Molecular differences of angiogenic versus vessel co-opting colorectal cancer liver metastases at single-cell resolution.

BACKGROUND: Colorectal cancer liver metastases (CRCLM) are associated with a poor prognosis, reflected by a five-year survival rate of 14%. Anti-angiogenic therapy through anti-VEGF antibody administration is one of the limited therapies available. However, only a subgroup of metastases uses sprouting angiogenesis to secure their nutrients and oxygen supply, while others rely on vessel co-option (VCO). The distinct mode of vascularization is reflected by specific histopathological growth patterns (HGPs), which have proven prognostic and predictive significance. Nevertheless, their molecular mechanisms are poorly understood. METHODS: We evaluated CRCLM from 225 patients regarding their HGP and clinical data. Moreover, we performed spatial (21,804 spots) and single-cell (22,419 cells) RNA sequencing analyses to explore molecular differences in detail, further validated in vitro through immunohistochemical analysis and patient-derived organoid cultures. RESULTS: We detected specific metabolic alterations and a signature of WNT signalling activation in metastatic cancer cells related to the VCO phenotype. Importantly, in the corresponding healthy liver of CRCLM displaying sprouting angiogenesis, we identified a predominantly expressed capillary subtype of endothelial cells, which could be further explored as a possible predictor for HGP relying on sprouting angiogenesis. CONCLUSION: These findings may prove to be novel therapeutic targets to the treatment of CRCLM, in special the ones relying on VCO.

Characterizing the mutational burden, DNA methylation landscape, and proteome of germ cell tumor-related somatic-type malignancies to identify the tissue-of-origin, mechanisms of therapy resistance, and druggable targets.

BACKGROUND: Germ cell tumors (GCT) might undergo transformation into a somatic-type malignancy (STM), resulting in a cell fate switch to tumors usually found in somatic tissues, such as rhabdomyosarcomas or adenocarcinomas. STM is associated with a poor prognosis, but the molecular and epigenetic mechanisms triggering STM are still enigmatic, the tissue-of-origin is under debate and biomarkers are lacking. METHODS: To address these questions, we characterized a unique cohort of STM tissues on mutational, epigenetic and protein level using modern and high-throughput methods like TSO assays, 850k DNA methylation arrays and mass spectrometry. RESULTS AND CONCLUSIONS: For the first time, we show that based on DNA methylation and proteome data carcinoma-related STM more closely resemble yolk-sac tumors, while sarcoma-related STM resemble teratoma. STM harbor mutations in FGF signaling factors (FGF6/23, FGFR1/4) highlighting the corresponding pathway as a therapeutic target. Furthermore, STM utilize signaling pathways, like AKT, FGF, MAPK, and WNT to mediate molecular functions coping with oxidative stress, toxin transport, DNA helicase activity, apoptosis and the cell cycle. Collectively, these data might explain the high therapy resistance of STM. Finally, we identified putative novel biomarkers secreted by STM, like EFEMP1, MIF, and DNA methylation at specific CpG dinucleotides.

Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options.

BACKGROUND: Being the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting CLDN6 (CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST. METHODS: Protein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay. RESULTS: We demonstrated that treatment with a CLDN6-ADC enhanced apoptosis induction specifically in CLDN6+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance. CONCLUSIONS: In summary, this study offers a novel CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.

Proteomic analysis identifies argininosuccinate synthetase 1 and special AT-rich sequence binding protein 1 as reliable markers for the immunohistochemical distinction between WHO types A and B3 thymomas.

AIMS: The reliable classification of type A versus type B3 thymomas has prognostic and therapeutic relevance, but can be problematic due to considerably overlapping morphology. No immunohistochemical markers aiding in this distinction have been published so far. METHODS AND RESULTS: We identified and quantified numerous differentially expressed proteins using an unbiased proteomic screen by mass spectrometry in pooled protein lysates from three type A and three type B3 thymomas. From these, candidates were validated in a larger series of paraffin-embedded type A and B3 thymomas. We identified argininosuccinate synthetase 1 (ASS1) and special AT-rich sequence binding protein 1 (SATB1) as highly discriminatory between 34 type A and 20 type B3 thymomas (94% sensitivity, 98% specificity and 96% accuracy). Although not the focus of this study, the same markers also proved helpful in the diagnosis of type AB (n = 14), B1 (n = 4) and B2 thymomas (n = 10). CONCLUSIONS: Mutually exclusive epithelial expression of ASS1 in 100% of type B3 thymomas and ectopic nuclear expression of SATB1 in 92% of type A thymomas support the distinction between type A and type B3 thymomas with 94% sensitivity, 98% specificity and 96% accuracy.

Dynamics of intestinal and intratumoral microbiome signatures in genetically engineered mice and human pancreatic ductal adenocarcinoma.

BACKGROUND: Emerging evidence has recently revealed a prominent role of the microbiome in pancreatic ductal adenocarcinoma (PDAC). However, while most observations were made in patients, mouse models still require a precise characterization of their disease-related microbiome to employ them for mechanistic and interventional preclinical studies. METHODS: To investigate the fecal and tumoral microbiome of LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) and control (CTRL) mice, Oxford Nanopore sequencing was applied. Feces were collected from 10 KPC mice and 10 CTRLs at 3 timepoints (6 weeks, 12 weeks, and when tumor-bearing (KPC) or 6 months (CTRL), respectively). Metagenomic sequencing was performed on feces DNA. KPC tumor and healthy pancreas DNA samples were subjected to 16S rRNA gene sequencing. Bacterial marker components were detected in KPC tumor tissue over time by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). RESULTS: Murine fecal samples showed a significantly different microbiome compared to age-matched healthy CTRLs regarding beta diversity (p = 0.001, R2 = 0.2-0.25 for Bray-Curtis). Adjusted human PDAC classifiers predicted disease status from feces of KPC mice achieving area under the receiver operating characteristic (AUROC) values of 80%. Furthermore, KPC tumors harbored significantly more bacterial components than healthy pancreas. Also the microbial composition differs significantly between KPC tumors and healthy pancreas tissue (p = 0.042 for Bray-Curtis). Microbiota found highly abundant in human PDAC samples were considerably more abundant in KPC tumors as compared to healthy pancreas samples (p-value <0.001). CONCLUSION: KPC fecal samples show similarities with the microbial composition of stool samples from human PDAC patients.

Primary mediastinal germ cell tumours: an immunohistochemical and molecular diagnostic approach.

AIMS: Primary mediastinal germ cell tumours (PMGCTs) are rare mediastinal neoplasms, and their diagnosis can be challenging, owing to small biopsy samples. The aim of this study was to develop a diagnostic algorithm using immunohistochemical staining, with a focus on novel markers, and molecular analysis of isochromosome 12p [i(12p)]. METHODS AND RESULTS: Paraffin-embedded tissues of 32 mediastinal tumours were analysed with immunohistochemical staining for sal-like transcription factor 4 (SALL4), Lin-28 homologue A (LIN28), octamer-binding transcription factor 3/4 (OCT3/4), D2-40, cluster of differentiation 117 (CD117), sex-determining region Y-box 17, sex-determining region Y-box 2 (SOX2), cluster of differentiation 30, the ß-subunit of human chorionic gonadotropin (ß-hCG), GATA-binding protein 3 (GATA3), forkhead box protein A2 (FOXA2), glypican-3 (GPC3), α-fetoprotein (AFP), terminal deoxynucleotidyl transferase (TdT), nuclear protein of the testis (NUT), and pan-cytokeratin. Quantitative real-time polymerase chain reaction was performed to investigate the i(12p) status. Fifteen seminomas, seven teratomas, one yolk sac tumour, one choriocarcinoma and seven mixed PMGCTs were diagnosed. Each entity had different immunohistochemical staining patterns, which helped to distinguish them: OCT3/4, D2-40, CD117 and TdT for seminoma; OCT3/4 and SOX2 for embryonal carcinoma; FOXA2, GPC3 and AFP for yolk sac tumour; and ß-hCG and GATA3 for choriocarcinoma. Mature teratomas stained positively for pan-cytokeratin in epithelial components and focally for SALL4, SOX2, GATA3, D2-40, and FOXA2. Furthermore, a NUT carcinoma mimicking a PMGCT was diagnosed, showing strong nuclear SOX2 staining and speckled nuclear NUT staining. i(12p) was detected in 24 of 27 PMGCTs (89%). CONCLUSION: A diagnostic algorithm is of great importance for a reliable diagnosis of PMGCT in, usually small, tissue biopsy samples. Therefore, a combination of three to four antibodies to identify the correct histological subtype is usually necessary, in addition to morphological features. The i(12p) status serves as an additional option to indicate a germ cell origin in selected cases.

Functional apoptosis profiling identifies MCL-1 and BCL-xL as prognostic markers and therapeutic targets in advanced thymomas and thymic carcinomas.

BACKGROUND: Multi-omics studies have shown a high and lack of common driver mutations in most thymomas (TH) and thymic carcinomas (TC) that hamper the development of novel treatment approaches. However, deregulation of apoptosis has been proposed as a common hallmark of TH and TC. BH3 profiling can be utilized to study the readiness of living cancer cells to undergo apoptosis and their dependency on pro-survival BCL-2 family proteins. METHODS: We screened a cohort of 62 TH and TC patient samples for expression of BCL-2 family proteins and used the TC cell line 1889c and native TH for dynamic BH3 profiling and treatment with BH3 mimetics. RESULTS: Immunohistochemical overexpression of MCL-1 and BCL-xL was a strong prognostic marker of TH and TC, and BH3 profiling indicated a strong dependency on MCL-1 and BCL-xL in TH. Single inhibition of MCL-1 resulted in increased binding of BIM to BCL-xL as an escape mechanism that the combined inhibition of both factors could overcome. Indeed, the inhibition of MCL-1 and BCL-xL in combination induced apoptosis in a caspase-dependent manner in untreated and MCL-1-resistant 1889c cells. CONCLUSION: TH and TC are exquisitely dependent on the pro-survival factors MCL-1 and BCL-xL, making them ideal candidates for co-inhibition by BH3 mimetics. Since TH show a heterogeneous dependency on BCL-2 family proteins, upfront BH3 profiling could select patients and tailor the optimal therapy with the least possible toxicity.

The detection of isochromosome i(12p) in malignant germ cell tumours and tumours with somatic malignant transformation by the use of quantitative real-time polymerase chain reaction.

AIMS: Malignant germ cell tumours (GCTs) of the testis are rare neoplasms, but the most common solid malignancies in young men. World Health Organization guidelines divide GCTs into five types, for which numerous immunohistochemical markers allow exact histological subtyping in the majority of cases. In contrast, a germ cell origin is often hard to prove in metastatic GCTs that have developed so-called somatic malignant transformation. A high percentage, up to 89%, of GCTs are characterised by the appearance of isochromosome 12p [i(12p)]. Fluorescence in-situ hybridisation has been the most common diagnostic method for the detection of i(12p) so far, but has the disadvantages of being time-consuming, demanding, and not being a stand-alone method. The aim of the present study was to establish a quantitative real-time polymerase chain reaction assay as an independent method for detecting i(12p) and regional amplifications of the short arm of chromosome 12 by using DNA extracted from formalin-fixed paraffin-embedded tissue. METHODS AND RESULTS: A cut-off value to distinguish between the presence and absence of i(12p) was established in a control set consisting of 36 tumour-free samples. In a training set of 149 GCT samples, i(12p) was detectable in 133 tumours (89%), but not in 16 tumours (11%). In a test set containing 27 primary and metastatic GCTs, all 16 tumours with metastatic spread and/or somatic malignant transformation were successfully identified by the detection of i(12p). CONCLUSION: In summary, the qPCR assay presented here can help to identify, further characterise and assign a large proportion of histologically inconclusive malignancies to a GCT origin.

Role of cancer stem cell markers ALDH1, BCL11B, BMI-1, and CD44 in the prognosis of advanced HNSCC.

PURPOSE: Cancer stem cells (CSCs) are held accountable for the progress of head and neck squamous cell carcinoma (HNSCC). In the presented study, the authors evaluated the prognostic value of CSC markers in two particular HNSCC cohorts. METHODS: This two cohort study consisted of 85 patients with advanced stage HNSCC, treated with primary radio(chemo)therapy (pRCT), and 95 patients with HNSCC, treated with surgery and partially adjuvant radio(chemo)therapy. Overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) were assessed. Samples were assessed for the expression of different molecular stem cell markers (ALDH1, BCL11B, BMI­1, and CD44). RESULTS: In the pRCT cohort, none of the baseline patient and tumor features exhibited a statistically significant relation with survival in either the cohort or the human papillomavirus (HPV)-stratified subcohorts. High expression of BMI­1 significantly decreased OS and DFS, while high expression of CD44 decreased all modes of survival. Multivariate analysis showed significant prognostic influence for all tested CSC markers, with high BMI­1 and CD44 decreasing survival (BMI-1: OS, DFS, DSS; CD44: OS, DFS) and high ALDH1 and BCL11B showing a beneficial effect on survival (ALDH1: OS, DFS; BCL11B: OS, DSS). In the surgical cohort, classical prognosticators such as HPV status, R1 resection, and nodal status in HPV-negative HNSCC played a significant role, but the tested CSC markers showed no significant effect on prognosis. CONCLUSION: Although validation in independent cohorts is still needed, testing for CSC markers in patients with advanced or late stage HNSCC might be beneficial, especially if many comorbidities exist or disease is irresectable. The findings might guide the development and earlier use of targeted therapies in the future.

Prognostic impact of additional HPV diagnostics in 102 patients with p16-stratified advanced oropharyngeal squamous cell carcinoma.

PURPOSE: p16 overexpression was considered as surrogate marker to identify human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCCs). METHODS: 102 patients with advanced stage OPSCCs treated primarily by transoral lasermicrosurgery were included. Prognostic associations of p16- and HPV-status were analyzed separately and combined. RESULTS: In contrast to p16, the HPV-status resulted in no significant survival discrepancies (5-year overall survival (OS) HPV-positive 64.9%, HPV-negative 78.7%). Combining both markers, p16-positive (p16-positive/HPV-positive, p16-positive/HPV-negative) and p16-negative/HPV-negative groups demonstrated comparable high survival (OS 78.1% vs. 85.6% vs. 73.6%). Lowest survival was observed for patients with p16-negative/HPV-positive OPSCCs (OS 40.8%). Never smoking patients with p16-positive OPSCCs demonstrated the highest survival, whereas within former/current smokers with p16-positive and p16-negative disease it was comparable low (OS 90.0% vs. 63.0% vs. 57.4%). CONCLUSIONS: p16- and HPV-status should not be considered as equivalent markers for a better prognosis. Furthermore, they should not generally predominate patient associated factors like smoking.

EWSR1 translocation in primary hyalinising clear cell carcinoma of the thymus.

AIMS: In thymic carcinomas, focal clear cell change is a frequent finding. In addition to a prominent, diffuse clear cell morphology, some of these carcinomas show an exuberant hyalinised extracellular matrix, and therefore probably represent a separate entity. However, a characteristic genomic alteration remains elusive. We hypothesised that, analogous to hyalinising clear cell carcinomas of the salivary gland, hyalinising clear cell carcinomas of the thymus might also harbour EWSR1 translocations. METHODS AND RESULTS: We identified nine archived cases of thymic carcinoma with focal clear cell features and two cases that showed remarkable hyalinised stroma and prominent, diffuse clear cell morphology. These two cases expressed p40 and were negative for Pax8, CD5, and CD117. Programmed death-ligand 1 was highly positive in one case (70%), and negative in the other one. EWSR1 translocation was identified in both cases of hyalinising clear cell carcinoma, and was absent in all nine carcinomas that showed clear cell features without substantial hyalinisation. In one of the EWSR1-translocated cases, a fusion between exon 13 and exon 6 of EWSR1 and ATF1, respectively was identified by next-generation sequencing. CONCLUSIONS: These findings suggest that the EWSR1 translocation and possibly the EWSR1-ATF1 fusion might be unifying genomic alterations for thymic clear cell carcinomas with prominent hyalinised stroma, for which we propose the term 'hyalinising clear cell carcinoma of the thymus'. Because the immunophenotype is unspecific, testing for the EWSR1 translocation might be helpful in discriminating this entity from other thymic neoplasms or metastases, in particular those with clear cell change.

Optimized expression-based microdissection of formalin-fixed lung cancer tissue.

Analysis of specific DNA alterations in precision medicine of tumors is crucially important for molecular targeted treatments. Lung cancer is a prototypic example and one of the leading causes of cancer-related deaths worldwide. One major technical problem of detecting DNA alterations in tissue samples is cellular heterogeneity, that is, mixture of tumor and normal cells. Microdissection is an important tool to enrich tumor cells from heterogeneous tissue samples. However, conventional laser capture microdissection has several disadvantages like user-dependent selection of regions of interest (ROI), high costs for dissection systems and long processing times. ROI selection in expression-based microdissection (xMD) directly relies on cancer cell-specific immunostaining. Whole-slide irradiation leads to localized energy absorption at the sites of most intensive staining and melting of a membrane covering the slide, so that tumor cells can be isolated by removing the complete membrane. In this study, we optimized xMD of lung cancer tissue by enhancing staining intensity of tumor cell-specific immunostaining and processing of the stained samples. This optimized procedure did not alter DNA quality and resulted in enrichment of mutated EGFR DNA from lung adenocarcinoma specimens after xMD. We here also introduce a quality control protocol based on digital whole-slide scanning and image analysis before and after xMD to quantify selectivity and efficiency of the procedure. In summary, this study provides a workflow for xMD, adapted and tested for lung cancer tissue that can be used for lung tumor cell dissection before diagnostic or investigatory analyses.

Metastatic type A thymoma: morphological and genetic correlation.

AIMS: The vast majority of type A thymomas are diagnosed in tumour stages 1 or 2, and metastatic cases are exceedingly rare. The histological and genetic features of such metastatic type A thymomas have not been described in detail. METHODS AND RESULTS: Five metastatic type A thymomas in tumour stage Masaoka IVb that had been reviewed by a panel of expert pathologists were analysed using comparative genomic hybridization (CGH). Cases 1, 2 and 3 showed the prototypical morphology of type A thymomas with mainly solid growth patterns. These cases displayed only very subtle nuclear irregularities and slight nuclear crowding, but no other atypical features. Mitoses were absent. Cases 3 and 4, in contrast, had a distinctly atypical morphology. CGH revealed partially recurrent alterations in four cases (with and without atypical morphology), including gains on chromosome 1q (one case), 17q (two cases), chromosome 19 (three cases) and 22q (one case) and losses on chromosome 17p (two cases) and 22q (one case). CONCLUSION: Rare metastatic type A thymomas, both with typical and 'atypical' histological features, show partially recurrent genomic alterations that differ from the much more frequent localized and indolent tumours. The fact that these alterations were recurring points to a link between clinical behaviour and molecular features. Our findings may have implications for the management and treatment of such tumours.

Phylogenetic analysis of multiple FISH markers in oral tongue squamous cell carcinoma suggests that a diverse distribution of copy number changes is associated with poor prognosis.

Oral tongue squamous cell carcinoma (OTSCC) is associated with poor prognosis. To improve prognostication, we analyzed four gene probes (TERC, CCND1, EGFR and TP53) and the centromere probe CEP4 as a marker of chromosomal instability, using fluorescence in situ hybridization (FISH) in single cells from the tumors of sixty-five OTSCC patients (Stage I, n = 15; Stage II, n = 30; Stage III, n = 7; Stage IV, n = 13). Unsupervised hierarchical clustering of the FISH data distinguished three clusters related to smoking status. Copy number increases of all five markers were found to be correlated to non-smoking habits, while smokers in this cohort had low-level copy number gains. Using the phylogenetic modeling software FISHtrees, we constructed models of tumor progression for each patient based on the four gene probes. Then, we derived test statistics on the models that are significant predictors of disease-free and overall survival, independent of tumor stage and smoking status in multivariate analysis. The patients whose tumors were modeled as progressing by a more diverse distribution of copy number changes across the four genes have poorer prognosis. This is consistent with the view that multiple genetic pathways need to become deregulated in order for cancer to progress.

High-grade acute organ toxicity and p16(INK4A) expression as positive prognostic factors in primary radio(chemo)therapy for patients with head and neck squamous cell carcinoma.

BACKGROUND: Superior treatment response and survival for patients with human papilloma virus (HPV)-positive head and neck cancer (HNSCC) are documented in clinical studies. However, the relevance of high-grade acute organ toxicity (HGAOT), which has also been correlated with improved prognosis, has attracted scant attention in HPV-positive HNSCC patients. Hence we tested the hypothesis that both parameters, HPV and HGAOT, are positive prognostic factors in patients with HNSCC treated with definite radiotherapy (RT) or radiochemotherapy (RCT). PATIENTS AND METHODS: Pretreatment tumor tissue and clinical records were available from 233 patients receiving definite RT (62 patients) or RCT (171 patients). HPV infection was analysed by means of HPV DNA detection or p16(INK4A) expression; HGAOT was defined as the occurrence of acute organ toxicity >grade 2 according to the Common Toxicity Criteria. Both variables were correlated with overall survival (OS) using Cox proportional hazards regression. RESULTS: Positivity for HPV DNA (44 samples, 18.9 %) and p16(INK4A) expression (102 samples, 43.8 %) were significantly correlated (p < 0.01), and HGAOT occurred in 77 (33 %) patients. Overall, the 5-year OS was 23 %; stratified for p16(INK4A) expression and HGAOT, OS rates were 47 %, 42 %, 20 % and 10 % for patients with p16(INK4A) expression and HGAOT, patients with HGAOT only, patients with p16(INK4A) expression only, and patients without p16(INK4A) expression or HGAOT, respectively. After multivariate testing p16(INK4A) expression (p = 0.003) and HGAOT (p < 0.001) were significantly associated with OS. CONCLUSION: P16(INK4A) expression and HGAOT are independent prognostic factors for OS of patients with HNSCC, whereas p16(INK4A) expression is particularly important for patients without HGAOT.

High frequency of MYC gene amplification is a common feature of radiation-induced sarcomas. Further results from EORTC STBSG TL 01/01.

Irradiation is a major causative factor among the small subgroup of sarcomas with a known etiology. The prognosis of radiation-induced sarcomas (RIS) is significantly worse than that of their spontaneous counterparts. The most frequent histological subtypes include undifferentiated pleomorphic sarcomas, angiosarcomas, and leiomyosarcomas. A high frequency of MYC amplifications in radiation-induced angiosarcomas, but not in primary angiosarcomas, has recently been described. To investigate whether MYC amplifications are also frequent in RIS other than angiosarcomas, we analyzed the MYC amplification status of 83 RIS and 192 sporadic sarcomas by fluorescence in situ hybridization. We found significantly higher numbers of MYC amplifications in RIS than in sporadic sarcomas (P < 0.0001), especially in angiosarcomas, undifferentiated pleomorphic sarcomas, and leiomyosarcomas. Angiosarcomas were special in that MYC amplifications were particularly frequent and always high level, while other RIS showed low-level amplifications. We conclude that MYC amplifications are a frequent feature of RIS as a group and may contribute to the biology of these tumors.

Non-Mutational Key Features in the Biology of Thymomas.

Thymomas (THs) are a unique group of heterogeneous tumors of the thymic epithelium. In particular, the subtypes B2 and B3 tend to be aggressive and metastatic. Radical tumor resection remains the only curative option for localized tumors, while more advanced THs require multimodal treatment. Deep sequencing analyses have failed to identify known oncogenic driver mutations in TH, with the notable exception of the GTF2I mutation, which occurs predominantly in type A and AB THs. However, there are multiple alternative non-mutational mechanisms (e.g., perturbed thymic developmental programs, metabolism, non-coding RNA networks) that control cellular behavior and tumorigenesis through the deregulation of critical molecular pathways. Here, we attempted to show how the results of studies investigating such alternative mechanisms could be integrated into a current model of TH biology. This model could be used to focus ongoing research and therapeutic strategies.

Association between spatial distribution of leukocyte subsets and clinical presentation of head and neck squamous cell carcinoma.

Background: Interactions between tumor cells and cells in the microenvironment contribute to tumor development and metastasis. The spatial arrangement of individual cells in relation to each other influences the likelihood of whether and how these cells interact with each other. Methods: This study investigated the effect of spatial distribution on the function of leukocyte subsets in the microenvironment of human head and neck squamous cell carcinoma (HNSCC) using multiplex immunohistochemistry (IHC). Leukocyte subsets were further classified based on analysis of two previously published HNSCC single-cell RNA datasets and flow cytometry (FC). Results: IHC revealed distinct distribution patterns of leukocytes differentiated by CD68 and CD163. While CD68hiCD163lo and CD68hiCD163hi cells accumulated near tumor sites, CD68loCD163hi cells were more evenly distributed in the tumor stroma. PD-L1hi and PD-1hi cells accumulated predominantly around tumor sites. High cell density of PD-L1hi CD68hiCD163hi cells or PD-1hi T cells near the tumor site correlated with improved survival. FC and single cell RNA revealed high variability within the CD68/CD163 subsets. CD68hiCD163lo and CD68hiCD163hi cells were predominantly macrophages (MΦ), whereas CD68loCD163hi cells appeared to be predominantly dendritic cells (DCs). Differentiation based on CD64, CD80, CD163, and CD206 revealed that TAM in HNSCC occupy a broad spectrum within the classical M1/M2 polarization. Notably, the MΦ subsets expressed predominantly CD206 and little CD80. The opposite was observed in the DC subsets. Conclusion: The distribution patterns and their distinct interactions via the PD-L1/PD-1 pathway suggest divergent roles of CD68/CD163 subsets in the HNSCC microenvironment. PD-L1/PD-1 interactions appear to occur primarily between specific cell types close to the tumor site. Whether PD-L1/PD-1 interactions have a positive or negative impact on patient survival appears to depend on both the spatial localization and the entity of the interacting cells. Co-expression of other markers, particularly CD80 and CD206, supports the hypothesis that CD68/CD163 IHC subsets have distinct functions. These results highlight the association between spatial leukocyte distribution patterns and the clinical presentation of HNSCC.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup's biology better. METHODS: A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. RESULTS: High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. CONCLUSIONS: Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.

Regulation and Therapeutic Targeting of MTHFD2 and EZH2 in KRAS-Mutated Human Pulmonary Adenocarcinoma.

Activating KRAS mutations occur in about 30% of pulmonary adenocarcinoma (AC) cases and the discovery of specific inhibitors of G12C-mutated KRAS has considerably improved the prognosis for a subgroup of about 14% of non-small cell lung cancer (NSCLC) patients. However, even in patients with a KRAS G12C mutation, the overall response rate only reaches about 40% and mutations other than G12C still cannot be targeted. Despite the fact that one-carbon metabolism (1CM) and epigenetic regulation are known to be dysregulated by aberrant KRAS activity, we still lack evidence that co-treatment with drugs that regulate these factors might ameliorate response rates and patient prognosis. In this study, we show a direct dependency of Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and Enhancer of Zeste Homolog 2 (EZH2) expression on mutationally activated KRAS and their prognostic relevance in KRAS-mutated AC. We show that aberrant KRAS activity generates a vulnerability of AC cancer cell lines to both MTHFD2 and EZH2 inhibitors. Importantly, co-inhibition of both factors was synergistically effective and comparable to KRASG12C inhibition alone, paving the way for their use in a therapeutic approach for NSCLC cancer patients.