Genetic profiles of oligometastatic non-small cell lung cancer and corresponding brain metastases.

OBJECTIVES: In patients with oligometastatic non-small cell lung cancer, systemic therapy in combination with local ablative treatment of the primary tumor and all metastatic sites is associated with improved prognosis. For patient selection and treatment allocation, further knowledge about the molecular characteristics of the oligometastatic state is necessary. Here, we performed a genetic characterization of primary non-small cell lung cancer and corresponding brain metastases. METHODS: We retrospectively identified patients with oligometastatic non-small cell lung cancer and synchronous (<3 months) or metachronous (>3 months) brain metastases who underwent surgical resection of both primary tumor and brain metastases. Mutation profiling of formalin-fixed paraffin-embedded tumor cell blocks was performed by targeted next generation sequencing using the oncomine focus assay panel. RESULTS: Sequencing was successful in 46 paired samples. An oncogenic alteration was present in 31 primary tumors (67.4%) and 40 brain metastases (86.9%). The alteration of the primary tumors was preserved in the corresponding brain metastases in 29 out of 31 cases (93.5%). The most prevalent oncogenic driver in both primary tumors and brain metastases was a KRAS mutation (n = 21). In 16 patients (34.8%), the brain metastasis harbored additional oncogenic alterations. The presence of a private genetic alteration in the brain metastasis was an independent predictor of shorter overall survival. CONCLUSIONS: In oligometastatic non-small cell lung cancer, brain metastases retain the main genetic alterations of the primary tumors. Patients may profit from targeted inhibition of mutated KRAS. Additional private genetic alterations in the brain metastases are dismal.

Fast-Track Development and Multi-institutional Clinical Validation of an Artificial Intelligence Algorithm for Detection of Lymph Node Metastasis in Colorectal Cancer.

Lymph node metastasis (LNM) detection can be automated using artificial intelligence (AI)-based diagnostic tools. Only limited studies have addressed this task for colorectal cancer (CRC). This study aimed to develop of a clinical-grade digital pathology tool for LNM detection in CRC using the original fast-track framework. The training cohort included 432 slides from one department. A segmentation algorithm detecting 8 relevant tissue classes was trained. The test cohorts consisted of materials from 5 pathology departments digitized by 4 different scanning systems. A high-quality, large training data set was generated within 7 days and a minimal amount of annotation work using fast-track principles. The AI tool showed very high accuracy for LNM detection in all cohorts, with sensitivity, negative predictive value, and specificity ranges of 0.980 to 1.000, 0.997 to 1.000, and 0.913 to 0.990, correspondingly. Only 5 of 14,460 analyzed test slides with tumor cells over all cohorts were classified as false negative (3/5 representing clusters of tumor cells in lymphatic vessels). A clinical-grade tool was trained in a short time using fast-track development principles and validated using the largest international, multi-institutional, multiscanner cohort of cases to date, showing very high precision for LNM detection in CRC. We are releasing a part of the test data sets to facilitate academic research.

Neoadjuvant nivolumab with or without relatlimab in resectable non-small-cell lung cancer: a randomized phase 2 trial.

Antibodies targeting the immune checkpoint molecules PD-1, PD-L1 and CTLA-4, administered alone or in combination with chemotherapy, are the standard of care in most patients with metastatic non-small-cell lung cancers. When given before curative surgery, tumor responses and improved event-free survival are achieved. New antibody combinations may be more efficacious and tolerable. In an ongoing, open-label phase 2 study, 60 biomarker-unselected, treatment-naive patients with resectable non-small-cell lung cancer were randomized to receive two preoperative doses of nivolumab (anti-PD-1) with or without relatlimab (anti-LAG-3) antibody therapy. The primary study endpoint was the feasibility of surgery within 43 days, which was met by all patients. Curative resection was achieved in 95% of patients. Secondary endpoints included pathological and radiographic response rates, pathologically complete resection rates, disease-free and overall survival rates, and safety. Major pathological (≤10% viable tumor cells) and objective radiographic responses were achieved in 27% and 10% (nivolumab) and in 30% and 27% (nivolumab and relatlimab) of patients, respectively. In 100% (nivolumab) and 90% (nivolumab and relatlimab) of patients, tumors and lymph nodes were pathologically completely resected. With 12 months median duration of follow-up, disease-free survival and overall survival rates at 12 months were 89% and 93% (nivolumab), and 93% and 100% (nivolumab and relatlimab). Both treatments were safe with grade ≥3 treatment-emergent adverse events reported in 10% and 13% of patients per study arm. Exploratory analyses provided insights into biological processes triggered by preoperative immunotherapy. This study establishes the feasibility and safety of dual targeting of PD-1 and LAG-3 before lung cancer surgery.ClinicalTrials.gov Indentifier: NCT04205552 .

The Impact of Cancer-Associated Fibroblasts on the Biology and Progression of Colorectal Carcinomas.

(1) Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. Cancer-associated fibroblasts (CAFs) are major components of CRC's tumour microenvironment (TME), but their biological background and interplay with the TME remain poorly understood. This study investigates CAF biology and its impact on CRC progression. (2) The cohort comprises 155 cases, including CRC, with diverse localizations, adenomas, inflammations, and controls. Digital gene expression analysis examines genes associated with signalling pathways (MAPK, PI3K/Akt, TGF-ß, WNT, p53), while next-generation sequencing (NGS) determines CRC mutational profiles. Immunohistochemical FAP scoring assesses CAF density and activity. (3) FAP expression is found in 81 of 150 samples, prevalent in CRC (98.4%), adenomas (27.5%), and inflammatory disease (38.9%). Several key genes show significant associations with FAP-positive fibroblasts. Gene set enrichment analysis (GSEA) highlights PI3K and MAPK pathway enrichment alongside the activation of immune response pathways like natural killer (NK)-cell-mediated cytotoxicity via CAFs. (4) The findings suggest an interplay between CAFs and cancer cells, influencing growth, invasiveness, angiogenesis, and immunogenicity. Notably, TGF-ß, CDKs, and the Wnt pathway are affected. In conclusion, CAFs play a significant role in CRC and impact the TME throughout development.

PDGF, NGF, and EGF as main contributors to tumorigenesis in high-risk retinoblastoma.

Retinoblastoma (RB) is the most common form of eye cancer experienced in childhood. Its aggressive malignancy is associated with excellent survival rates in high-income countries; however, the prognosis in third-world countries is less favorable. Early diagnosis can maximize the patient's visual outcomes and their survival rate. Therapy should be conducted in highly specialized treatment centers. Intravenous chemotherapy (IVC) in bilaterally affected children currently forms the majority of therapy. Local destructive procedures and local chemotherapies such as intra-arterial chemotherapy (IAC) or intravitreal chemotherapy can be taken into consideration depending on the extent and size of the tumor. Nonetheless, children and parents remain under constant stress, revisiting doctors for medical treatment and fearing vision loss and even enucleation of the eye. Adequate molecular patient stratification to improve targeted therapy is still lacking. This retrospective study analyzed formalin-fixed paraffin-embedded specimens from a cohort of 21 RB samples. A total of 11 of those samples showed undifferentiated retinoblastoma (URB) histopathological risk features, and the other 10 showed differentiated retinoblastoma (DRB) histopathological grading. RNA from all samples was isolated and analyzed via digital gene expression patterns. Conductors of cell survival and DNA repair were dominant in the DRB samples. In contrast, the agents responsible for cell-cycle progression and apoptosis were overexpressed in URB samples. Our work reveals the importance of molecular mechanisms within the immune system subjected to histologic subtypes of RB, providing more detailed background on their genetic behavior. This is of great interest for therapeutic strategies, such as targeted immune- and gene-based therapies, for retinoblastoma.

Tumor cell cytoplasmic metallothionein expression associates with differential tumor immunogenicity and prognostic outcome in high-grade serous ovarian carcinoma.

Background: The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. Methods: A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. Results: MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT-positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. Conclusion: This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells.

Induction of metallothionein expression by supplementation of zinc induces resistance against platinum-based treatment in malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Currently, multimodality treatment including chemotherapy with cisplatin or carboplatin in combination with pemetrexed offers the best options. Detoxification of heavy metals in the cell by metallothioneins (MT) is associated with early failure to platin-based chemotherapy. The induction of MTs gene expression or its enzyme results in saturation by exposure to metal ions such as zinc or cadmium. Its therapeutically effect is still not analyzed in depth. Methods: In our study, we investigated three MPM cell lines and one fibroblast cell line in the course of cisplatin treatment and supplementation of zinc. Cell state analyses via an enzyme-activity based assay were performed. With this, we were able to analyze apoptosis, necrosis and viability of cells. Additionally, we tested treated cells for changes in metallothionein IIA (MT2A) expression by using quantitative realtime polymerase chain reaction. Results: Zinc supplementation induces gene expression of MT2A. Overall, a zinc dose-dependent induction of apoptosis under platin-based treatment could be observed. This effect could be verified in all analyzed cell lines in varying intensity. Conclusions: MT expression is induced by zinc in a dose-dependent manner and inhibits a successful cisplatin therapy. Therefore, heavy metal exposure during cisplatin therapy, e.g., via cigarette smoke, might be an important factor. This should be considered in further therapeutic approaches.

Cancer-Associated Fibroblasts Influence Survival in Pleural Mesothelioma: Digital Gene Expression Analysis and Supervised Machine Learning Model.

The exact mechanism of desmoplastic stromal reaction (DSR) formation is still unclear. The interaction between cancer cells and cancer-associated fibroblasts (CAFs) has an important role in tumor progression, while stromal changes are a poor prognostic factor in pleural mesothelioma (PM). We aimed to assess the impact of CAFs paracrine signaling within the tumor microenvironment and the DSR presence on survival, in a cohort of 77 PM patients. DSR formation was evaluated morphologically and by immunohistochemistry for Fibroblast activation protein alpha (FAP). Digital gene expression was analyzed using a custom-designed CodeSet (NanoString). Decision-tree-based analysis using the "conditional inference tree" (CIT) machine learning algorithm was performed on the obtained results. A significant association between FAP gene expression levels and the appearance of DSR was found (p = 0.025). DSR-high samples demonstrated a statistically significant prolonged median survival time. The elevated expression of MYT1, KDR, PIK3R1, PIK3R4, and SOS1 was associated with shortened OS, whereas the upregulation of VEGFC, FAP, and CDK4 was associated with prolonged OS. CIT revealed a three-tier system based on FAP, NF1, and RPTOR expressions. We could outline the prognostic value of CAFs-induced PI3K signaling pathway activation together with FAP-dependent CDK4 mediated cell cycle progression in PM, where prognostic and predictive biomarkers are urgently needed to introduce new therapeutic strategies.

Integrated Clinical, Molecular and Immunological Characterization of Pulmonary Sarcomatoid Carcinomas Reveals an Immune Escape Mechanism That May Influence Therapeutic Strategies.

Pulmonary sarcomatoid carcinoma (PSC) has highly aggressive biological behaviour and poor clinical outcomes, raising expectations for new therapeutic strategies. We characterized 179 PSC by immunohistochemistry, next-generation sequencing and in silico analysis using a deep learning algorithm with respect to clinical, immunological and molecular features. PSC was more common in men, older ages and smokers. Surgery was an independent factor (p < 0.01) of overall survival (OS). PD-L1 expression was detected in 82.1% of all patients. PSC patients displaying altered epitopes due to processing mutations showed another PD-L1-independent immune escape mechanism, which also significantly influenced OS (p < 0.02). The effect was also maintained when only advanced tumour stages were considered (p < 0.01). These patients also showed improved survival with a significant correlation for immunotherapy (p < 0.05) when few or no processing mutations were detected, although this should be interpreted with caution due to the small number of patients studied. Genomic alterations for which there are already approved drugs were present in 35.4% of patients. Met exon 14 skipping was found more frequently (13.7%) and EGFR mutations less frequently (1.7%) than in other NSCLC. In summary, in addition to the divergent genomic landscape of PSC, the specific immunological features of this prognostically poor subtype should be considered in therapy stratification.

One Third of Malignant Pleural Mesothelioma Shows High Immunohistochemical Expression of MSLN or CXCR4 Which Indicates Potent Candidates for Endo-Radiotherapy.

Malignant pleural mesothelioma (MPM) is a mainly asbestos-related tumour associated with a very poor prognosis. Therapeutic approaches include multimodal therapy and chemotherapeutics, with cisplatin being the drug of choice, but response rates of only up to 14% indicate very poor outcomes. Effective treatment options are lacking. Besides the diagnostic usage of radioligands in positron emission tomography (PET)/computed tomography (CT), the endo-radioligand therapy with Lu177 has been proven as a powerful tool in cancer therapy. Mesothelin (MSLN) and C-XC chemokine receptor 4 (CXCR4) are membrane-bound proteins, expressed in certain cancers, and thus are promising targets for endo-radiotherapy. A significant portion of high MSLN- or CXCR4-expressing tumors within the MPM may open the field for this sophisticated treatment approach in the near future. Formalin-fixed, paraffin-embedded (FFPE) tumour specimens from 105 patients suffering from MPM and treated at the Lung Cancer Centre of Essen and at the Helios Klinikum Emil von Behring Berlin were screened. The tumour samples were arranged in tissue microarrays. We immunohistochemically stained the tumour samples against MSLN and CXCR4. The protein expressions of the stainings were scored by a pathologist by using a semiquantitative method. The data obtained were correlated with the clinical outcome. Overall, 77.1% of the analysed tumours showed CXCR4 protein expression (25.7% of them at high expression level (Score 3)). 48.6% of all samples showed an overall strong staining (Score ≥ 2), 59% of the investigated tumours showed MSLN protein expression (10.5% of them at high expression (Score 3)), and 36.2% of all samples showed an overall strong staining (Score ≥ 2). Our results show significant tissue expression levels, for both CXCR4 and MSLN protein, in a major portion of clinical MPM samples. One-third of patients showed outstanding immunoexpression of at least one of these markers, making them interesting candidates for radioligand-based PET/CT diagnostics and follow-up and furthermore may profit from endo-radiotherapy.

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. MATERIALS AND METHODS: TROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. RESULTS: TROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. CONCLUSION: TROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Predictive value of molecular subtypes and APOBEC3G for adjuvant chemotherapy in urothelial bladder cancer.

OBJECTIVE: Although targeted approaches have become available in second- and third-line settings, platinum-based chemotherapy remains the standard first-line treatment for advanced muscle-invasive bladder cancer (MIBC). Therefore, the prediction of platinum resistance is of utmost clinical importance. METHODS: In this study, we established a routine compatible method for the molecular classification of MIBC samples according to various classification systems and applied this method to evaluate the impact of subtypes on survival after adjuvant chemotherapy. This retrospective study included 191 patients with advanced MIBC (pT≥3 or pN+) who underwent radical cystectomy, with or without adjuvant chemotherapy. A 48-gene panel and classifier rule set were established to determine molecular subtypes according to TCGA, MDA, LundTax, and Consensus classifications. Additionally, 12 single platinum-predictive candidate genes were assessed. The results were correlated with patients' clinicopathological and follow-up data and were validated using independent data sets. RESULTS: Our final evaluation of 159 patients demonstrated better survival in the luminal groups for those who received chemotherapy compared with those who did not. In contrast, no such differences were observed in basal subtypes. The use of chemotherapy was associated with better survival in patients with high APOBEC3G expression (p < 0.002). This association was confirmed using an independent data set of patients who received neoadjuvant platinum therapy. CONCLUSIONS: The proposed method robustly replicates the most commonly used transcriptome-based subtype classifications from paraffin-embedded tissue samples. The luminal, but not basal, molecular subtypes had the greatest benefit from adjuvant platinum therapy. We identified and validated APOBEC3G as a novel predictive marker for platinum-treated patients.

Lung adenocarcinoma patients with malignant pleural effusions in hot adaptive immunity status have a longer overall survival.

Malignant pleural effusion (MPE) is a common complication of lung adenocarcinoma (LADC) which is associated with a dismal prognosis. We investigated the prognostic role of PD-L1 and other immunomodulators expression in the immune compartment of MPE immune composition. MPE cytologic cell blocks of 83 LADC patients were analysed for the mRNA expression of 770 cancer-immune genes by the NanoString nCounter platform. The expression of relevant immune cell lineage markers was validated by immunohistochemistry (IHC) using quantitative pathology. The mRNA immune profiling identified four MPE patient clusters (C). C1/2 (adaptive+, hot) showed better overall survival (OS) than C3/4 (adaptive-, cold). Additionally, cold immunity profiles (adaptive-), C4 (innate+) were associated with worse OS than C3 (innate-). High PD-L1 expression was linked to the regulation of T cell activation and interferon signalling pathways. Genes of pattern recognition receptor and type I interferon signalling pathways were specifically upregulated in the long-survival (≥90 days) patient group. Moreover, immunomodulators were co-activated and highly expressed in hot adaptive immunity patient clusters, whereas CD274 (PD-L1), TNFRSF9 (4-1BB), VEGFA (VEGF-A) and CD276 (B7-H3) were upregulated in the groups referred as cold. The patient cluster, age and PD-L1 expression were independent prognosticators for LADC MPE patients (p-value < 0.05). Our study sheds light on the variances of immune contexture regarding different PD-L1 expression and survival conditions. It revealed four distinct prognostic patient clusters with specific immune cell components and immunomodulator expression profiles, which, collectively, is supportive for future therapeutic and prognosis for cancer management.

Novel Insights into Pathophysiology of Orbital Inflammatory Diseases and Progression to Orbital Lymphoma by Pathway Enrichment Analysis.

Non-specific orbital inflammation (NSOI) and IgG4-related orbital disease (IgG4-ROD) are currently treated with non-specific immunosuppressive agents based on non-randomized, uncontrolled studies. Therefore, relapses and prolongated courses are common and remain challenging. For a more specific therapy, a better understanding of the underlying pathophysiology is crucial. Therefore, we aimed to analyze signaling pathways to expand the knowledge on the pathophysiology and possibly identify specific targets in the future, as occurred recently in Graves' orbitopathy with the IGF-1 receptor. Furthermore, we analyzed potential mechanisms for the described potential progression to orbital MALT (mucosa-associated lymphoid tissue) lymphoma. The investigation cohort for this screening study comprised of 12 patients with either typical NSOI (n = 6), IgG4-ROD or MALT lymphoma (n = 3 each). Mean age was 56.4 ± 17 years. MALT samples, in contrast with IgG4-ROD and NSOI, showed overall upregulation for extracellular matrix receptor interaction (ECM) and adipocytokine signaling. Investigating signaling compounds for MALT samples, differentially expressed genes were re-identified as targets with relevant expression. Even though pathway analysis showed differentially altered products when comparing IgG4-ROD with MALT, main conductors of differentiation in B- and T-cell signaling were commonly altered when observing the microenvironment of examined tissues. Our data reveal the characteristic differences and similarities in genetic-expression-based pathway profiles between MALT lymphoma, IgG4-ROD and NSOI, which may be useful for elucidating the associated pathogenic mechanisms and developing specific treatments for these orbital diseases.

Evaluation of Orbital Lymphoproliferative and Inflammatory Disorders by Gene Expression Analysis.

Non-specific orbital inflammation (NSOI) and IgG4-related orbital disease (IgG4-ROD) are often challenging to differentiate. Furthermore, it is still uncertain how chronic inflammation, such as IgG4-ROD, can lead to mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, we aimed to evaluate the diagnostic value of gene expression analysis to differentiate orbital autoimmune diseases and elucidate genetic overlaps. First, we established a database of NSOI, relapsing NSOI, IgG4-ROD and MALT lymphoma patients of our orbital center (2000−2019). In a consensus process, three typical patients of the above mentioned three groups (mean age 56.4 ± 17 years) at similar locations were selected. Afterwards, RNA was isolated using the RNeasy FFPE kit (Qiagen) from archived paraffin-embedded tissues. The RNA of these 12 patients were then subjected to gene expression analysis (NanoString nCounter®), including a total of 1364 target genes. The most significantly upregulated and downregulated genes were used for a machine learning algorithm to distinguish entities. This was possible with a high probability (p < 0.0001). Interestingly, gene expression patterns showed a characteristic overlap of lymphoma with IgG4-ROD and NSOI. In contrast, IgG4-ROD shared only altered expression of one gene regarding NSOI. To validate our potential biomarker genes, we isolated the RNA of a further 48 patients (24 NSOI, 11 IgG4-ROD, 13 lymphoma patients). Then, gene expression pattern analysis of the 35 identified target genes was performed using a custom-designed CodeSet to assess the prediction accuracy of the multi-parameter scoring algorithms. They showed high accuracy and good performance (AUC ROC: IgG4-ROD 0.81, MALT 0.82, NSOI 0.67). To conclude, genetic expression analysis has the potential for faster and more secure differentiation between NSOI and IgG4-ROD. MALT-lymphoma and IgG4-ROD showed more genetic similarities, which points towards progression to lymphoma.

Cancer-Associated Fibroblasts Regulate Kinase Activity in Mesothelioma Cell Lines via Paracrine Signaling and Thereby Dictate Cell Faith and Behavior.

BACKGROUND: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro. METHODS: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients.

Proteasomal Processing Immune Escape Mechanisms in Platinum-Treated Advanced Bladder Cancer.

In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host's anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens' characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased ("processing escapes"). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.

CAF-Associated Paracrine Signaling Worsens Outcome and Potentially Contributes to Chemoresistance in Epithelial Ovarian Cancer.

Background: High-grade serous ovarian cancer (HGSOC) is the predominant and deadliest form of ovarian cancer. Some of its histological subtypes can be distinguished by frequent occurrence of cancer-associated myofibroblasts (CAFs) and desmoplastic stroma reaction (DSR). In this study, we want to explore the relationship between therapy outcome and the activity of CAF-associated signaling pathways in a homogeneous HGSOC patient collective. Furthermore, we want to validate these findings in a general Epithelial ovarian cancer (EOC) cohort. Methods: The investigation cohort consists of 24 HGSOC patients. All of them were treated with platinum-based components and clinical follow-up was available. The validation cohort was comprised of 303 patients. Sequencing data (whole transcriptome) and clinical data were extracted from The Cancer Genome Atlas (TCGA). RNA of HGSOC patients was isolated using a Maxwell RSC instrument and the appropriate RNA isolation kit. For digital expression analysis a custom-designed gene panel was employed. All genes were linked to various DSR- and CAF- associated pathways. Expression analysis was performed on the NanoString nCounter platform. Finally, data were explored using the R programming environment (v. 4.0.3). Result: In total, 15 CAF-associated genes were associated with patients' survival. More specifically, 6 genes (MMP13, CGA, EPHA3, PSMD9, PITX2, PHLPP1) were linked to poor therapy outcome. Though a variety of different pathways appeared to be associated with therapy failure, many were related to CAF paracrine signaling, including MAPK, Ras and TGF-ß pathways. Similar results were obtained from the validation cohort. Discussion: In this study, we could successfully link CAF-associated pathways, as shown by increased Ras, MAPK and PI3K-Akt signaling to therapy failure (chemotherapy) in HGSOC and EOCs in general. As platinum-based chemotherapy has been the state-of-the-art therapy to treat HGSOC for decades, it is necessary to unveil the reasons behind resistance developments and poor outcome. In this work, CAF-associated signaling is shown to compromise therapy response. In the validation cohort, CAF-associated signaling is also associated with therapy failure in general EOC, possibly hinting towards a conserved mechanism. Therefore, it may be helpful to stratify HGSOC patients for CAF activity and consider alternative treatment options.

Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure. METHODS: We identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort. RESULTS: The two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity. CONCLUSION: Pressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.

Mitogen signal-associated pathways, energy metabolism regulation, and mediation of tumor immunogenicity play essential roles in the cellular response of malignant pleural mesotheliomas to platinum-based treatment: a retrospective study.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare malignant tumor associated with asbestos exposure, with infaust prognosis and overall survival below 20 months in treated patients. Platinum is still the backbone of the chemotherapy protocols, and the reasons for the rather poor efficacy of platinum compounds in MPM remain largely unknown. Therefore, we aimed to analyze differences in key signaling pathways and biological mechanisms in therapy-naïve samples and samples after chemotherapy in order to evaluate the effect of platinum-based chemotherapy. METHODS: The study cohort comprised 24 MPM tumor specimens, 12 from therapy-naïve and 12 from patients after platinum-based therapy. Tumor samples were screened using the NanoString nCounter platform for digital gene expression analysis with an appurtenant custom-designed panel comprising a total of 366 mRNAs covering the most important tumor signaling pathways. Significant pathway associations were identified by gene set enrichment analysis using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt). RESULTS: We have found reduced activity of TNF (normalized enrichment score: 2.03), IL-17 (normalized enrichment score: 1.93), MAPK (normalized enrichment score: 1.51), and relaxin signaling pathways (normalized enrichment score: 1.42) in the samples obtained after platinum-based therapy. In contrast, AMPK (normalized enrichment score: -1.58), mTOR (normalized enrichment score: -1.50), Wnt (normalized enrichment score: -1.38), and longevity regulating pathway (normalized enrichment score: -1.31) showed significantly elevated expression in the same samples. CONCLUSIONS: We could identify deregulated signaling pathways due to a directed cellular response to platinum-induced cell stress. Our results are paving the ground for a better understanding of cellular responses and escape mechanisms, carrying a high potential for improved clinical management of patients with MPM.