Mitochondrial DNA Changes in Respiratory Complex I Genes in Brain Gliomas.

Mitochondria are organelles necessary for oxidative phosphorylation. The interest in the role of mitochondria in the process of carcinogenesis results from the fact that a respiratory deficit is found in dividing cells, especially in cells with accelerated proliferation. The study included tumor and blood material from 30 patients diagnosed with glioma grade II, III and IV according to WHO (World Health Organization). DNA was isolated from the collected material and next-generation sequencing was performed on the MiSeqFGx apparatus (Illumina). The study searched for a possible relationship between the occurrence of specific mitochondrial DNA polymorphisms in the respiratory complex I genes and brain gliomas of grade II, III and IV. The impact of missense changes on the biochemical properties, structure and functioning of the encoded protein, as well as their potential harmfulness, were assessed in silico along with their belonging to a given mitochondrial subgroup. The A3505G, C3992T, A4024G, T4216C, G5046A, G7444A, T11253C, G12406A and G13604C polymorphisms were assessed as deleterious changes in silico, indicating their association with carcinogenesis.

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.

Attempting to Identify Bacterial Allies in Immunotherapy of NSCLC Patients.

Introduction: Factors other than PD-L1 (Programmed Death Ligand 1) are being sought as predictors for cancer immuno- or chemoimmunotherapy in ongoing studies and long-term observations. Despite high PD-L1 expression on tumor cells, some patients do not benefit from immunotherapy, while others, without the expression of this molecule, respond to immunotherapy. Attention has been paid to the composition of the gut microbiome as a potential predictive factor for immunotherapy effectiveness. Materials and Methods: Our study enrolled 47 Caucasian patients with stage IIIB or IV non-small cell lung cancer (NSCLC). They were eligible for treatment with first- or second-line immunotherapy or chemoimmunotherapy. We collected stool samples before the administration of immunotherapy. We performed next-generation sequencing (NGS) on DNA isolated from the stool sample and analyzed bacterial V3 and V4 of the 16S rRNA gene. Results: We found that bacteria from the families Barnesiellaceae, Ruminococcaceae, Tannerellaceae, and Clostridiaceae could modulate immunotherapy effectiveness. A high abundance of Bacteroidaaceae, Barnesiellaceae, and Tannerellaceae could extend progression-free survival (PFS). Moreover, the risk of death was significantly higher in patients with a high content of Ruminococcaceae family (HR = 6.3, 95% CI: 2.6 to 15.3, p < 0.0001) and in patients with a low abundance of Clostridia UCG-014 (HR = 3.8, 95% CI: 1.5 to 9.8, p = 0.005) regardless of the immunotherapy line. Conclusions: The Clostridia class in gut microbiota could affect the effectiveness of immunotherapy, as well as the length of survival of NSCLC patients who received this method of treatment.

Pathway-level mutation analysis in primary high-grade serous ovarian cancer and matched brain metastases.

Brain metastases (BMs) in ovarian cancer (OC) are a rare event. BMs occur most frequently in high-grade serous (HGS) OC. The molecular features of BMs in HGSOC are poorly understood. We performed a whole-exome sequencing analysis of ten matched pairs of formalin-fixed paraffin-embedded samples from primary HGSOC and corresponding BMs. Enrichment significance (p value; false discovery rate) was computed using the Reactome, the Kyoto Encyclopedia of Genes and Genomes pathway collections, and the Gene Ontology Biological Processes. Germline DNA damage repair variants were found in seven cases (70%) and involved the BRCA1, BRCA2, ATM, RAD50, ERCC4, RPA1, MLHI, and ATR genes. Somatic mutations of TP53 were found in nine cases (90%) and were the only stable mutations between the primary tumor and BMs. Disturbed pathways in BMs versus primary HGSOC constituted a complex network and included the cell cycle, the degradation of the extracellular matrix, cell junction organization, nucleotide metabolism, lipid metabolism, the immune system, G-protein-coupled receptors, intracellular vesicular transport, and reaction to chemical stimuli (Golgi vesicle transport and olfactory signaling). Pathway analysis approaches allow for a more intuitive interpretation of the data as compared to considering single-gene aberrations and provide an opportunity to identify clinically informative alterations in HGSOC BM.

Mitochondrial DNA Changes in Genes of Respiratory Complexes III, IV and V Could Be Related to Brain Tumours in Humans.

Mitochondrial DNA changes can contribute to both an increased and decreased likelihood of cancer. This process is complex and not fully understood. Polymorphisms and mutations, especially those of the missense type, can affect mitochondrial functions, particularly if the conservative domain of the protein is concerned. This study aimed to identify the possible relationships between brain gliomas and the occurrence of specific mitochondrial DNA polymorphisms and mutations in respiratory complexes III, IV and V. The investigated material included blood and tumour material collected from 30 Caucasian patients diagnosed with WHO grade II, III or IV glioma. The mitochondrial genetic variants were investigated across the mitochondrial genome using next-generation sequencing (MiSeq/FGx system-Illumina). The study investigated, in silico, the effects of missense mutations on the biochemical properties, structure and functioning of the encoded protein, as well as their potential harmfulness. The A14793G (MTCYB), A15758G, (MT-CYB), A15218G (MT-CYB), G7444A (MT-CO1) polymorphisms, and the T15663C (MT-CYB) and G8959A (ATP6) mutations were assessed in silico as harmful alterations that could be involved in oncogenesis. The G8959A (E145K) ATP6 missense mutation has not been described in the literature so far. In light of these results, further research into the role of mtDNA changes in brain tumours should be conducted.

Presence of Akkermansiaceae in gut microbiome and immunotherapy effectiveness in patients with advanced non-small cell lung cancer.

The significance of Akkermansia bacteria presence in gut micobiome, mainly Akkermansia mucinifila, is currently being investigated in the context of supporting therapy and marker for response to immunotherapy in cancer patients. It is indicated that patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs) respond better to treatment if this bacterium is present in the intestine.We performed next-generation sequencing of the gut microbiome from patients treated in the first or second line therapy with anti-PD-1 (anti-programmed death 1) or anti-PD-L1 (anti-programmed death ligand 1) monoclonal antibodies. In our study group of 47 NSCLC patients, the percentage of Akkermansiaceae was higher in patients with disease stabilization and with partial response to immunotherapy compared to patients with disease progression. Moreover, we found that a higher percentage of Akkermansiaceae was present in patients with squamous cell carcinoma compared to adenocarcinoma. Our study showed that Akkermansiaceae could be supporting marker for response to immunotherapies in NSCLC patients, nonetheless further in-depth studies should be conducted in the role of Akkermansiaceae in cancer immunotherapy.

Profiling Glioblastoma Cases with an Expression of DCX, OLIG2 and NES.

Glioblastoma (GBM) remains the leading cause of cancer-related deaths with the lowest five-year survival rates among all of the human cancers. Multiple factors contribute to its poor outcome, including intratumor heterogeneity, along with migratory and invasive capacities of tumour cells. Over the last several years Doublecortin (DCX) has been one of the debatable factors influencing GBM cells' migration. To resolve DCX's ambiguous role in GBM cells' migration, we set to analyse the expression patterns of DCX along with Nestin (NES) and Oligodendrocyte lineage transcription factor 2 (OLIG2) in 17 cases of GBM, using immunohistochemistry, followed by an analysis of single-cell RNA-seq data. Our results showed that only a small subset of DCX positive (DCX+) cells was present in the tumour. Moreover, no particular pattern emerged when analysing DCX+ cells relative position to the tumour margin. By looking into single-cell RNA-seq data, the majority of DCX+ cells were classified as non-cancerous, with a small subset of cells that could be regarded as glioma stem cells. In conclusion, our findings support the notion that glioma cells express DCX; however, there is no clear evidence to prove that DCX participates in GBM cell migration.

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.

Epidermal Growth Factor Receptor and its Oncogenic EGFRvIII Variant in Benign and Malignant Brain Tumors.

BACKGROUND/AIM: Tumorigenesis and cancer progression might be driven by abnormal activation of growth factor receptors. Importantly, molecular changes in EGFR-dependent signaling is one of the most common characteristics of brain tumors. PATIENTS AND METHODS: HER1 and EGFRvIII variants in meningiomas and glioblastomas were evaluated at the RNA level. RESULTS: EGFRvIII was found in 18.6% of glioblastomas (GBM), whereas 25% of EGFRvIII positive tumors express wild-type EGFR as well. HER1 was over-expressed in benign meningiomas compared to glioblastomas, whereas HER1 expression in meningiomas differed significantly between sub-types of meningiomas. EGFRvIII and HER1 where positively correlated in glioblastomas. Yet, the patient overall survival did not differ between high- and low-HER1 expressing glioblastomas or between EGFRvIII positive and negative GBMs. CONCLUSION: HER1 may be considered as an independent factor for classification of benign meningiomas. The mRNA levels of HER1 or EGFRvIII should not be used as independent prognostic factors for patients with gliomas.

A Case Report of a Female Patient With Hodgkin Lymphoma Localized in the Central Nervous System and With Concomitant Pulmonary Lymphomatoid Granulomatosis.

The involvement of the central nervous system (CNS) in Hodgkin lymphoma (HL) has been rarely reported, especially in its primary isolated form. Herein, we present a case of a 33-year-old woman, who received immunosuppressive treatment due to ulcerative colitis (at the beginning azathioprine and sulfasalazine, changed to mesalazine), with repetitive episodes of loss of consciousness for a few weeks and with no other symptoms. Magnetic resonance imaging scans of the head revealed a tumor in the lateral part of the left temporal lobe and in the cerebellum. Moreover, a subsequent computed tomographic scan of the chest revealed diffuse tumorous lesions in the lungs. The brain tumor was resected and a tumorous lesion resected from the lungs was biopsied. The histopathological analysis confirmed the final diagnosis of HL localized in the CNS with concomitant pulmonary lymphomatoid granulomatosis (LYG) grade 1. After the patient underwent radiotherapy and chemotherapy, the patient showed complete regression of lesions in the CNS and lungs, which was confirmed by positron emission tomographic scan. LYG and CNS-HL are rare proliferative disease derived from lymphocytes B and associated with EBV infections. An association between LYG and other autoimmune disorders has been reported, but to the best of our knowledge, this is the first case of the CNS-HL associated with lymphatoid granulomatosis.

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.

Safety and Efficacy of Bendamustine Monotherapy in the First-Line Treatment of Patients with Chronic Lymphocytic Leukemia: Polish Lymphoma Research Group Real-Life Analysis.

Bendamustine is a cytostatic drug with a unique structure, combining the features of purine nucleoside analogs and alkylating agents. In patients with chronic lymphocytic leukemia (CLL) it is commonly used in combination with rituximab (BR protocol) both in the first-line as well as subsequent lines of therapy, and in clinical trials it is often combined with new targeted therapies. Therefore, the data on its real-life safety and efficacy are of clinical significance. As the Polish Lymphoma Research Group (PLRG), we retrospectively analyzed the efficacy and tolerability of bendamustine monotherapy in 96 patients with CLL. The median number of bendamustine cycles was 5, and 44 patients did not complete the planned 6 cycles (46%). Among the adverse events associated with the earlier termination of bendamustine treatment, infections were the most common (20.5%), followed by neutropenia (15.9%) and thrombocytopenia (15.9%). Dose reductions and/or delays occurred in 31% of treatment cycles (132 of 425) with neutropenia (17.9%) as the most frequent cause. Efficacy analysis showed an overall response rate of 88.2% with complete remission and partial remission achieved in 43.8 and 41.7% of patients, respectively. At the 24th month of follow-up, progression-free survival was 52% and overall survival was 69.7%. Bendamustine in monotherapy was found to be safe and efficacious, at least in terms of early response. Special attention should be paid to infectious complications, and especially that immune disorders are characteristic in the clinical course of CLL. Our observations suggest efforts must be made to ensure the proper timing and proper dose in the administration of the drug, and to avoid the premature termination of the treatment.

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.

MicroRNAs aid the assessment of programmed death ligand 1 expression in patients with non-small cell lung cancer.

The qualification of patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) antibody therapy is based on an immunohistochemistry (IHC) assessment of PD-L1 expression. Immunological checkpoint inhibitors improve the overall survival of patients with expression of PD-L1; however certain PD-L1-negative patients may also benefit from immunotherapy. This indicates the requirement for novel predictive factors for the qualification of immunotherapy. It is also necessary to understand the mechanisms that effect the expression of PD-L1 in tumor cells. The expression of PD-L1 in 47 formalin-fixed, paraffin-embedded, NSCLC specimens was assessed using IHC and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of 8 microRNAs (miRNAs, miRs) complementary to PD-L1-mRNA was also evaluated using RT-qPCR. A positive correlation was revealed between the expression level of PD-L1-mRNA and 2 miRs, miR-141 (R=0.533; P=0.0029) and miR-1184 (R=0.463; P=0.049). There was also a positive correlation between the percentage of PD-L1-positive tumor cells and the expression levels of miR-141 (R=0.441; P=0.0024), miR-200b (R=0.372; P=0.011) and miR-429 (R=0.430; P=0.0028), and between the percentage of the tumor area with immune cell infiltration and the expression levels of miR-141 (R=0.333; P=0.03) and miR-200b (R=0.312; P=0.046). Additionally, the percentage of tumor cells expressing PD-L1 positively correlated with miR-141 expression (R=0.407; P=0.0055). Correlations between the expression of the investigated miRs (particularly miR-141) and PD-L1 indicated that miRs may regulate PD-L1 expression at a post-transcriptional level.

The diagnostic quality of needle brain biopsy specimens obtained with different sampling methods - Experimental study.

The aim is to examine whether brain tissue samples obtained through needle biopsy are better for histopathological evaluation when obtained with defined vacuum pressure, a novel needle rotation method, and using different needle type - Laitinen or Nashold. Moreover the paper aims to answer the question: Does vacuum and mechanical injury resulting from different sampling methods damage the tissue specimen challenging the diagnosis?. Eight hundred biopsy specimens from fresh swine brains were obtained using Nashold and Laitinen brain biopsy needles through inner cannula cutting or needle rotation sampling at vacuum pressure, from 0 to 0.06 MPa. The specimen weight and tissue quality for microscopic assessment were evaluated using the Mair score. Rising aspiration pressure increased the biopsy sample weight. Needle rotation delivered larger biopsy samples than the standard method. Laitinen provided larger samples than the Nashold needle, with the same sampling method or vacuum pressure. A higher histopathological diagnostic quality of tissue was obtained with the Laitinen needle than with Nashold, with higher vacuum pressure than lower pressure and finally with needle rotation than the standard method. No tissue damage caused by higher suction pressure or method of tissue separation was documented. Brain tissue samples obtained through needle biopsy are better for histopathological evaluation when obtained with higher vacuum pressure, a novel needle rotation method and with Laitinen needle. Higher suction pressure and sampling methods did not cause tissue damage.

The First Case of Elbow Bursitis Caused by Dirofilaria repens in Humans.

Our case report involves a 28-year-old man who was diagnosed with left elbow bursitis. After thorough macroscopic and microscopic examinations and serological and molecular tests, it was found that the inflammation had been caused by a Dirofilaria repens infection. This case report is the world's first documented description.

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.

Screening for ALK abnormalities in central nervous system metastases of non-small-cell lung cancer.

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%-7% of primary non-small-cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non-smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ2 = 16.783) in former-smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.