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.

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.

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.

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.

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.

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.

Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice.

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not 'normoxic' (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS-MS revealed 52 proteins differentially abundant in sEVs from hypoxic and 'normoxic' MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix-receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix-receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

Genetic knockdown of Klk8 has sex-specific multi-targeted therapeutic effects on Alzheimer's pathology in mice.

AIMS: Previous work in our lab has identified the protease kallikrein-8 (KLK8) as a potential upstream mover in the pathogenesis of Alzheimer's disease (AD). We showed pathologically elevated levels of KLK8 in the cerebrospinal fluid and blood of patients with mild cognitive impairment or dementia due to AD, and in brains of patients and transgenic CRND8 (TgCRND8) mice in incipient stages of the disease. Furthermore, short-term antibody-mediated KLK8 inhibition in moderate stage disease alleviated AD pathology in female mice. However, it remains to be shown whether long-term reversal of KLK8 overexpression can also counteract AD. Therefore, the effects of genetic Klk8-knockdown were determined in TgCRND8 mice. METHODS: The effects of heterozygous ablation of murine Klk8 (mKlk8) gene on AD pathology of both sexes were examined by crossbreeding TgCRND8 [hAPP+/-] with mKlk8-knockdown [mKlk8+/-] mice resulting in animals with or without AD pathology which revealed pathologically elevated or normal KLK8 levels. RESULTS: mKlk8-knockdown had negligible effects on wildtype animals but led to significant decline of amyloid beta (Aß) and tau pathology as well as an improvement of structural neuroplasticity in a sex-specific manner in transgenics. These changes were mediated by a shift to non-amyloidogenic cleavage of the human amyloid precursor protein (APP), recovery of the neurovascular unit and maintaining microglial metabolic fitness. Mechanistically, Klk8-knockdown improved Aß phagocytosis in primary glia and Aß resistance in primary neurons. Most importantly, transgenic mice revealed less anxiety and a better memory performance. CONCLUSIONS: These results reinforce the potential of KLK8 as a therapeutic target in AD.

Dissimilar patterns of tumor-infiltrating immune cells at the invasive tumor front and tumor center are associated with response to neoadjuvant chemotherapy in primary breast cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) are described as an important immune modulator in the tumor microenvironment and are associated with breast cancer (BC) outcome. The spatial analysis of TILs and TIL subtype distribution at the invasive tumor front (ITF) and the tumor center (TC) might provide further insights into tumor progression. METHODS: We analyzed core biopsies from 87 pre-therapeutic BC patients for total TILs and the following subtypes: CD3+, CD4+, CD8+, CD20+ and CD68+ cells in correlation to clinicopathological parameters and disseminated tumor cells (DTCs) in the bone marrow. RESULTS: TILs and TIL subtypes showed significantly different spatial distribution among both tumor areas. TILs, especially CD3+ T cells were associated with the tumor status and tumor grading. BC patients responding to neoadjuvant chemotherapy had significantly more TILs and CD3+ T cells at the TC. The presence of DTCs after NACT was related to CD4+ infiltration at the TC. CONCLUSION: The dissimilar spatial association of TILs and TIL subtypes with clinicopathological parameters, NACT response and minimal residual disease underlines the necessity of detailed TIL analysis for a better understanding of immune modulatory processes.

Gene expression profiling of homologous recombination repair pathway indicates susceptibility for olaparib treatment in malignant pleural mesothelioma in vitro.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a tumour arising from pleural cavities with poor prognosis. Multimodality treatment with pemetrexed combined with cisplatin shows unsatisfying response-rates of 40%. The reasons for the rather poor efficacy of chemotherapeutic treatment are largely unknown. However, it is conceivable that DNA repair mechanisms lead to an impaired therapy response. We hypothesize a major role of homologous recombination (HR) for genome stability and survival of this tumour. Therefore, we analysed genes compiled under the term "BRCAness". An inhibition of this pathway with olaparib might abrogate this effect and induce apoptosis. METHODS: We investigated the response of three MPM cell lines and lung fibroblasts serving as a control to treatment with pemetrexed, cisplatin and olaparib. Furthermore, we aimed to find possible correlations between response and gene expression patterns associated with BRCAness phenotype. Therefore, 91 clinical MPM samples were digitally screened for gene expression patterns of HR members. RESULTS: A BRCAness-dependent increase of apoptosis and senescence during olaparib-based treatment of BRCA-associated-protein 1 (BAP1)-mutated cell lines was observed. The gene expression pattern identified could be found in approx. 10% of patient samples. Against this background, patients could be grouped according to their defects in the HR system. Gene expression levels of Aurora Kinase A (AURKA), RAD50 as well as DNA damage-binding protein 2 (DDB2) could be identified as prognostic markers in MPM. CONCLUSIONS: Defects in HR compiled under the term BRCAness are a common event in MPM. The present data can lead to a better understanding of the underlaying cellular mechanisms and leave the door wide open for new therapeutic approaches for this severe disease with infaust prognosis. Response to Poly (ADP-ribose)-Polymerase (PARP)-Inhibition could be demonstrated in the BAP1-mutated NCI-H2452 cells, especially when combined with cisplatin. Thus, this combination therapy might be effective for up to 2/3 of patients, promising to enhance patients' clinical management and outcome.

Prognostic significance of PD-1 and PD-L1 positive tumor-infiltrating immune cells in ovarian carcinoma.

INTRODUCTION: Ovarian carcinoma is associated with the highest mortality of all gynecologic malignancies. Even after optimal treatment, prognosis remains poor. There is no established biomarker to predict individual patient outcome. OBJECTIVE: To evaluate the prognostic significance of PD-1 and PD-L1 expression in tumor tissues from patients with ovarian cancer. METHODS: Tissue micro-arrays were prepared from routinely formalin-fixed, paraffin-embedded tumor tissues and examined immunohistochemically for the expression of programed cell death protein 1 (PD-1) and one of its ligands (PD-L1) on epithelial tumor cells, as well as on tumor- and stroma-infiltrating immune cells. RESULTS: The presence of PD-1 positive tumor-infiltrating immune cells was significantly associated with prolonged overall survival. PD-1 and PD-L1 positive tumor-infiltrating immune cells were associated with the presence of lymph node metastases and higher tumor grade. Interestingly, the amount of PD-1/PD-L1 positive tumor- and stroma-infiltrating immune cells independent of PD-1 or PD-L1 expression did not show any significant correlation with prognostic variables. CONCLUSION: Our results highlight the prognostic value of PD-1 and PD-L1 positive tumor-infiltrating immune cells in ovarian carcinoma. Their association with favorable prognosis supports the hypothesis that the expression of PD-1 and PD-L1 on tumor-infiltrating immune cells represents a strong immune response.

Myelination in Multiple Sclerosis Lesions Is Associated with Regulation of Bone Morphogenetic Protein 4 and Its Antagonist Noggin.

Remyelination is a central aspect of new multiple sclerosis (MS) therapies, in which one aims to alleviate disease symptoms by improving axonal protection. However, a central problem is mediators expressed in MS lesions that prevent effective remyelination. Bone morphogenetic protein4 (BMP4) inhibits the development of mature oligodendrocytes in cell culture and also blocks the expression of myelin proteins. Additionally, numerous studies have shown that Noggin (SYM1)-among other physiological antagonists of BMP4-plays a prominent role in myelin formation in the developing but also the adult central nervous system. Nonetheless, neither BMP4 nor Noggin have been systematically studied in human MS lesions. In this study, we demonstrated by transcript analysis and immunohistochemistry that BMP4 is expressed by astrocytes and microglia/macrophages in association with inflammatory infiltrates in MS lesions, and that astrocytes also express BMP4 in chronic inactive lesions that failed to remyelinate. Furthermore, the demonstration of an increased expression of Noggin in so-called shadow plaques (i.e., remyelinated lesions with thinner myelin sheaths) in comparison to chronically inactive demyelinated lesions implies that antagonizing BMP4 is associated with successful remyelination in MS plaques in humans. However, although BMP4 is strongly overexpressed in inflammatory lesion areas, its levels are also elevated in remyelinated lesion areas, which raises the possibility that BMP4 signaling itself may be required for remyelination. Therefore, remyelination might be influenced by a small number of key factors. Manipulating these molecules, i.e., BMP4 and Noggin, could be a promising therapeutic approach for effective remyelination.

Mutational analysis of pulmonary tumours with neuroendocrine features using targeted massive parallel sequencing: a comparison of a neglected tumour group.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows. METHODS: A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes. RESULTS: After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020). CONCLUSIONS: The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

SOX4, SOX11 and PAX6 mRNA expression was identified as a (prognostic) marker for the aggressiveness of neuroendocrine tumors of the lung by using next-generation expression analysis (NanoString).

BACKGROUND: Neuroendocrine tumors of the lung (NELC) account for 25% of all lung cancer cases and transcription factors may drive dedifferentiation of these tumors. This study was conducted to identify supportive diagnostic and prognostic biomarkers. MATERIALS & METHODS: A total of 16 TC, 13 AC, 16 large cell neuroendocrine carcinomas and 15 small cell lung cancer were investigated for the mRNA expression of 11 transcription factors and related genes (MYB, MYBBP1A, OCT4, PAX6, PCDHB, RBP1, SDCBP, SOX2, SOX4, SOX11, TEAD2). RESULTS: SOX4 (p = 0.0002), SOX11 (p < 0.0001) and PAX6 (p = 0.0002) were significant for tumor type. Elevated PAX6 and SOX11 expression correlated with poor outcome in large cell neuroendocrine carcinomas and small cell lung cancer (p < 0.0001 and p = 0.0232, respectively) based on survival data of 34 patients (57%). CONCLUSION: Aggressiveness of NELC correlated with increasing expression of transcription factors. SOX11 seems to be a highly valuable diagnostic and prognostic marker for aggressive NELC.

Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumors of the lung: results of a profiling study.

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides), non-coding, highly conserved single-stranded RNAs with posttranscriptional regulatory features, including the regulation of cell proliferation, differentiation, survival, and apoptosis. They are deregulated in a broad variety of tumors showing characteristic expression patterns and can, thus, be used as a diagnostic tool. In contrast to non-small cell carcinoma of the lung neuroendocrine lung tumors, encompassing typical and atypical carcinoids, small cell lung cancer and large cell neuroendocrine lung cancer, no data about deregulation of tumor entity-specific miRNAs are available to date. miRNA expression differences might give useful information about the biological characteristics of these tumors, as well as serve as helpful markers.In 12 pulmonary neuroendocrine tumors classified as either typical carcinoid, atypical, large cell neuroendocrine or small cell lung cancer, screening for 763 miRNAs known to be involved in pulmonary cancerogenesis was conducted by performing 384-well TaqMan low-density array real-time qPCR. In the entire cohort, 44 miRNAs were identified, which showed a significantly different miRNA expression. For 12 miRNAs, the difference was highly significant (P<0.01). Eight miRNAs showed a negative (miR-22, miR-29a, miR-29b, miR-29c, miR-367*; miR-504, miR-513C, miR-1200) and four miRNAs a positive (miR-18a, miR-15b*, miR-335*, miR-1201) correlation to the grade of tumor biology. The miRNAs let-7d; miR-19; miR-576-5p; miR-340*; miR-1286 are significantly associated with survival. Members of the miR-29 family seem to be extremely important in this group of tumors. We found a number of miRNAs, which showed a highly significant deregulation in pulmonary neuroendocrine tumors. Moreover, some of these deregulated miRNAs seem to allow discrimination of the various subtypes of pulmonary neuroendocrine tumors. Thus, the analysis of specific sets of miRNAs can be proposed as diagnostic and/or predictive markers in this group of neoplasias.

Mdm2 protein expression is strongly associated with survival in malignant pleural mesothelioma.

AIMS: TP53 mutations are extremely rare in malignant pleural mesothelioma (MPM). In TP53 wild-type tumors, the functional p53 protein can be inactivated by MDM2. MATERIALS & METHODS: A total of 61 patient samples were tested for their Mdm2 and p53 protein expression levels via immunohistochemistry. RESULTS: This study demonstrates nuclear Mdm2 expression in three out of four mesothelioma cell lines and 21.3% of the MPM specimens investigated. After silencing of the MDM2 gene by siRNA in MPM cell lines, Mdm2 immunoexpression is lost and cells show changes indicative of severe damage. Mdm2 protein expression in MPM is detected in epithelioid and biphasic subtypes only and is significantly associated with poor survival compared with Mdm2-negative tumors. This may be explained by increased Mdm2 levels possibly leading to an increased ubiquitilation and proteasomal degradation of functional p53 protein. CONCLUSION: Expression of Mdm2 is a strong prognostic factor associated with shortened overall survival in MPM.

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

OBJECTIVES: In patients with oligometastatic non-small-cell lung cancer (NSCLC), systemic therapy in combination with local ablative treatment of the primary tumour 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 NSCLC and corresponding brain metastases (BM). METHODS: We retrospectively identified patients with oligometastatic NSCLC and synchronous (<3 months) or metachronous (>3 months) BM who underwent surgical resection of both primary tumour and BM. Mutation profiling of formalin-fixed paraffin-embedded tumour 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 tumours (67.4%) and 40 BM (86.9%). The alteration of the primary tumours was preserved in the corresponding BM in 29 out of 31 cases (93.5%). The most prevalent oncogenic driver in both primary tumours and BM was a KRAS (Kirsten rat sarcoma viral oncogene) mutation (s = 21). In 16 patients (34.8%), the BM harboured additional oncogenic alterations. The presence of a private genetic alteration in the BM was an independent predictor of shorter overall survival. CONCLUSIONS: In oligometastatic NSCLC, BM retain the main genetic alterations of the primary tumours. Patients may profit from targeted inhibition of mutated KRAS. Additional private genetic alterations in the BM are dismal.

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.

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 .