NRG1 Gene Fusions-What Promise Remains Behind These Rare Genetic Alterations? A Comprehensive Review of Biology, Diagnostic Approaches, and Clinical Implications.

Non-small cell lung cancer (NSCLC) presents a variety of druggable genetic alterations that revolutionized the treatment approaches. However, identifying new alterations may broaden the group of patients benefitting from such novel treatment options. Recently, the interest focused on the neuregulin-1 gene (NRG1), whose fusions may have become a potential predictive factor. To date, the occurrence of NRG1 fusions has been considered a negative prognostic marker in NSCLC treatment; however, many premises remain behind the targetability of signaling pathways affected by the NRG1 gene. The role of NRG1 fusions in ErbB-mediated cell proliferation especially seems to be considered as a main target of treatment. Hence, NSCLC patients harboring NRG1 fusions may benefit from targeted therapies such as pan-HER family inhibitors, which have shown efficacy in previous studies in various cancers, and anti-HER monoclonal antibodies. Considering the increased interest in the NRG1 gene as a potential clinical target, in the following review, we highlight its biology, as well as the potential clinical implications that were evaluated in clinics or remained under consideration in clinical trials.

Immunological insights: assessing immune parameters in medical professionals exposed to SARS-CoV-2.

BACKGROUND: The immunological background responsible for the severe course of COVID-19 and the immune factors that protect against SARS-CoV-2 infection are still unclear. The aim of this study was to investigate immune system status in persons with high exposure to SARS-CoV-2 infection. METHODS: Seventy-one persons employed in the observation and infectious diseases unit were qualified for the study between November 2020 and October 2021. Symptomatic COVID-19 was diagnosed in 35 persons. Anti-SARS-CoV-2 antibodies were also found in 8 persons. Peripheral blood mononuclear cells subpopulations were analyzed by flow cytometry, and the concentrations of cytokines and anti-SARS-CoV-2 antibodies were determined by ELISA. RESULTS: The percentages of cytotoxic T lymphocytes (CTLs), CD28+ and T helper (Th) cells with invariant T-cell receptors were significantly higher in persons with symptomatic COVID-19 than in those who did not develop COVID-19' symptoms. Conversely, symptomatic COVID-19 persons had significantly lower percentages of: a) CTLs in the late stage of activation (CD8+/CD95+), b) NK cells, c) regulatory-like Th cells (CD4+/CTLA-4+), and d) Th17-like cells (CD4+/CD161+) compared to asymptomatic COVID-19' persons. Additionally, persons with anti-SARS-CoV-2 antibodies had a significantly higher lymphocyte count and IL-6 concentration than persons without these antibodies. CONCLUSION: Numerous lymphocyte populations are permanently altered by SARS-CoV-2 infection. High percentages of both populations: NK cells-as a part of the non-specific response, and T helper cells' as those regulating the immune response, could protect against the acute COVID-19 symptoms development. Understanding the immune background of COVID-19 may improve the prevention of this disease by identifying people at risk of a severe course of infection. TRIAL REGISTRATION: This is a retrospective observational study without a trial registration number.

Obesity and lung cancer - is programmed death ligand-1 (PD-1L) expression a connection?

Introduction: In this retrospective study, the authors evaluated whether obesity-induced immunomodulation impacts the expression of programmed death ligand-1 (PD-1L) in lung cancer cells.Methods: A retrospective cross-sectional study was performed. The study included 67 patients. The data did not have a normal distribution. Results: For women, using ANOVA test (p = 0.050) with post-hoc analysis, a statistically significant difference in expression was found between women with overweight and women with normal weight (p = 0.040). Conclusions: Apart from the above, the authors did not find any statistically significant correlation between PD-1L expression on lung cancer cells and body mass index, either in the whole group or in histological subgroups.

Creating an Innovative Artificial Intelligence-Based Technology (TCRact) for Designing and Optimizing T Cell Receptors for Use in Cancer Immunotherapies: Protocol for an Observational Trial.

BACKGROUND: Cancer continues to be the leading cause of mortality in high-income countries, necessitating the development of more precise and effective treatment modalities. Immunotherapy, specifically adoptive cell transfer of T cell receptor (TCR)-engineered T cells (TCR-T therapy), has shown promise in engaging the immune system for cancer treatment. One of the biggest challenges in the development of TCR-T therapies is the proper prediction of the pairing between TCRs and peptide-human leukocyte antigen (pHLAs). Modern computational immunology, using artificial intelligence (AI)-based platforms, provides the means to optimize the speed and accuracy of TCR screening and discovery. OBJECTIVE: This study proposes an observational clinical trial protocol to collect patient samples and generate a database of pHLA:TCR sequences to aid the development of an AI-based platform for efficient selection of specific TCRs. METHODS: The multicenter observational study, involving 8 participating hospitals, aims to enroll patients diagnosed with stage II, III, or IV colorectal cancer adenocarcinoma. RESULTS: Patient recruitment has recently been completed, with 100 participants enrolled. Primary tumor tissue and peripheral blood samples have been obtained, and peripheral blood mononuclear cells have been isolated and cryopreserved. Nucleic acid extraction (DNA and RNA) has been performed in 86 cases. Additionally, 57 samples underwent whole exome sequencing to determine the presence of somatic mutations and RNA sequencing for gene expression profiling. CONCLUSIONS: The results of this study may have a significant impact on the treatment of patients with colorectal cancer. The comprehensive database of pHLA:TCR sequences generated through this observational clinical trial will facilitate the development of the AI-based platform for TCR selection. The results obtained thus far demonstrate successful patient recruitment and sample collection, laying the foundation for further analysis and the development of an innovative tool to expedite and enhance TCR selection for precision cancer treatments. TRIAL REGISTRATION: ClinicalTrials.gov NCT04994093; https://clinicaltrials.gov/ct2/show/NCT04994093. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/45872.

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.

RET Proto-Oncogene-Not Such an Obvious Starting Point in Cancer Therapy.

Mutations and fusions of RET (rearranged during transfection) gene are detected in a few common types of tumors including thyroid or non-small cells lung cancers. Multiple kinase inhibitors (MKIs) do not show spectacular effectiveness in patients with RET-altered tumors. Hence, recently, two novel RET-specific inhibitors were registered in the US and in Europe. Selpercatinib and pralsetinib showed high efficacy in clinical trials, with fewer adverse effects, in comparison to previously used MKIs. However, the effectiveness of these new drugs may be reduced by the emergence of resistance mutations in RET gene and activation of different activating signaling pathways. This review presents the function of the normal RET receptor, types of molecular disturbances of the RET gene in patients with various cancers, methods of detecting these abnormalities, and the effectiveness of modern anticancer therapies (ranging from immunotherapies, through MKIs, to RET-specific inhibitors).

Immunotherapy in Non-Small-Cell Lung Cancer Patients with Driver Alterations: A New Strategy?

For many years, researchers have been trying to develop the most effective ways to fight lung cancer, which is the cause of the largest number of cancer-related deaths among men and women worldwide. The most advanced treatments for nearly all non-small-cell lung cancer (NSCLC) types include immunotherapy with immune checkpoint inhibitors (ICIs), mainly anti-programmed death 1/anti-programmed death ligand 1 monoclonal antibodies (anti-PD-1/PD-L1 mAbs) in monotherapy or in combination with other strategies. Despite significant advances, long survival is not achievable in most cases, so new solutions are constantly being sought. One of the questions raised by oncologists is the efficacy of ICIs in patients with molecular driver alterations, especially when the possibilities of using molecularly targeted therapies are exhausted (e.g., due to resistance to tyrosine kinase inhibitors). There are studies investigating this problem, but it is still poorly described. Among probable immunotherapy' failures reasons, low immunogenicity of tumors with one driver mutation is listed. Nevertheless, in some cases, the therapy is efficient, and more research is required to establish the management of NSCLC patients with oncogenic driver abnormalities. The aim of this article is to review current discoveries in this matter.

Deep learning-based tumor microenvironment segmentation is predictive of tumor mutations and patient survival in non-small-cell lung cancer.

BACKGROUND: Despite the fact that tumor microenvironment (TME) and gene mutations are the main determinants of progression of the deadliest cancer in the world - lung cancer, their interrelations are not well understood. Digital pathology data provides a unique insight into the spatial composition of the TME. Various spatial metrics and machine learning approaches were proposed for prediction of either patient survival or gene mutations from this data. Still, these approaches are limited in the scope of analyzed features and in their explainability, and as such fail to transfer to clinical practice. METHODS: Here, we generated 23,199 image patches from 26 hematoxylin-and-eosin (H&E)-stained lung cancer tissue sections and annotated them into 9 different tissue classes. Using this dataset, we trained a deep neural network ARA-CNN. Next, we applied the trained network to segment 467 lung cancer H&E images from The Cancer Genome Atlas (TCGA) database. We used the segmented images to compute human-interpretable features reflecting the heterogeneous composition of the TME, and successfully utilized them to predict patient survival and cancer gene mutations. RESULTS: We achieved per-class AUC ranging from 0.72 to 0.99 for classifying tissue types in lung cancer with ARA-CNN. Machine learning models trained on the proposed human-interpretable features achieved a c-index of 0.723 in the task of survival prediction and AUC up to 73.5% for PDGFRB in the task of mutation classification. CONCLUSIONS: We presented a framework that accurately predicted survival and gene mutations in lung adenocarcinoma patients based on human-interpretable features extracted from H&E slides. Our approach can provide important insights for designing novel cancer treatments, by linking the spatial structure of the TME in lung adenocarcinoma to gene mutations and patient survival. It can also expand our understanding of the effects that the TME has on tumor evolutionary processes. Our approach can be generalized to different cancer types to inform precision medicine strategies.

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.

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.

Immunoprofiling: An Encouraging Method for Predictive Factors Examination in Lung Cancer Patients Treated with Immunotherapy.

The efficiency of immunotherapy using monoclonal antibodies that inhibit immune checkpoints has been proven in many clinical studies and well documented by numerous registration approaches. To date, PD-L1 expression on tumor and immune cells, tumor mutation burden (TMB), and microsatellite instability (MSI) are the only validated predictive factors used for the qualification of cancer patients for immunotherapy. However, they are not the ideal predictive factors. No response to immunotherapy could be observed in patients with high PD-L1 expression, TMB, or MSI. On the other hand, the effectiveness of this treatment method also may occur in patients without PD-L1 expression or with low TMB and with microsatellite stability. When considering the best predictive factor, we should remember that the effectiveness of immunotherapy relies on an overly complex process depending on many factors. To specifically stimulate lymphocytes, not only should their activity in the tumor microenvironment be unlocked, but above all, they should recognize tumor antigens. The proper functioning of the anticancer immune system requires the proper interaction of many elements of the specific and non-specific responses. For these reasons, a multi-parameter analysis of the immune system at its different activity levels is considered a very future-oriented predictive marker. Such complex immunological analysis is performed using modern molecular biology techniques. Based on the gene expression studies, we can determine the content of individual immune cells within the tumor, its stroma, and beyond. This includes all cell types from active memory cytotoxic T cells, M1 macrophages, to exhausted T cells, regulatory T cells, and M2 macrophages. In this article, we summarize the possibilities of using an immune system analysis to predict immunotherapy efficacy in cancer patients. Moreover, we present the advantages and disadvantages of immunoprofiling as well as a proposed future direction for this new method of immune system analysis in cancer patients who receive immunotherapy.

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.

Acute hypersensitivity pneumonitis in woodworkers caused by inhalation of birch dust contaminated with Pantoea agglomerans and Microbacterium barkeri.

CASE DESCRIPTION: Five workers (2 males and 3 females) employed in a furniture factory located in eastern Poland developed hypersensitivity pneumonitis (HP) after the pine wood used for furniture production was replaced by birch wood. All of them reported onset of respiratory and general symptoms (cough, shortness of breath, general malaise) after inhalation exposure to birch dust, showed crackles at auscultation, ground-glass attenuations in HRCT examination, and lymphocytosis in the BAL examination. The diagnosis of acute HP was set in 4 persons and the diagnosis of subacute HP in one. IDENTIFICATION OF SPECIFIC ALLERGEN: Samples of birch wood associated with evoking disease symptoms were subjected to microbiological analysis with the conventional and molecular methods. Two bacterial isolates were found to occur in large quantities (of the order 108 CFU/g) in examined samples: Gram-negative bacterium of the species Pantoea agglomerans and a non-filamentous Gram-positive actinobacterium of the species Microbacterium barkeri. In the test for inhibition of leukocyte migration, 4 out of 5 examined patients showed a positive reaction in the presence of P. agglomerans and 2 in the presence of M. barkeri. Only one person showed the presence of precipitins to P. agglomerans and none to M. barkeri. In the inhalation challenge, which is the most relevant allergological test in the HP diagnostics, all patients reacted positively to P. agglomerans and only one to M. barkeri. The results indicate that P. agglomerans developing in birch wood was the main agent causing HP in the workers exposed to the inhalation of dust from this wood, while the etiologic role of M. barkeri is probably secondary. CONCLUSION: The results demonstrate that apart from fungi and filamentous actinobacteria, regarded until recently as causative agents of HP in woodworkers, Gram-negative bacteria and non-filamentous actinobacteria may also elicit disease symptoms in the workers processing wood infected with large amounts of these microorganisms. The results obtained also seem to indicate that cellular-mediated reactions are more significant for causing disease symptoms compared to those that are precipitin-mediated.

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.

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.

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).

The Effectiveness of Pemetrexed Monotherapy Depending on Polymorphisms in TS and MTHFR Genes as Well as Clinical Factors in Advanced NSCLC Patients.

In NSCLC, second-line chemotherapy using pemetrexed or docetaxel has limited efficacy and should be dedicated to selected groups of patients. Pemetrexed is an antifolate compound with the ability to inhibit enzymes (TS, DHFR and GARFT) involved in pyrimidine and purine synthesis. The objective of this study was to evaluate the association between polymorphisms of TS and MHFR genes and clinical outcomes in NSCLC patients treated with pemetrexed monotherapy. DNA was isolated from peripheral blood of 72 non-squamous NSCLC patients treated with pemetrexed. Using PCR and RFLP methods, the variable number of tandem repeats (VNTR), the G > C SNP in these repeats and insertion/deletion polymorphism of TS gene as well as 677C > T SNP in MTHFR gene were analyzed and correlated with disease control rate, progression-free survival and overall survival (OS) of NSCLC patients. Carriers of 2R/3R(G), 3R(C)/3R(G), 3R(G)/3R(G) genotypes showed significantly more frequent early progression than carriers of 2R/2R, 2R/3R(C), 3R(C)/3R(C) genotypes of TS gene (p < 0.05). Among carriers of triple 28 bp tandem repeats (3R) in TS gene and C/C genotype of MTHFR gene a significantly shorter OS was observed (HR = 3.07; p = 0.003). In multivariate analysis, significantly higher risk of death was observed in carriers of both 3R/3R genotype in TS and C/C genotype in 677C > T SNP in MTHFR (HR = 3.85; p < 0.005) as well as in patients with short duration of response to first-line chemotherapy (HR = 2.09; p < 0.005). Results of our study suggested that genetic factors may have a high predictive and prognostic value (even greater than clinical factors) for patients treated with pemetrexed monotherapy.