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Evgeny Imyanitov
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P37 - Pathology - Biomarker Testing (ID 107)
- Event: WCLC 2020
- Type: Posters
- Track: Pathology, Molecular Pathology and Diagnostic Biomarkers
- Presentations: 2
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P37.03 - Analysis of Drug-Induced RNA Expression Changes in NSCLC Patient-Derived Explants as a Potential Tool for Personalized Therapy Choice (ID 2085)
00:00 - 00:00 | Presenting Author(s): Evgeny Imyanitov
- Abstract
Introduction
Tumor tissue has a complex 3D-structure, which includes different cellular and matrix components. Ex vivo tissue explants retain the individual features of original neoplasms and may be used for personalized testing of the drug sensitivity. The existing morphological methods for evaluation of drug response in alive tissue sections are extraordinarily time-consuming and have poor interlaboratory reproducibility. We attempted to establish RNA-based expression assays, which may supplement or replace the existing pathology-bases analyses.
Methods
Fresh tumor tissues were obtained from patients with non-small cell lung cancer (NSCLC) undergoing surgery. Tumor 0.4-mm-thick slices were generated with a tissue chopper. NSCLC explants were cultured for 24 hours in a standard media containing either cisplatin (30 mkg/ml) or gefitinib (4 mkg/ml) or no drug (control). Samples from the same tumor sections were analyzed in parallel both with RNA-expression tests (PCR, RNAseq) and immunohistochemistry (IHC).
Results
234 individual explants from 16 NSCLCs were obtained. The expression level of Ki-67 determined by PCR showed only limited correlation with percentage of Ki-67 positive cells identified by IHC (R2=0.27). MKI-67 (Ki-67 encoding gene) and TPX2 were chosen as proliferation markers for RNA expression tests. They showed nearly identical changes in expression upon drug exposure. As expected, EGFR-mutated tumors demonstrated more pronounced decline of expression of RNA proliferation markers in response to gefitinib as compared to EGFR wild-type NSCLCs. Interestingly, EGFR-mutated cancers appeared to be more sensitive to cisplatin as well. Selected tumor sets were further subjected to transcriptome analysis using RNAseq. EGFR-mutated explants had significantly higher expression level of MAGEA3 than wild-type samples. Furthermore, MAGEA3 expression in EGFR-positive tumors was decreasing during incubation with gefitinib but not with cisplatin. Thirty-five genes were responsive to cisplatin exposure. HEXIM1, NXF1, POLG2 and CCL7 appeared to be the most promising markers of cisplatin sensitivity.
Conclusion
RNA expression markers are promising reporters of individual tumor drug responsiveness.
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P37.21 - Improvement of PCR-Based Detection of ALK Rearrangements (ID 1132)
00:00 - 00:00 | Presenting Author(s): Evgeny Imyanitov
- Abstract
Introduction
ALK fusions can be detected by FISH (florescence is situ hybridization), IHC (immunohistochemistry), RT-PCR (reverse transcription polymerase chain reaction) and NGS (next generation sequencing). All these methods have advantages and drawbacks. We aimed to improve the performance of PCR testing, which is cost-efficient and requires minimal amount of material.
Methods
We assessed various PCR test designs and evaluated the spectrum of ALK gene fusions in 2620 EGFR mutation-negative formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma samples.
Results
We found that RT-PCR results could be significantly improved by using ALK-specific primers instead of random hexamers for cDNA synthesis. In addition, we revealed that the decrease of PCR fragment size below 100 bp is also helpful in preventing failures of the test. Single-tube variant-specific PCR for 4 most common rearrangements (V.1, V.2, V.3 and V.9) identified ALK fusions in 150 (6.8%) tumors. These ALK-positive samples were further analyzed by the test for unbalanced 5’/3’-end ALK expression, and the range of the variations for the 5’/3’ ratio was evaluated in comparison to 261 control ALK fusion-negative cDNAs; the obtained data allowed reliable calculation of the threshold for this PCR assay. Subsequently, the remaining lung adenocarcinomas were analyzed by the PCR for unbalanced 5’/3’-end ALK expression; this test revealed additional 109 samples, which had evidence for ALK activation and thus required validation by an independent method. Thirteen of these cases were tested by NGS analysis at the time of the abstract submission; five of these samples were proven to have ALK rearrangement, each containing distinct translocation variant (EML4ex6-ALKex19, EML4ex21-ALKex20, EML4ex13-ALKex3-ALKex20, GCC2ex13-ALKex20 and SFTPBex2-ALKex18).
Conclusion
This study provides a roadmap for adjustment of PCR assays to the detection of actionable gene fusions.
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P57 - Tumor Biology and Systems Biology - Basic and Translational Science - DNA Repair (ID 195)
- Event: WCLC 2020
- Type: Posters
- Track: Tumor Biology and Systems Biology - Basic and Translational Science
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P57.02 - High Frequency of Heterozygous Truncating Germ-Line Mutations in DNA Repair Genes in Young-Onset and/or ALK-Rearranged Lung Cancer Patients (ID 1136)
00:00 - 00:00 | Presenting Author(s): Evgeny Imyanitov
- Abstract
Introduction
While the majority of lung cancer (LC) cases arise in subjects with a long-time smoking history, there is a subset of LCs with distinct mode of the disease presentation. For example, some LCs are driven by ALK/ROS1/RET rearrangements, and these tumors are characterized by the lack of history of tobacco use and by an unusually young age at onset. There are also instances of ALK/ROS1/RET fusion-negative LCs arising in young non-smokers. We hypothesized that the development of these unusual subtypes of LC is attributed to genetic causes.
Methods
We performed whole exome sequencing of blood-derived DNA for 20 adenocarcinoma patients [15 ALK fusion-positive LCs (age range: 24-71 years; 7/15 (47%) subjects aged < 45 years); 3 young-onset (< 40 years) LCs with negative or unknown ALK/ROS1/RET fusion status; 2 women with multiple primary tumors (bilateral LC, 47 years and LC + cervical cancer, 43 years)].
Results
Exome sequencing revealed 6 heterozygous truncating germ-line mutations in DNA repair genes (ATM p. Q1478X, BARD1 c. 2300_2301del, BRCA2 p.Q1371X, CHEK2 c.1046dupA, XPA c.268_269insA and XRCC2 c.96delT). Four individuals carried single mutations and a woman with LC + cervical cancer had inactivating lesions in XPA and XRCC2 genes. There were no other plausible candidates for the dominant model of inheritance of cancer predisposition. Given that young-onset and ALK fusion-positive LC patients rarely report cancer family history, we analyzed the exome data for candidate recessive mutations. One patient carried biallelic c.C883T (p.T278I) substitution in the NEIL1 gene. This mutation is very likely to be pathogenic according to in silico predictions. We tested this mutation in 554 LC patients and 250 healthy controls. The frequency of NEIL1 c.C883T (p.T278I) heterozygotes was equal in both groups (3.2%) and no new instances homozygotes for this genotype was identified.
Conclusion
The results of this study justify an extended analysis of germ-line mutations in DNA repair genes in young-onset and/or ALK/ROS1/RET fusion-positive lung cancer patients.
