DETECTION OF MUTATIONS AND FUSIONS IN LUNG ADENOCARCINOMA US-ING ION TORRENT SEQUENCING TECHNOLOGY

Introduction. Adenocarcinomas are the most common lung tumors and are often diagnosed in advanced stages when the tumor has a polyclonal form with a wide variety of genetic alterations and activated mutational processes. Comprehensive analysis of mutational status from FFPE tissue samples in such patients can provide a therapeutic perspective and contribute considerably to clinical decisions thereby increasing the overall survival rate. Material and meth-ods. 22 genes were analyzed for mutations and 4 genes for fusion transcripts using two Ion AmpliSeq panels. DNA was isolated from paraffin-embedded tissue sections while RNA analysis was performed using two types of samples: paraffin blocks and fresh tumor tissue. Key variant detection and data analysis was performed using next platforms: Ion Reporter, ONCOMINE, R language. Results. The study of 30 tumor samples allowed the detection of 147 mutations and 4 fusions in 19 genes, and the therapeutically actionable variants were associated with different drugs clinically approved or in the phase of clinical trials. The most genetic variants were identified in the TP53, EGFR and NOTCH1 genes with a prevalence of over 50% in the TP53 gene, while all 4 detected fusions (one fusion per sample) represent the association of the ALK gene with other partners: EML4(13)-ALK(20) – present in 2 samples; EML4(6)-ALK(20); and an ALK fusion with an unknown partner gene. Conclusions

Although lung adenocarcinomas (LUAD) are the most common lung tumors, they are often diagnosed late when local tissue invasion is well advanced and metastasis is present. Genetic alterations such as somatic mutations or gene fusions are often associated with the development of these tumors and molecular profiling allows to approach the correct treatment strategy. According to The Cancer Genome Atlas (1), patients with this type of tumors show mutations in the tumor suppressor TP53 overlapping with other oncogenic driver alterations such as KRAS, EGFR, BRAF, ERBB2 mutations or ALK, RET, ROS1 gene fusions all of which have potential therapeutic implications. The complete genetic profile of LUAD is not simple to establish in routine clinical practice since this often involves the use of invasive techniques, the availability of a well-equipped molecular biology laboratory with qualified personnel and, last but not least, involves high costs when performing the analyses. Although a few years ago experts in the field recommended only EGFR testing in advanced cancers for this type of tumor, more recent recommendations include testing for EML4-ALK fusions. However, advances in molecular profiling suggest the need to increase the number of molecular targets that need to be tested in any stage of lung adenocarcinoma (2). This will provide a more comprehensive genetic picture of the tumor and, respectively, a more effective treatment strategy.
Aim: in our study we proposed the use of Ion Torrent sequencing technology for the detection of mutations and fusions in the tumors of patients with lung adenocarcinomas, and for fusion transcripts -two types of RNA: the first, isolated from FFPE samples (Formalin-fixed, paraffin-embedded), and the second -from fresh tumor tissue. The research was carried out in the Cancer Biology Scientific Laboratory within the Oncological Institute of Moldova during the years 2021-2022.

MATERIAL AND METHODS
The study analyzed 30 tumors taken from 30 patients with the histological diagnosis of lung adenocarcinoma with a content of more than 20% tumor cells per sample. DNA was isolated from paraffin-embedded tumor tissue sections (3 10

RESULTS
Following the isolation of the RNA samples, a mean concentration of 20.92 ng/µL (non-normal distribution, SD=13.73; Median=15.90; IQR= 12.80) was obtained for the paraffin-embedded tissue samples and a mean 11.17 ng/µL (non-normal distribution, SD=7.12; Median=10.20; IQR=4.64) for fresh tissue. The mean DNA concentration was 7.57 ng/µL (non-normal distribution, SD=7.26; Median=4.40; IQR=8.38). Regarding the average read length, this was 69 nucleotides (non-normal distribution, SD=15.95; Me-dian=72; IQR=29) for the RNA extracted from  A summary of the identified mutations is presented in Table 1. Most variants were identified in the TP53 gene with 51% of the total number of mutations detected. The EGFR gene follows with 17.7% and NOTCH1 with 7.5% ( fig. 3).
The top of the key variants by the number of samples in which they appear is shown in Figure 4.
The NOTCH1 V1578del deletion that is the most frequent key variant, according to the ONCOMINE platform, is not associated with relevant therapies both in lung adenocarcinoma and in other types of cancer, however, from the results obtained, a major frequency of this mutation is observed with tumors positive for ALK fusions, NOTCH1 V1578del being present in 3 of 4 ALK positive samples.    (5).
The research carried out allowed the evaluation of the most important gene mutations and fusions in 30 samples of lung adenocarcinoma as well as their association with different drugs clinically approved or in the phase of clinical trials (5). Analysis of RNA from two types of samples (FFPE and fresh tissue) was performed and the same results were obtained. We can mention that Ion Torrent sequencing is a suitable technique for evaluating genetic alterations in lung adenocarcinoma and can contribute to the best clinical decisions. Advances in tumor genome sequencing and the identification of druggable molecular targets favor the selection of the most effective therapies in the treatment of cancer, however there are some cost barriers to the widespread translation of these technologies into clinical practice. In order to obtain maximum benefits from the exploration of the tumor genome, well-equipped molecular biology laboratories are needed, with qualified personnel both in terms of laboratory analysis and for the use of bioinformatics algorithms so that the molecular data can be effectively interpreted, integrated with the clinical profile and reflected in clinical reports.

CONCLUSIONS
1. Research allowed reproducibility of results for fusion transcripts in both types of RNA samples (FFPE and fresh tissue).
2. The obtained results confirm the need for clinical testing for EML4-ALK fusions in patients with lung adenocarcinoma.
3. As KRAS mutations are among the top key variants after EGFR mutations, testing for actionable genetic variants of the KRAS gene in patients with lung adenocarcinoma is of increased interest.
4. Further studies are needed to confirm or refute a possible relationship between ALK gene fusions and the NOTCH1 V1578del mutation.