Unlocking molecular mechanisms and identifying druggable targets in matched-paired brain metastasis of breast and lung cancers

Introduction: The incidence of brain metastases in cancer patients is increasing, with lung and breast cancer being the most common sources. Despite advancements in targeted therapies, the prognosis remains poor, highlighting the importance to investigate the underlying mechanisms in brain metastases. The aim of this study was to investigate the differences in the molecular mechanisms involved in brain metastasis of breast and lung cancers. In addition, we aimed to identify cancer lineage-specific druggable targets in the brain metastasis. Methods: To that aim, a cohort of 44 FFPE tissue samples, including 22 breast cancer and 22 lung adenocarcinoma (LUAD) and their matched-paired brain metastases were collected. Targeted gene expression profiles of primary tumors were compared to their matched-paired brain metastases samples using nCounter PanCancer IO 360™ Panel of NanoString technologies. Pathway analysis was performed using gene set analysis (GSA) and gene set enrichment analysis (GSEA). The validation was performed by using Immunohistochemistry (IHC) to confirm the expression of immune checkpoint inhibitors. Results:Our results revealed the significant upregulation of cancer-related genes in primary tumors compared to their matched-paired brain metastases (adj. p ≤ 0.05). We found that upregulated differentially expressed genes in breast cancer brain metastasis (BM-BC) and brain metastasis from lung adenocarcinoma (BM-LUAD) were associated with the metabolic stress pathway, particularly related to the glycolysis. Additionally, we found that the upregulated genes in BM-BC and BM-LUAD played roles in immune response regulation, tumor growth, and proliferation. Importantly, we identified high expression of the immune checkpoint VTCN1 in BM-BC, and VISTA, IDO1, NT5E, and HDAC3 in BM-LUAD. Validation using immunohistochemistry further supported these findings. Conclusion: In conclusion, the findings highlight the significance of using matched-paired samples to identify cancer lineage-specific therapies that may improve brain metastasis patients outcomes.</p

Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2

In previous studies we found expression of the protein colligin 2 (heat shock protein 47 (HSP47), SERPINH1) in glioma neovasculature while not in normal brain tissue. Generally, the regulation of heat shock gene expression in eukaryotes is mediated by heat shock factors (HSF). In mammals, three heat shock transcription factors, HSF-1, -2, and -4, have been isolated. Here we investigated the relation between the expression of colligin 2 and these heat shock factors at the mRNA level using real-time reverse transcriptase PCR (qRT-PCR) in different grades of astrocytic tumorigenesis, viz., low-grade glioma and glioblastoma. Endometrium samples, representing physiological angiogenesis, were included as controls. Since colligin 2 is a chaperon for collagens, the gene expression of collagen I (COL1A1) was also investigated. The blood vessel density of the samples was monitored by expression of the endothelial marker CD31 (PECAM1). Because NG2-immunopositive pericytic cells are involved in glioma neovascularization, the expression of NG2 (CSPG4) was also measured

Non-Small Cell Lung Cancer Brain Metastasis: The Link between Molecular Mechanisms and Novel Therapeutic Approaches

The prognosis of patients suffering from non-small cell lung carcinomas (NSCLC) worsens significantly when brain metastasis occurs. Seeding to the brain usually happens relatively early in the course of disease and therefore, new therapies anticipating this complication would result in considerable improvement in outcomes. In this review, we address recent molecular data of NSCLC with a focus on the risk of the formation of brain metastasis. Included is new data on the involvement of miRNAs and lncRNAs in the rise of the cerebral seeding of NSCLC. We summarize novel therapeutic approaches developed in the light of these recent molecular discoveries

Analyzing Flow Cytometry or Targeted Gene Expression Data Influences Clinical Discoveries—Profiling Blood Samples of Pancreatic Ductal Adenocarcinoma Patients

Introduction:Monitoring the therapeutic response of pancreatic ductal adenocarcinoma (PDAC) patients is crucial to determine treatment strategies. Several studies have examined the effectiveness of FOLFIRINOX as a first-line treatment in patients with locally advanced pancreatic cancer, but little attention has been paid to the immunologic alterations in peripheral blood caused by this chemotherapy regimen. Furthermore, the influence of the measurement type (e.g., flow cytometry and targeted gene expression) on the clinical discoveries is unknown. Therefore, we aimed to scrutinize the influence of using flow cytometry or targeted immune gene expression to study the immunological changes in blood samples of PDAC patients who were treated with a single-cycle FOLFIRINOX combined with lipegfilgrastim (FFX-Lipeg). Material and Methods: Whole-blood samples from 44 PDAC patients were collected at two time points: before the first FOLFIRINOX cycle and 14 days after the first cycle. EDTA blood tubes were used for multiplex flow cytometry analyses to quantify 18 immune cell populations and for complete blood count tests as the standard clinical routine. The flow cytometry data were analyzed with FlowJo software. In addition, Tempus blood tubes were used to isolate RNA and measure 1230 immune-related genes using NanoString Technology®. Data quality control, normalization, and analysis were performed using nSolver™ software and the Advanced Analysis module. Results: FFX-Lipeg treatment increased the number of neutrophils and monocytes, as shown by flow cytometry and complete blood count in concordance with elevated gene expression measured via targeted gene expression profiling analysis. Interestingly, flow cytometry analysis showed an increase in the number of B and T cells after treatment, while targeted gene expression analysis showed a decrease in B and T cell-specific gene expression. Conclusions: Targeted gene expression complements flow cytometry analysis to provide a comprehensive understanding of the effects of FFX-Lipeg. Flow cytometry and targeted gene expression showed increases in neutrophils and monocytes after FFX-Lipeg. The number of lymphocytes is increased after treatment; nevertheless, their cell-specific gene expression levels are downregulated. This highlights that different techniques influence clinical discoveries. Therefore, it is important to carefully select the measurement technique used to study the effect of a treatment.</p

Unraveling Differences in Molecular Mechanisms and Immunological Contrasts between Squamous Cell Carcinoma and Adenocarcinoma of the Cervix

This study aims to refine our understanding of the inherent heterogeneity in cervical cancer by exploring differential gene expression profiles, immune cell infiltration dynamics, and implicated signaling pathways in the two predominant histological types of cervix carcinoma, Squamous Cell Carcinoma (SCC) and Adenocarcinoma (ADC). Targeted gene expression data that were previously generated from samples of primary cervical cancer were re-analyzed. The samples were grouped based on their histopathology, comparing SCC to ADC. Each tumor in the study was confirmed to be high risk human papilloma virus (hrHPV) positive. A total of 21 cervical cancer samples were included, with 11 cases of SCC and 10 of ADC. Data analysis revealed a total of 26 differentially expressed genes, with 19 genes being overexpressed in SCC compared to ADC (Benjamini–Hochberg (BH)-adjusted p-value &lt; 0.05). Importantly, the immune checkpoint markers CD274 and CTLA4 demonstrated significantly higher expression in SCC compared to ADC. In addition, SCC showed a higher infiltration of immune cells, including B and T cells, and cytotoxic cells. Higher activation of a variety of pathways was found in SCC samples including cytotoxicity, interferon signaling, metabolic stress, lymphoid compartment, hypoxia, PI3k-AKT, hedgehog signaling and Notch signaling pathways. Our findings show distinctive gene expression patterns, signaling pathway activations, and trends in immune cell infiltration between SCC and ADC in cervical cancer. This study underscores the heterogeneity within primary cervical cancer, emphasizing the potential benefits of subdividing these tumours based on histological and molecular differences.</p

Clinical and Molecular Profiling to Develop a Potential Prediction Model for the Response to Alemtuzumab Therapy for Acute Kidney Transplant Rejection

Alemtuzumab, a monoclonal antibody that depletes CD52‐bearing immune cells, is an effective drug for the treatment of severe or glucocorticoid‐resistant acute kidney transplant rejection (AR). Patient‐specific predictions on treatment response are, however, urgently needed, given the severe side effects of alemtuzumab. This study developed a multidimensional prediction model with the aim of generating clinically useful prognostic scores for the response to alemtuzumab. Clinical and histological characteristics were collected retrospectively from patients who were treated with alemtuzumab for AR. In addition, targeted gene expression profiling of AR biopsy tissues was performed. Least absolute shrinkage and selection operator (LASSO) logistic regression modeling was used to construct the ALEMtuzumab for Acute Rejection (ALEMAR) prognostic score. Response to alemtuzumab was defined as patient and allograft survival and at least once an estimated glomerular filtration rate (eGFR) > 30 mL/min/1.73 m(2) during the first 6 months after treatment. One hundred fifteen patients were included, of which 84 (73%) had a response to alemtuzumab. The ALEMAR‐score accurately predicted the chance of response. Gene expression analysis identified 13 differentially expressed genes between responders and nonresponders. The combination of the ALEMAR‐score and selected genes resulted in improved predictions of treatment response. The present preliminary prediction model is potentially helpful for the development of stratified alemtuzumab treatment for acute kidney transplant rejection but requires validation

FOLFIRINOX chemotherapy modulates the peripheral immune landscape in pancreatic cancer:Implications for combination therapies and early response prediction

Background: FOLFIRINOX chemotherapy has improved outcomes for pancreatic cancer patients, but poor long-term survival outcomes and high toxicity remain challenges. This study investigates the impact of FOLFIRINOX on plasma proteins and peripheral immune cells to guide immune-based combination therapies and, ideally, to identify a potential biomarker to predict early disease progression during FOLFIRINOX. Methods: Blood samples were collected from 86 pancreatic cancer patients before and two weeks after the first FOLFIRINOX cycle and subjected to comprehensive immune cell and proteome profiling. Principal Component Analysis and Linear Mixed Effect Regression models were used for data analysis. FOLFIRINOX efficacy was radiologically evaluated after the fourth cycle. Results: One cycle of FOLFIRINOX diminished tumour-cell-related pathways and enhanced pathways related to immune activation, illustrated by an increase in pro-inflammatory IL–18, IL–15, and TNFRSF4. Similarly, FOLFIRINOX promoted the activation of CD4 + and CD8 + T cells, the proliferation of NK(T), and the activation of antigen-presenting cells. Furthermore, high pre-treatment levels of VEGFA and PRDX3 and an elevation in FCRL3 levels after one cycle predicted early progression under FOLFIRINOX. Finally, patients with progressive disease exhibited high levels of inhibitory markers on B cells and CD8 + T cells, while responding patients exhibited high levels of activation markers on CD4 + and CD8 + T cell subsets. Conclusion: FOLFIRINOX has immunomodulatory effects, providing a foundation for clinical trials exploring immune-based combination therapies that harness the immune system to treat pancreatic cancer. In addition, several plasma proteins hold potential as circulating predictive biomarkers for early prediction of FOLFIRINOX response in patients with pancreatic cancer.</p

A method to correlate mRNA expression datasets obtained from fresh frozen and formalin-fixed, paraffin-embedded tissue samples: A matter of thresholds

Background: Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF) tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE) material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible. Aim: To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform. Methods: Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples. Results: Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samp

The challenge of gene expression profiling in heterogeneous clinical samples

Almost all samples used in tumor biology, such as tissues and bodily fluids, are heterogeneous, i.e., consist of different cell types. Evaluating the degree of heterogeneity in samples can increase our knowledge on processes such as clonal selection and metastasis. In addition, generating expression profiles from specific sub populations of cells can reveal their distinct functions. Tissue heterogeneity also poses a challenge, as it can confound the interpretation of gene expression data. This chapter will (1) give a brief overview on how heterogeneity may influence gene expression profiling data and (2) describe the methods that are currently available to assess transcriptional biomarkers in a heterogeneous cell population.</p

Fat-Soluble Vitamin Deficiencies and Disruption of the Immune System in Pancreatic Cancer:A Vicious Cycle

Pancreatic ductal adenocarcinoma (PDAC) is currently an increasing contributor to cancer-related mortality. Despite advances in cancer treatment, PDAC survival rates have remained roughly unchanged over the years. Specifically, late diagnosis and insensitivity to currently available therapeutic regimens have been identified as the main causes for its poor survival. Pancreatic exocrine insufficiency (PEI) is a typical complication associated with PDAC diagnosis and pancreatic surgery. Pancreatic exocrine insufficiency, a major contributor to maldigestion in PDAC, is often not treated because it remains undetected because of lack of overt signs and symptoms. In this review, we will focus on the major consequences of PEI, including the inadequacy of lipase excretion, which results in deficiency of fat-soluble vitamins. Because PDAC is known for its immune-high jacking mechanisms, we describe key features in which deficiencies of fat-soluble vitamins may contribute to the aggressive biological behavior and immune evasion in PDAC. Because PEI has been shown to worsen survival rates in patients with PDAC, detecting PEI and the related fat-soluble vitamin deficits at the time of PDAC diagnosis is critical. Moreover, timely supplementation of pancreatic enzymes and fat-soluble vitamins may improve outcomes for PDAC patients.</p