We would like to check, whether the expression of Drosha and Dicer mRNA is detectable in plasma of NSCLC patients, and whether it can differentiate early and advanced stages of this disease.
Method
We enrolled 59 (43.1%) NSCLC patients in early (I-IIIA) stages and 78 (56.9%) in locally advanced or advanced (IIIB-IV) stages. We isolated total mRNA and reverse transcription PCR (RT-PCR) was performed. RT-PCR was made using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR (qPCR) was performed for assessment of Drosha and Dicer mRNA expression on Eco Illumina Real-Time PCR system device (Illumina Inc.). We used TaqMan probe Hs00203008_m1 (Applied Biosystems) for Drosha and Hs00229023_ m1 (Applied Biosystems) for Dicer mRNA expression measurement. GAPDH was used a housekeeping gene. Statistical analysis were performed with use of Statistica 13.1 software (Tibco).
Result
Drosha mRNA expression was detectable in plasma of 57 (41.6%) patients. 17 (28.8%) patients with Drosha mRNA expression were in early stages and 40 (51.3%) patients were in stages IIIB or IV (χ2=6.98, p=0.008).
Expression of Dicer mRNA was detectable in plasma of 71 (57.8%) patients. 15 (25.4%) of patients from this group were in early stages and 56 (71.8%) – in IIIB or IV stages (χ2=28.93, p<0.0001).
Significant higher expression of Drosha mRNA was observed in group of patients with lymph node involvement compared with group of patients without lymph node metastases (p=0.001).
Moreover, significantly higher expression of Dicer mRNA was observed in group of patients with distant metastases compared with group without metastases (p=0.0002).
Furthermore, we found statistically nonsignificant (p=0.07) lower expression of Drosha mRNA in stages IIIB-IV compared with early stages.
We did not find any differences between Drosha or Dicer mRNAs expression in patients stratified by age, tumor size or histopathological diagnosis (p<0.1).
Conclusion
Plasma expression of Drosha and Dicer mRNAs is detected more often in advanced stages of NSCLC. Probably, different mRNAs from more damaged tumor cells in more advanced disease stages are present in higher expression in blood stream. However, this proves that free mRNAs of Drosha and Dicer are mainly produced by cancer cells in NSCLC patients. indirectly, it can be concluded that cancer cells have disturbed production of microRNAs. There are necessity to use more sensitive tools (i.e. Next Generation Sequencing method) to asses expression of Dicer and Drosha mRNAs in early stages of NSCLC.
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P2.01 - Advanced NSCLC (ID 159)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Advanced NSCLC
- Presentations: 2
- Now Available
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.01-44 - Promoter Polymorphisms of TOP2A and ERCC1 Genes as Predictive Factors for Chemotherapy in Non-Small Cell Lung Cancer Patients (Now Available) (ID 654)
10:15 - 18:15 | Author(s): Marek Sawicki
- Abstract
Background
TOP2A is an enzyme that control topologic changes in DNA during transcription and replication. ERCC1 is an enzyme takes part in DNA repair processes. Purpose of our studies was to assess predictive role of particular single nucleotide polymorphisms (SNPs) in promoter regions of TOP2A and ERCC1 genes in non-small cell lung cancer patients (NSCLC) treated with chemotherapy.
Method
We enrolled 116 NSCLC patients qualified to first line chemotherapy. Information on the chemotherapy regimens was available in 106 patients. All chemotherapy regimens were based on platinum compounds. 66 (62%) patients received additionally inhibitors of cell divisions (vinorelbine, taxanes). 40 (38%) patients were treated with nucleoside analogs or antimetabolites (gemcytabine, pemetrexed). DNA was isolated from whole blood with Qiamp DNA Blood Mini kit (Qiagen, Germany) according to the manufacture’s instruction. We examined five SNPs: rs11615 (ERCC1), rs3212986 (CD3EAP), rs13695 (TOP2A), rs34300454 (TOP2A), rs11540720 (TOP2A). Quantitative PCR using TaqMan probe (ThermoFisher, USA) was performed on Eco Illumina Real-Time PCR system device (Illumina Inc., USA). Statistical analysis were performed with MedCalc and Statistica 13.1 softwares.
Result
In whole group of patients, median of progression free survival (PFS) was 3 months. Patients with CC genotype in rs34300454 had significantly higher median PFS (8 months) compared to patients with CT genotype (4 months, p=0.0026; HR=0.36 with 95% CI: 0,19 to 0,7). We did not detect patients with TT genotype of this SNP. However, the differences in median PFS between patients with different genotypes of the TOP2A gene were significant only in the group receiving inhibitors of cell divisions (p=0.011). In second group of patients, we did not observed such significant relationship. Control of disease (response to chemotherapy or stable disease) were observed insignificantly more often in patients with AA genotype in rs11615 of ERCC1 gene than in patients with AG genotype of this SNP (X2=3.453, p=0.063).
Conclusion
Polymorphism of TOP2A gene could influence PFS in NSCLC patients treated witch chemotherapy and CC genotype of this polymorphism may be a good predictive factor for chemotherapy regimens containing cell division inhibitors.
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P2.01-66 - Genomic Landscapes of DNA Copy Number Alterations in Primary Lung Cancers and Matched Brain Metastases (Now Available) (ID 1726)
10:15 - 18:15 | Author(s): Marek Sawicki
- Abstract
Background
Lung cancer (LC) is the leading cause of cancer mortality worldwide. The majority of LC patients will develop distant metastases at some point during their disease, and brain is among the most common sites of relapse. However, little is known about the mutational landscape of brain metastases (BM) and their potential inter-tumor heterogeneity. Better knowledge on this subject may pave the way to new therapeutic strategies. Here, we map the DNA copy number alterations (CNAs) by sequencing a cohort of primary LC samples and matched BM.
Method
The study group included 57 patients (21 females and 36 males, median age 61±8 years; 35 adenocarcinomas, 18 squamous-cell carcinomas, 2 large-cell carcinomas, 1 adenosquamous carcinoma and 1 small-cell lung cancer). From all patients pair-matched tissue samples from primary tumor and corresponding BM were collected, fixed in formalin and embedded in paraffin. All patients were therapy-naïve at the time of primary tumor collection. Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany), followed by NGS library preparation using the NEBNext Ultra II DNA kit (NEB, USA). Samples were sequenced shallowly (average depth 26 Mreads) on the NextSeq 500 system (Illumina, USA). The R package QDNAseq was used to call and visualize DNA copy number levels. The P value of <0.05 (Wilcoxon paired test) was considered statistically significant.
Result
The median time between primary LC diagnosis and BM occurrence was 13 months range, 0 to 91 months), and synchronous BM were diagnosed in 12% of patients. Overall survival in the entire group was 22.5 months. The number of CNA was significantly higher in BM than in primary tumor, regardless of clinical/demographic data or type of aneuploidy (gains/losses). Primary tumors harbored significantly more gains and almost no losses. In both tumor sites, the most frequent gains affected 1q, 5p, 7p, 8q and 20q, whereas gains of 17q and 19q, and losses of 4p, 4q, 5q, 8p, 9p, 16q, 17p, 18q, 22q were identified only in BM. The fraction of the genome affected by mutational events in BM correlated positively with time to BM development. Three the top altered genes (IL7R, MLT11, SETDB1) were identical in both primary lesions and BM.
Conclusion
Our results indicate that while primary LC lesions harbor frequent amplifications, the CNA landscape of BM is dominated by deletion events. Higher number of CNA harbored by late compared to synchronous BM suggests high levels of genomic instability.
