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. 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. 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. 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.

Purpose RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. Materials We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. Results 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Conclusion Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

Small cell lung cancer (SCLC) shows an aggressive course with early metastases to distant organs. The main treatment regimen for SCLC patients involves platinum based chemotherapy (cisplatin or carboplatin) and etoposide. Genetic alternations, as single-nucleotide polymorphisms (SNPs) in TOP2A (topoisomerase II alpha) and in ERCC1 (endonuclease non-catalytic subunit) genes, were tested as a prognostic and predictive factors in patients with non-small cell lung cancer (NSCLC) treated with chemotherapy. However, there is limited data about clinical relevance of these genetic alternations in SCLC. Therefore, we undertook the present retrospective study to determine the influence of SNPs in TOP2A (rs34300454; rs13695; rs11540720) and ERCC1 (rs11615; rs3212986) genes on efficiency and toxicity of chemotherapy with platinum and etoposide in patients with SCLC. The studied group included 103 Caucasian SCLC patients (65 male, 38 female, median age 65 ± 7,5 years). We collected detailed clinical-demographical data including: smoking history, environmental/occupational exposure to carcinogens, performance status, disease stage, and the presence of distant metastases. The response to the treatment was carefully monitored according to RECIST criteria, as well as side effects as anemia, neutropenia or weight loss were noted. For SNPs genotyping, we used DNA isolated from peripheral blood leukocytes using Qiamp DNA Mini Kit (Qiagen, Germany) and TaqMan hydrolyzing probes (Applied Biosystem, USA) in real-time PCR technique on Eco Illumina (Illumina, USA) device. Patients with C/C genotype in rs13695 of TOP2A gene had significantly lower risk of neutropenia during chemotherapy than C/T heterozygous patients (p=0,01894; χ2=5.51; OR=2,676; 95% CI=1,165-6,143). Patients harbouring homozygous C/C genotype in rs3212986 of ERCC1 gene had significantly higher risk of anaemia during chemotherapy than heterozygous C/A patients (p=0,04531; χ2=4,01; OR=0,417; 95% CI=0,175-0,991). Furthermore, homozygous A/A genotype in rs11615 of ERCC1 gene was associated with significant prolongation of overall survival (12 vs. 9 months) compared to heterozygous G/A genotype of this gene (p=0,0120; χ2=6,3063; HR=1,657; 95% CI=1,0710-2,5633). SNPs in ERCC1 and TOP2 genes are associated with the toxicities and overall survival of SCLC patients treated with platinum and etoposide based chemotherapy.