Genomic Profiling Identifies Putative Pathogenic Alterations in NSCLC Brain Metastases.

Introduction: Brain metastases (BM) severely affect the prognosis and quality of life of patients with NSCLC. Recently, molecularly targeted agents were found to have promising activity against BM in patients with NSCLC whose primary tumors carry "druggable" mutations. Nevertheless, it remains critical to identify specific pathogenic alterations that drive NSCLC-BM and that can provide novel and more effective therapeutic targets. Methods: To identify potentially targetable pathogenic alterations in NSCLC-BM, we profiled somatic copy number alterations (SCNAs) in 51 matched pairs of primary NSCLC and BM samples from 33 patients with lung adenocarcinoma and 18 patients with lung squamous cell carcinoma. In addition, we performed multiregion copy number profiling on 15 BM samples and whole-exome sequencing on 40 of 51 NSCLC-BM pairs. Results: BM consistently had a higher burden of SCNAs compared with the matched primary tumors, and SCNAs were typically homogeneously distributed within BM, suggesting BM do not undergo extensive evolution once formed. By comparing focal SCNAs in matched NSCLC-BM pairs, we identified putative BM-driving alterations affecting multiple cancer genes, including several potentially targetable alterations in genes such as CDK12, DDR2, ERBB2, and NTRK1, which we validated in an independent cohort of 84 BM samples. Finally, we identified putative pathogenic alterations in multiple cancer genes, including genes involved in epigenome editing and 3D genome organization, such as EP300, CTCF, and STAG2, which we validated by targeted sequencing of an independent cohort of 115 BM samples. Conclusions: Our study represents the most comprehensive genomic characterization of NSCLC-BM available to date, paving the way to functional studies aimed at assessing the potential of the identified pathogenic alterations as clinical biomarkers and targets.

Correlation between KRAS, NRAS and BRAF mutations and tumor localizations in patients with primary and metastatic colorectal cancer.

Introduction: Detection of abnormalities in the KRAS, NRAS and BRAF genes is extremely important for proper qualification of colorectal cancer (CRC) patients for therapy with anti-EGFR (epidermal growth factor receptor) monoclonal antibodies. However, data about prevalence of mutations in these genes, in different localizations of CRC tumors, are limited. Material and methods: We examined the frequency of mutations in the KRAS, NRAS and BRAF genes in 500 Caucasian CRC patients (200 women and 300 men, median age 66 years). DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissues using a Qiagen QIAamp DNA FFPE-kit. Analysis of mutations was carried out using the KRAS/BRAF, NRAS and BRAF Mutation Analysis Kit for Real-Time PCR (EntroGen) with the Cobas 480 real-time PCR apparatus (Roche Diagnostics). Results: KRAS mutations were detected in 190 (38%) patients, NRAS mutations in 20 (4%) patients, and BRAF mutations in 24 (4.8%) patients. There were no associations between age of CRC patients and frequency of KRAS, NRAS and BRAF gene mutations. These mutations were significantly more often diagnosed in women (55.5%) than in men (41%, p < 0.005). Tumors of the rectum and sigmoideum were the most often observed in both groups of CRC patients - with and without KRAS, NRAS and BRAF gene mutations. However, transverse colon, ascending colon and cecum cancers were the most often affected by mutations. Conclusions: Our study showed that the occurrence of mutations in the KRAS, NRAS and BRAF genes is not accidental and depends on the location of CRC tumors.

The efficacy of T790M mutation testing in liquid biopsy-Real clinic data.

Osimertnib is still widely used in the treatment of NSCLC patients who have previously received erlotinib, gefitinib or afatinib and have developed resistance to these drugs mediated by the T790M mutation in exon 20 of EGFR gene. We assessed the results of T790M mutation testing in liquid biopsy by Entrogen test and real-time PCR technique in routine clinical practice. Analysis was conducted in 73 plasma samples from 41 patients with locally advanced or metastatic lung adenocarcinoma treated with first- or second-generation of EGFR TKIs. We detected T790M mutation in 18 patients (43.9% of patients, 24.6% positive tests in 73 samples). The incidence of T790M mutation in liquid biopsy was significantly higher in patients with T3-T4 tumors compared to patients with T0-T2 tumors (p = 0.0368, χ2 = 4.36). Median PFS at the time of progression according to RECIST was significantly (p = 0.0444) higher in patients with T790M mutation than in patients without this mutation (22.5 vs. 15 months). Our results confirmed that T790M mutation is more often detected in patients with a large tumor spreading in the chest and with the long duration of response to first- or second generation of EGFR TKIs. The low sensitivity of the real-time PCR technique in T790M mutation detection could be partially compensated by repeating the tests.

Crizotinib efficacy in advanced non-squamous NSCLC patients with ALK or ROS1 rearrangement.

In patients with advanced non-small cell lung cancer (NSCLC), comprehensive genetic diagnostics is currently carried out in order to qualify for molecularly targeted therapies and immunotherapy. The aim of the study was to assess the usefulness of the reverse transcriptase (RT-PCR) method in the diagnosis of gene rearrangements, the effectiveness of EGFR, ALK, ROS1, and PD-L1 inhibitors in first-line treatment in NSCLC patients. We enrolled 95 non-squamous NSCLC patients with known status of EGFR, ALK, ROS1, MET and RET genes and PD-L1 protein expression. We used the real time PCR, fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and RT-PCR techniques for determination of predictive factors. In patients with ALK and ROS1 genes alteration, the median overall survival was 34 months in crizotinib treated patients and 6 months in patients who received chemotherapy (HR = 0.266, p = 0.0056). The risk of death was lower in patients treated with molecularly targeted therapies or immunotherapy compared to patients with predictive factors without personalized treatment (HR = 0.265, 95% CI 0.116-0.606) and to patient without predictive factors who received chemotherapy (HR = 0.42, 95% CI 0.162-1.09). Diagnosis of predictive factors and implementation of personalized treatment are key to prolonging the survival in advanced NSCLC patients.

Impact of copy number variant and single nucleotide polymorphism of the programmed death-ligand 1 gene, programmed death-ligand 1 protein expression and therapy regimens on overall survival in a large group of Caucasian patients with non-small cell lung carcinoma.

Anti-programmed death-1 or anti-programmed death-ligand 1 (PD-L1) blockade may be ineffective in some patients with non-small cell lung cancer (NSCLC) with high percentage of tumor cells with PD-L1 expression. In addition, immunotherapy may provide great benefits in patients without PD-L1 expression. The present study assessed PD-L1 protein expression by immunohistochemistry, copy number variation (CNV) of PD-L1 and two single nucleotide polymorphisms (SNPs), rs822335 and rs822336, in the promoter of PD-L1 by quantitative PCR in 673 patients with NSCLC. Overall survival time of patients with NSCLC depending on the assessed predictive factors (PD-L1 CNV or SNP) and the treatment methods (immunotherapy in first/second line of treatment or chemotherapy) was analyzed. The present study revealed significantly higher PD-L1 copies number in patients with ≥10% and ≥50% of tumor cells with PD-L1 expression compared to patients with lower percentage of PD-L1-positive tumor cells (P=0.02 and P=0.0002, respectively). There was a significant positive correlation (R=0.2; P=0.01) between number of PD-L1 copies and percentage of tumor cells with PD-L1 protein expression. Percentage of tumor cells with PD-L1 expression was lower in patients with TT genotype of the rs822335 polymorphism compared to those with CC genotype (P=0.03). The present study observed significantly higher risk of death in patients treated with chemotherapy compared to those treated with immunotherapy (P<0.0001; hazard ratio=2.4768; 95% confidence interval, 2.0120-3.0490). The present study demonstrated a close relationship between PD-L1 copies number, genotype of rs822335 PD-L1 polymorphism and PD-L1 protein expression on tumor cells. However, the impact of CNV and SNPs of PD-L1 on overall survival of patients with NSCLC requires further investigation.

Tissue MicroRNA Expression as a Predictor of Response to Immunotherapy in NSCLC Patients.

INTRODUCTION: Expression of PD-L1 protein on tumor cells, which is so far the only validated predictive factor for immunotherapy, is regulated by epigenetic and genetic factors. Among the most important ones that regulate gene expression are microRNAs. MATERIALS AND METHODS: The study included 60 patients with NSCLC who underwent first or second line immunotherapy with pembrolizumab or nivolumab. FFPE materials were collected before the start of immunotherapy. We examined relative expression of microRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429, miR-508-3p, miR-1184, miR-1255a) and PD-L1 mRNA expression. Copy number variation (CNV) of PD-L1 gene by qPCR and FISH methods were assessed. Two single nucleotide polymorphisms (SNPs) in promoter region of PD-L1 gene (rs822335 and rs822336) were examined. Expression of PD-L1 protein on tumor cells was assessed by immunohistochemistry (IHC). The response rate to immunotherapy and progression free survival (PFS) measured in weeks and overall survival (OS) measured in months from the start of immunotherapy were evaluated. RESULTS: Response to immunotherapy was observed in nine patients (15%, including one complete response), disease stabilization in 22 patients (36.7%), and progression in 29 patients (48.3%). Significantly higher (p=0.015) expression of miR-200b and significantly lower (p=0.043) expression of miR-429 were observed in responders compared to patients who did not respond to immunotherapy. The median PFS in the whole group of patients was 16 weeks, and the median OS was 10.5 month. In univariate analysis, the median PFS was significantly higher in patients with high miR-200b expression (HR=0.4253, 95%CI: 0.1737-1.0417, p=0.05) and high miR-508 expression (HR=0.4401, 95%CI: 0.1903-1.0178, p=0.05) and with low expression of miR-429 (HR=0.1288, 95%CI: 0.01727-0.9606, p=0.0456) compared to patients with low and high expression of these molecules, respectively. The median OS was higher in patients with low expression of miR-429 (HR=0,6288, 95%CI: 0,3053-1,2949, p=0.06) compared with patients with high expression of this microRNA. In multivariate analysis, we found that patients with PD-L1 expression on ≥1% of tumor cells compared to patients without PD-L1 expression on cancer cells had a significantly lower risk of progression (HR=0.3857, 95%CI: 0.1612-0.9226, p=0.0323) and death (HR=0.377, 95%CI: 0.1636-0.8688, p=0.022). CONCLUSION: The miR-200b and miR-429 molecules in tumor cells seem to have greatest impact on the effectiveness of immunotherapy in NSCLC patients.

PD-L1 gene copy number and promoter polymorphisms regulate PD-L1 expression in tumor cells of non-small cell lung cancer patients.

Most drugs targeting PD-1 or PD-L1 are more effective when cancer cells of non-small cell lung cancer (NSCLC) patients express PD-L1 protein. The polymorphisms of PD-L1 gene and PD-L1 gene copy number could be responsible for PD-L1 mRNA and protein expression. We analyzed PD-L1 protein expression using two IHC assays, mRNA (PD-L1) expression by qRT-PCR, PD-L1 gene promoter region polymorphisms (rs822335 and rs822336) by qPCR and PD-L1 gene copy number by fluorescence in situ hybridization method. Patients with CC genotype in rs822335 had significantly (p = 0.043) higher percentage of tumor cells with PD-L1 expression (test with 22C3 antibody) than patients with CT or TT genotypes. PD-L1 gene copy number significantly positively correlated with percentage of tumor cells with PD-L1 expression detected in tests with 22C3 antibody (p = 0.005, R = +0.442) and with SP142 antibody (p = 0.021, R = +0.369). PD-L1 gene copy number did not correlate with PD-L1 mRNA expression. Patients with PD-L1 expression tested with 22C3 antibody had significantly higher expression of PD-L1 mRNA (p = 0.023), number of chromsosme 9 centromeres (p = 0.023) and PD-L1 gene copy number (p = 0.003) than patients without PD-L1 expression on tumor cells PD-L1 gene polymorphisms and PD-L1 gene copy number may be a predictor for PD-L1 protein expression on tumor cells.

Analysis of KRAS, NRAS, BRAF, and PIK3CA mutations could predict metastases in colorectal cancer: A preliminary study.

BACKGROUND: Colorectal cancer (CRC) is usually diagnosed in the metastatic stage, when chemotherapy and molecularly-targeted therapies, instead of surgery, play the most important therapeutic role. Application of anti-epidermal growth factor receptor (EGFR) therapy requires the analysis of RAS mutation status and only RAS wild-type (wt) patients are qualified for the therapy. OBJECTIVES: The objective of this study was to analyze driver mutations in KRAS, NRAS, BRAF, and PIK3CA genes in CRC patients. MATERIAL AND METHODS: We assessed the KRAS, NRAS, BRAF, and PIK3CA genes in 102 inoperable, locally advanced and advanced CRC patients. Real-time polymerase chain reaction (RT-PCR) and high resolution melt PCR (HRM-PCR) techniques with DNA intercalating dye were applied in the study. RESULTS: Forty-six patients demonstrated the presence of examined mutations (45.1%). No significant differences in driver mutation occurrence between men and women, as well as between younger (<65 years) and older (≥65 years) patients were found. The mutations were present significantly more frequently in metastatic than in primary tumors (p = 0.039) due to the high incidence of KRAS gene mutations in metastatic tissue. BRAF and PIK3CA mutations were found only in primary tumors. The incidence of PIK3CA mutations was significantly higher (11.77%) in early than in advanced stages of the disease (1.96%; p = 0.05); NRAS mutations were found only in metastatic cancer (7.85%; p = 0.041). Only a single mutation of the PIK3CA and no mutations of NRAS were found in rectal cancer. CONCLUSIONS: Our results have shown low occurrence of driver mutations in Polish CRC patients, involving also mutations in rarely tested genes. The extent of the research panel of additional mutations could contribute to creating a better method of qualifying patients for molecularly targeted therapies and obtaining a better outcome for these therapeutic strategies.

Discrepancies between ALK protein disruption and occurrence of ALK gene rearrangement in Polish NSCLC patients.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) rearrangement are predisposed to molecularly targeted therapies. Proper diagnostic is crucial for quick and correct patients qualification to optimal treatment method. Genetic tests to detect predictive factors could be performed sequentially. After excluding EGFR mutations, abnormal ALK protein expression should be tested using immunohistochemistry (IHC) method. In patients with disrupted ALK expression, the rearrangement of the ALK gene should be confirmed by FISH method. Despite few years of experience in analysis of these predictive factors, there are still problems in interpretation of diagnostic tests results. Especially, some recommendations for ALK IHC diagnosis are not precise. METHODS: Mutations in EGFR gene were examined using real-time PCR technique in 1,108 formalin-fixed paraffin-embedded (FFPE) tissues, 398 FFPE cell-blocks and 470 cytological specimens of NSCLC. The disrupted ALK protein expression was analysed in 1,100 samples including 782 histological and 306 cytological (cell-blocks) samples using IHC. Twelve materials (1.1%) were non-diagnostic in IHC. ALK gene rearrangement using FISH method was analysed in IHC positive cases. RESULTS: The frequency of EGFR mutations was 8.6%. EGFR mutations occurred significantly more often in females (P=0.00001, χ2=62.732) and in adenocarcinoma cases (P=0.0002, χ2=14.222). The exon 19 deletions (49%) and exon 21 Leu858Arg substitution (38%) were the most common, rare EGFR mutations occurred in 13% of patients. Any expression of abnormal ALK protein was detected in 202 cases (18.57%). ALK gene rearrangement was confirmed in 49 cases (4.5%). ALK gene rearrangement is significantly more common in female than in male (P=0.0105, χ2=6.541). In patients with ALK gene rearrangement, the median percentage of nuclei with ALK rearrangement was only 25.5%. The polysomy (≥4 gene copy number per nuclei) of ALK gene was observed in 39 cases (21.4% of patients with diagnostic result of FISH examination). Median number of ALK gene copy per nuclei was 2.9±0.77. Significant positive correlation between percentage of cells with abnormal ALK expression in IHC test and percentage of nuclei with ALK rearrangement in FISH method was detected (R=0.617, P<0.00001). Significant negative correlation between the number of copies of ALK gene and the percentage of cells with expression of abnormal ALK was observed (R=-0.2004, P<0.05). ALK gene rearrangement was significantly more frequently observed in the material with coarse-grained cytoplasmic and membranous IHC staining than in materials with light cytoplasmic stippling. The occurrence of cytoplasmic stippling correlated with the increase of ALK gene copy number. CONCLUSIONS: We indicated that diagnosis of ALK disruption in NSCLC patients should be notably careful using IHC and FISH methods. Recommendations for ALK diagnosis should include the way of interpretation of cases with low percentage of cells with abnormal ALK protein expression in IHC test, character of IHC reaction, and cases with ALK gene polysomy in FISH method.

Immunohistochemical assays incorporating SP142 and 22C3 monoclonal antibodies for detection of PD-L1 expression in NSCLC patients with known status of EGFR and ALK genes.

Different immunohistochemical (IHC) assays were approved for PD-L1 expression examination on tumor cells in qualification to immune-checkpoint inhibitors therapy in NSCLC patients. These assays have some similarities, but also very serious differences. We assessed 2 IHC tests for PD-L1 expression evaluation in NSCLC tumors with different pathological diagnoses and genetic abnormalities. We enrolled 48 NSCLC patients (median age: 65 years) with known status of EGFR and ALK genes. We compared the effectiveness of PD-L1 expression examination of two IHC assays with 22C3 (Dako) and SP142 antibodies (Ventana). IHC tests were performed in resected tissue samples and in cellblocks from bronchoscopy biopsies (formalin-fixed paraffin-embedded). IHC staining was carried out on Dako Autostainer Link 48 and Ventana Benchmark GX. The percentage of tumors with PD-L1 expression of ≥5% and ≥50% on tumor cells was significantly (p<0.05) higher in assay with 22C3 (66.7% and 45.8%) than with SP142 antibody (39.6% and 22.9%). The median percentage of tumor cells with PD-L1 expression was significantly (p<0.0001) higher in test with 22C3 than with SP142 antibody. Percentage of squamous cell carcinoma (SCC) patients with PD-L1 expression was significantly higher than of non-SCC patients. Large group of patients without PD-L1 expression on tumor cells was identified among patients with common EGFR mutations and ALK rearrangement. Our results support that the highest PD-L1 expression on tumor cells occurs in SCC patients and in adenocarcinoma patients without common, druggable genetic abnormalities. The above mentioned results were clearly visible in IHC assay with 22C3 (strong cell staining).

Comparison of the effectiveness of erlotinib, gefitinib, and afatinib for treatment of non-small cell lung cancer in patients with common and rare EGFR gene mutations.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are routinely used to treat non-small cell lung cancer (NSCLC) in patients with common activating mutations of the EGFR gene. The aim of the study was to compare the efficacies of EGFR-TKIs in patients with common (exon 19 deletions and exon 21 p.Leu858Arg) and rare EGFR mutations. A retrospective analysis of 180 NSCLC patients with common (n=167) and rare (n=13) EGFR mutations treated with erlotinib (n=98), gefitinib (n=66) and afatinib (n=16) was performed. EGFR mutations were determined using RT-PCR and the EntroGen EGFR Mutations Analysis kit. Partial and complete response (PR and CR), progression-free survival (PFS), and overall survival (OS) were analyzed. Demographic and clinical factors had no impact on PFS or OS in patients treated with EGFR-TKIs. Erlotinib, gefitinib, and afatinib showed similar efficacies based on treatment response, median PFS, and OS. The type of EGFR mutation had no impact on median OS; however, median PFS was significantly longer in patients with the exon 19 deletion compared to patients with the exon 21 p.Leu858Arg substitution and rare EGFR gene mutations (P=0.013). Patients with common EGFR mutations showed significantly longer median PFS than those with rare EGFR mutations (10 vs. 5 months; P=0.009). Erlotinib, gefitinib, and afatinib show similar efficacies in NSCLC patients with both common and rare EGFR mutations. When undergoing EGFR-TKI treatment, patients with rare EGFR mutations showed similar OS but poorer PFS. Further investigation into the associations between particular rare EGFR mutations and EGFR-TKIs treatment outcomes is required.

Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.

AIMS AND BACKGROUND: Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. METHODS: Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. RESULTS: In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. CONCLUSIONS: We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.

[Influence of animal breeding manner on the occurence of internal parasites]. / Wplyw sposobu chowu zwierzat na wystepowanie pasozytów wewnetrznych.

On the turn of July and August the prevalence and intensity of internal parasites of cattle, deer, and primitive Polish horses were estimated. It was determined, that all groups of animals were infected with parasites. The prevalence and intensity of infection were diversified and depended on the animal species, breed, age, and even sex. For instance, dairy cows of lowland black-and-white breed were six times stronger infected than Polish red breed, despite using the same pasture and the same cowshed. Nematodes and coccidia were present in calves using small, frequently wet, calf-runs and at heifers grazed on pasture since early spring. Their parasites were gastrointestinal nematodes and tapeworms. Mares were infected solely with strongylids, while the sucking foals--additionally with ascarid nematodes. Mares of primitive Polish horses were infected by hookworm strongylids, ascarids, and tapeworms while stallions harboured only toothed strongylids. The animals surveyed were infected chiefly with nematodes and to a considerably smaller degree with tapeworms and coccidia.