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Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Surgical resection remains the gold standard of treatment for early-stage lung cancer. Several risk models exist to predict postoperative morbidity and mortality. Psoas muscle sarcopenia has already successfully been used for morbidity prediction in lung transplantation and is not yet included in the available risk scores for pulmonary resections. We hypothesized that the skeletal muscle index and mediastinal adipose tissue might also have an impact on postoperative outcomes after primary surgery for primary lung cancer. The institutional database was queried for patients with primary lung cancer who were treated with primary lobectomy or segmentectomy between February 2009 and November 2018. In total, 311 patients were included for analysis. Patients receiving neo-/adjuvant chemotherapy or with a positive nodal status were excluded to rule out any morbidity or mortality due to (neo-)adjuvant treatment. Sarcopenia was defined as a skeletal muscle index of 2/m2 for women and 2/m2 for men. Mediastinal adipose tissue was defined with a radiodensity of −150 to −30 Hounsfield units. Sarcopenia was diagnosed in 78 (25.1%) of the 311 patients. Male patients were significantly more likely to suffer from sarcopenia (31.5% vs. 18.1%, p = 0.009). Comorbidities, lung function, tumour histology, pathologic tumour staging, mediastinal adipose tissue and age did not differ between groups with or without sarcopenia. Sarcopenic patients had a significantly longer length of stay, with 13.0 days vs. 9.5 (p = 0.003), and a higher rate of any postoperative complications (59.0% vs. 44.6%, p = 0.036). There was no difference in recurrence rate. Five-year overall survival was significantly better in the patient cohort without sarcopenia (75.6% vs. 64.5%, p = 0.044). Mediastinal adipose tissue showed no significant impact on length of stay, postoperative complications, recurrence rate, morbidity or survival. Sarcopenia, quantified with the skeletal muscle index, is shown to be a risk factor for postoperative morbidity and reduced survival in primary lung cancer. Efforts should be taken to pre-emptively screen for sarcopenia and start countermeasures (e.g., physical prehabilitation, protein-rich nutrition, etc.) during the preoperative workup phase.

Drug resistance is a common cause of therapy failure in head and neck squamous cell carcinoma (HNSCC). One approach to tackling it is by targeting fundamental cellular processes, such as translation. The eukaryotic translation initiation factor 2α (EIF2α) is a key player in canonical translation initiation and integrates diverse stress signals; when phosphorylated, it curbs global protein synthesis. This study evaluates EIF2α expression and phosphorylation in HNSCC. A small-molecule inhibitor of EIF2α dephosphorylation, salubrinal, was tested in vitro, followed by viability assays, flow cytometry, and immunoblot analyses. Patient-derived 3D tumor spheres (PD3DS) were cultured with salubrinal and their viability assessed. Lastly, salubrinal was evaluated with standard-of-care chemotherapeutics. Our analysis of RNA and proteomics data shows elevated EIF2α expression in HNSCC. Immunohistochemical staining reveals increasing EIF2α abundance from premalignant lesions to invasive and metastatic carcinoma. In immunoblots from intraoperative samples, EIF2α expression and steady-state phosphorylation are higher in HNSCC than in neighboring normal tissue. Inhibition of EIF2α dephosphorylation decreases HNSCC cell viability and clonogenic survival and impairs the G1/S transition. Salubrinal also decreases the viability of PD3DS and acts synergistically with cisplatin, 5-fluorouracil, bleomycin, and proteasome inhibitors. Our results indicate that pharmacological inhibition of EIF2α dephosphorylation is a potential therapeutic strategy for HNSCC.

Background: Chronic inflammation is a significant factor in colorectal cancer (CRC) development, especially in colitis-associated CRC (CAC). T-cell exhaustion is known to influence inflammatory bowel disease (IBD) progression and antitumor immunity in IBD patients. This study aimed to identify unique immune infiltration characteristics in CAC patients. Methods: We studied 20 CAC and 20 sporadic CRC (sCRC) patients, who were matched by tumor stage, grade, and location. Immunohistochemical staining targeted various T-cell markers (CD3, CD4, CD8, and FOXP3), T-cell exhaustion markers (TOX and TIGIT), a B-cell marker (CD20), and a neutrophil marker (CD66b) in tumor and tumor-free mucosa from both groups. The quantification of the tumor immune stroma algorithm assessed immune-infiltrating cells. Results: CAC patients had significantly lower TOX+ cell infiltration than sCRC in tumors (p = 0.02) and paracancerous tissues (p < 0.01). Right-sided CAC showed increased infiltration of TOX+ cells (p = 0.01), FOXP3+ regulatory T-cells (p < 0.01), and CD20+ B-cells (p < 0.01) compared to left-sided CAC. In sCRC, higher tumor stages (III and IV) had significantly lower TIGIT+ infiltrate than stages I and II. In CAC, high CD3+ (p < 0.01) and CD20+ (p < 0.01) infiltrates correlated with improved overall survival. In sCRC, better survival was associated with decreased TIGIT+ cells (p < 0.038) and reduced CD8+ infiltrates (p = 0.02). Conclusion: In CAC, high CD3+ and CD20+ infiltrates relate to improved survival, while this association is absent in sCRC. The study revealed marked differences in TIGIT and TOX expression, emphasizing distinctions between CAC and sCRC. T-cell exhaustion appears to have a different role in CAC development.

Patients with locally advanced head and neck squamous cell carcinoma (HNSCC) frequently require primary radiochemotherapy (RCT). Despite intensity modulation, the desired radiation-induced effects observed in HNSCC may also be observed as side effects in healthy tissue, e.g., the sternocleidomastoid muscle (SCM). These side effects (e.g., tissue fibrosis) depend on the interval between the completion of RCT and restaging CT. For salvage surgery, the optimal time window for surgery is currently clinically postulated at between 6 and 12 weeks after completion of RCT. Thus, no extensive tissue fibrosis is to be expected. This interval is based on clinical studies exploring surgical complications. Studies directly exploring radiation-induced changes of the SCM in HNSCC patients are sparse. The present study quantified tissue alterations in the SCM and paravertebral musculature (PVM) after RCT, applying radiomics to determine the optimal time window for salvage surgery. Three radiomic key parameters, (1) volume, (2) mean positivity of pixels (MPP), and (3) uniformity, were extracted with mint LesionTM in the staging CTs and restaging CTs of 98 HNSCC patients. Of these, 25 were female, the mean age was 62 (±9.6) years, and 80.9% were UICC Stage IV. The mean restaging interval was 55 (±28; range 29–229) days. Only the mean volume significantly decreased after RCT, from 9.0 to 8.4 and 96.5 to 91.9 mL for the SCM and PVM, respectively (both p = 0.007, both Cohen’s d = 0.28). In addition, the mean body mass index (BMI) decreased from 23.9 (±4.2) to 21.0 (±3.6) kg/m² (p < 0.001; Cohen’s d = 0.9). The mean BMI decreased significantly and was correlated with the volume decrease for the SCM (r = 0.27; p = 0.007) and PVM (r = 0.41; p < 0.001). If t-test p-values were adjusted for the BMI decrease, no significant change in volumes for the SCM and PVM was observed (both p > 0.05). The present data support the clinically postulated optimal interval for salvage surgery of 6 to 12 weeks.

Cholangiocarcinoma (CCA) are characterized by their desmoplastic and hypervascularized tumor microenvironment (TME), which is mainly composed of tumor cells and cancer-associated fibroblasts (CAFs). CAFs play a pivotal role in general and CCA tumor progression, angiogenesis, metastasis, and the development of treatment resistance. To our knowledge, no continuous human in vivo-like co-culture model is available for research. Therefore, we aimed to establish a new model system (called MUG CCArly) that mimics the desmoplastic microenvironment typically seen in CCA. Proteomic data comparing the new CCA tumor cell line with our co-culture tumor model (CCTM) indicated a higher gene expression correlation of the CCTM with physiological CCA characteristics. A pro-angiogenic TME that is typically observed in CCA could also be better simulated in the CCTM group. Further analysis of secreted proteins revealed CAFs to be the main source of these angiogenic factors. Our CCTM MUG CCArly represents a new, reproducible, and easy-to-handle 3D CCA model for preclinical studies focusing on CCA-stromal crosstalk, tumor angiogenesis, and invasion, as well as the immunosuppressive microenvironment and the involvement of CAFs in the way that drug resistance develops.

Background: Surgery is the standard treatment in localized renal cell carcinoma (RCC). Pembrolizumab is now approved for adjuvant therapy in high-risk patients. However, inhomogeneity of studies gives ambiguity which patient benefit most from adjuvant therapy. A high infiltration of CD8+ T cells is known to be linked with poor prognosis in RCC. CXCR3 is a key player of CD8+ T cell differentiation and infiltration. We aimed to evaluate CXCR3 as a potential marker for predicting recurrence. Methods: CXCR3 and immune cell subsets (CD4, CD8, CD68 and FoXP3) were measured on RCC samples by multiplex immunofluorescence (mIF) staining. Cellular localization of CXCR3 was evaluated using single-cell RNA analysis on a publicly available dataset. Results: Tumor samples of 42 RCC patients were analyzed, from which 59.5% were classified as clear-cell RCC and of which 20 had recurrence. Single-cell RNA analysis revealed that CXCR3 was predominantly expressed in intratumoral T cells and dendritic cells. CXCR3 expression was higher in advanced tumors stages (p = 0.0044) and grade (p = 0.0518), correlating significantly with a higher CD8+ T cell expression (p < 0.001). Patients with CXCR3high RCCs had also a significant shorter RFS compared to CXCR3low (median: 78 vs. 147 months, p = 0.0213). In addition, also tumor stage pT3/4 (p < 0.0001) as well as grade G3/4 (p = 0.0008) negatively influenced RFS. Conclusion: CXCR3high cell density was associated with high T cell infiltration and advanced tumor stage, worsening RFS in surgically resected RCC patients. Beside its prognostic value, CXCR3 might be a predictive biomarker to guide therapy decision for adjuvant therapy in localized RCC.

The development and progression of colorectal cancer (CRC) are known to be affected by the interplay between tumor and immune cells. However, the impact of CRC cells on the systemic immunity has yet to be elucidated. We aimed to comprehensively evaluate the circulating immune subsets and transcriptional profiles of CRC patients. In contrast to healthy controls (HCs), CRC patients had a lower percentage of B and T lymphocytes, T helper (Th) cells, non-classical monocytes, dendritic cells, and a higher proportion of polymorphonuclear myeloid-derived suppressor cells, as well as a reduced expression of CD69 on NK cells. Therefore, CRC patients exhibit a more evident systemic immune suppression than HCs. A diagnostic model integrating seven immune subsets was constructed to distinguish CRC patients from HCs with an AUC of 1.000. Moreover, NR3C2, CAMK4, and TRAT1 were identified as candidate genes regulating the number of Th cells in CRC patients. The altered composition of circulating immune cells in CRC could complement the regional immune status of the tumor microenvironment and contribute to the discovery of immune-related biomarkers for the diagnosis of CRC.

Aim: The aim of this study was to assess the effects of including somatostatin receptor agonist (SSTR) PET imaging in meningioma radiotherapy planning by means of changes in inter-observer variability (IOV). Further, the possibility of using threshold-based delineation approaches for semiautomatic tumor volume definition was assessed. Patients and Methods: Sixteen patients with meningioma undergoing fractionated radiotherapy were delineated by five radiation oncologists. IOV was calculated by comparing each delineation to a consensus delineation, based on the simultaneous truth and performance level estimation (STAPLE) algorithm. The consensus delineation was used to adapt a threshold-based delineation, based on a maximization of the mean Dice coefficient. To test the threshold-based approach, seven patients with SSTR-positive meningioma were additionally evaluated as a validation group. Results: The average Dice coefficients for delineations based on MRI alone was 0.84 ± 0.12. For delineation based on MRI + PET, a significantly higher dice coefficient of 0.87 ± 0.08 was found (p < 0.001). The Hausdorff distance decreased from 10.96 ± 11.98 mm to 8.83 ± 12.21 mm (p < 0.001) when adding PET for the lesion delineation. The best threshold value for a threshold-based delineation was found to be 14.0% of the SUVmax, with an average Dice coefficient of 0.50 ± 0.19 compared to the consensus delineation. In the validation cohort, a Dice coefficient of 0.56 ± 0.29 and a Hausdorff coefficient of 27.15 ± 21.54 mm were found for the threshold-based approach. Conclusions: SSTR-PET added to standard imaging with CT and MRI reduces the IOV in radiotherapy planning for patients with meningioma. When using a threshold-based approach for PET-based delineation of meningioma, a relatively low threshold of 14.0% of the SUVmax was found to provide the best agreement with a consensus delineation.

Most metastatic colorectal cancer (mCRC) patients succumb to refractory disease due to secondary chemotherapy resistance. To elucidate the molecular changes associated with secondary resistance, we recruited 64 patients with mCRC and hepatic metastases before standard first-line chemotherapy between 2014 and 2018. We subjected DNA from primary tumor specimens (P), hepatic metastasis specimens after treatment (M), and liquid biopsies (L) taken prior to (pre), during (intra), and after (post) treatment to next generation sequencing. We performed Nanostring expression analysis in P and M specimens. Comparative bioinformatics and statistical analysis revealed typical mutational patterns with frequent alterations in TP53, APC, and KRAS in P specimens (n = 48). P and pre-L (n = 42), as well as matched P and M (n = 30), displayed a similar mutation spectrum. In contrast, gene expression profiles classified P (n = 31) and M (n = 23), distinguishable by up-regulation of immune/cytokine receptor and autophagy programs. Switching of consensus molecular subtypes from P to M occurred in 58.3% of cases. M signature genes SFRP2 and SPP1 associated with inferior survival, as validated in an independent cohort. Molecular changes during first-line treatment were detectable by expression profiling rather than by mutational tumor and liquid biopsy analyses. SFRP2 and SPP1 may serve as biomarkers and/or actionable targets.

Leukemia is the most frequent malignancy in children and adolescents, with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) as the most common subtypes. Minimal residual disease (MRD) measured by flow cytometry (FCM) has proven to be a strong prognostic factor in ALL as well as in AML. Machine learning techniques have been emerging in the field of automated MRD quantification with the objective of superseding subjective and time-consuming manual analysis of FCM-MRD data. In contrast to ALL, where supervised multi-class classification methods have been successfully deployed for MRD detection, AML poses new challenges: AML is rarer (with fewer available training data) than ALL and much more heterogeneous in its immunophenotypic appearance, where one-class classification (anomaly detection) methods seem more suitable. In this work, a new semi-supervised approach based on the UMAP algorithm for MRD detection utilizing only labels of blast free FCM samples is presented. The method is tested on a newly gathered set of AML FCM samples and results are compared to state-of-the-art methods. We reach a median F1-score of 0.794, while providing a transparent classification pipeline with explainable results that facilitates inter-disciplinary work between medical and technical experts. This work shows that despite several issues yet to overcome, the merits of automated MRD quantification can be fully exploited also in AML.

While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involvement. To this end, we employed a multi-omics data mining approach to define a multiplex imaging panel and developed DeepFLEX, a pipeline for subsequent multiplex image analysis, whereby we constructed a single-cell atlas of over 35,000 disseminated tumor cells (DTCs) and cells of their microenvironment in the metastatic bone marrow niche. Further, we independently profiled the transcriptome of a cohort of 38 patients with and without bone marrow metastasis. Our results revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. Importantly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most prominently an immature neutrophil/granulocytic myeloid-derived suppressor-like cell type. The presented findings indicate that metastatic tumor cells shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.

Resistance of tumor cells to chemoradiotherapy represents a fundamental problem in clinical oncology. The underlying mechanisms are actively debated. Here we show that blocking inflammatory cytokine receptor signaling via STAT3 re-sensitized treatment-refractory cancer cells and abolished tumor growth in a xenograft mouse model when applied together with chemoradiotherapy. STAT3 executed treatment resistance by triggering the expression of RBPJ, the key transcriptional regulator of the NOTCH pathway. The mandatory RBPJ interaction partner, NOTCH intracellular domain, was provided by tumor cell-intrinsic expression of NOTCH ligands that caused tonic NOTCH proteolysis. In fact, NOTCH inhibition phenocopied the effect of blocking STAT3 signaling. Moreover, genetic profiling of rectal cancer patients revealed the importance of the STAT3/NOTCH axis as NOTCH expression correlated with clinical outcome. Our data uncovered an unprecedented signal alliance between inflammation and cellular development that orchestrated resistance to chemoradiotherapy. Clinically, our findings allow for biomarker-driven patient stratification and offer novel treatment options.

The role of gut microbiota in colorectal cancer is subject to extensive research. Before usage of biorepositories for microbiome studies, it is crucial to evaluate technical feasibility of microbiome profiling from various biospecimens. The aim of this study was to assess the feasibility of DNA-extraction and microbiome profiling of samples from different sample sites, tissue sites and storage duration of a colorectal cancer biobank. Mucosa samples, mucosal scrapings and feces as well as different tissue sites (tumor, normal mucosa) were analyzed. 16S rRNA gene-based microbiome profiling with taxonomic assignment was performed on the Illumina MiSeq (Illumina, San Diego, USA) platform from stored snap frozen samples. For statistical analysis, α- and β-diversity measures, PCoA, permutational multivariate analysis of variance and graphical representation were performed. Microbiome analysis could be successfully performed in most of the samples (overall 93.3%) with sufficient numbers of high-quality reads. There were no differences between sample sites, while in some measures significant differences were found between tumor and normal mucosa (α-diversity, Shannon/Simpson Indices p = 0.028/0.027, respectively). Samples stored for up to eight years were used and storage conditions had no significant influence on the results. Tumor and tissue samples of a biobank stored long term can be successfully used for microbiome analysis. As large sample sizes are needed for association studies to evaluate microbial impact on tumorigenesis or progression of colorectal cancer, an already established biorepository may be a useful alternative to prospective clinical studies.

Pedicle clamping (PC) during liver resection for colorectal metastases (CRLM) is used to reduce blood loss and allogeneic blood transfusion (ABT). The effect on long-term oncologic outcomes is still under debate. A retrospective analysis of the impact of PC on ABT-demand regarding overall (OS) and recurrence-free survival (RFS) in 336 patients undergoing curative resection for CRLM was carried out. Survival analysis was performed by both univariate and multivariate methods and propensity-score (PS) matching. PC was employed in 75 patients (22%). No increased postoperative morbidity was monitored. While the overall ABT-rate was comparable (35% vs. 37%, p = 0.786), a reduced demand for more than two ABT-units was observed (p = 0.046). PC-patients had better median OS (78 vs. 47 months, p = 0.005) and RFS (36 vs. 23 months, p = 0.006). Multivariate analysis revealed PC as an independent prognostic factor for OS (HR = 0.60; p = 0.009) and RFS (HR = 0.67; p = 0.017). For PC-patients, 1:2 PS-matching (N = 174) showed no differences in the overall ABT-rate compared to no-PC-patients (35% vs. 40%, p = 0.619), but a trend towards reduced transfusion requirement (>2 ABT-units: 9% vs. 21%, p = 0.052; >4 ABT-units: 2% vs. 11%, p = 0.037) and better survival (OS: 78 vs. 44 months, p = 0.088; RFS: 36 vs. 24 months; p = 0.029). Favorable long-term outcomes and lower rates of increased transfusion demand were observed in patients with PC undergoing resection for CRLM. Further prospective evaluation of potential oncologic benefits of PC in these patients may be meaningful.

Tumor-selective replicating “oncolytic” viruses are novel and promising tools for immunotherapy of cancer. However, despite their first success in clinical trials, previous experience suggests that currently used oncolytic virus monotherapies will not be effective enough to achieve complete tumor responses and long-term cure in a broad spectrum of cancers. Nevertheless, there are reasonable arguments that suggest advanced oncolytic viruses will play an essential role as enablers of multi-stage immunotherapies including established systemic immunotherapies. Oncolytic adenoviruses (oAds) display several features to meet this therapeutic need. oAds potently lyse infected tumor cells and induce a strong immunogenic cell death associated with tumor inflammation and induction of antitumor immune responses. Furthermore, established and versatile platforms of oAds exist, which are well suited for the incorporation of heterologous genes to optimally exploit and amplify the immunostimulatory effect of viral oncolysis. A considerable spectrum of functional genes has already been integrated in oAds to optimize particular aspects of immune stimulation including antigen presentation, T cell priming, engagement of additional effector functions, and interference with immunosuppression. These advanced concepts have the potential to play a promising future role as enablers of multi-stage immunotherapies involving adoptive cell transfer and systemic immunotherapies.

Open orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these drawbacks, this study aimed to evaluate the establishment of a minimally invasive model using murine colonoscopy. CT26 and MC38 CRC cells of different concentrations were injected into BALB/C and C57BL/6J mice, respectively. Follow-up endoscopies were performed to assign an endoscopic score to tumor growth. Gross autopsy, histologic and immuno-histochemical evaluation, and immune scoring were performed. To describe the learning curve of the procedures, a performance score was given. Local tumor growth with colorectal wall infiltration, luminal ulceration, the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. The tumors showed cytoplasmic immuno-staining for CK20. Compared to the MC38/C57BL/6J model, tumorigenicity and immunogenicity of the CT26/BALB/C model were higher. Tumor volume correlated with the endoscopic score. This endoscopy-guided orthotopic mouse model is easy to learn and quick to establish. It features early metastasis and enables the study of interactions with the immune system. When specific cell concentrations and cell lines are applied, controlled local tumor growth and metastasis can be achieved within short observation periods.

Tumor heterogeneity is considered to be a hallmark of glioblastoma (GBM). Only more recently, it has become apparent that GBM is not only heterogeneous between patients (intertumoral heterogeneity) but more importantly, also within individual patients (intratumoral heterogeneity). In this study, we focused on assessing intratumoral heterogeneity. For this purpose, the heterogeneity of 38 treatment-naïve GBM was characterized by immunohistochemistry. Perceptible areas were rated for ALDH1A3, EGFR, GFAP, Iba1, Olig2, p53, and Mib1. By clustering methods, two distinct groups similar to subtypes described in literature were detected. The classical subtype featured a strong EGFR and Olig2 positivity, whereas the mesenchymal subtype displayed a strong ALDH1A3 expression and a high fraction of Iba1-positive microglia. 18 tumors exhibited both subtypes and were classified as “subtype-heterogeneous”, whereas the areas of the other tumors were all assigned to the same cluster and named “subtype-dominant”. Results of epigenomic analyses corroborated these findings. Strikingly, the subtype-heterogeneous tumors showed a clearly shorter overall survival compared to subtype-dominant tumors. Furthermore, 21 corresponding pairs of primary and recurrent GBM were compared, showing a dominance of the mesenchymal subtype in the recurrent tumors. Our study confirms the prognostic impact of intratumoral heterogeneity in GBM, and more importantly, makes this hallmark assessable by routine diagnostics.

While a shift in energy metabolism is essential to cancers, the knowledge about the involvement of the mitochondrial genome in tumorigenesis and progression in oral squamous cell carcinoma (OSCC) is still very limited. In this study, we evaluated 37 OSCC tumors and the corresponding benign mucosa tissue pairs by deep sequencing of the complete mitochondrial DNA (mtDNA). After extensive quality control, we identified 287 variants, 137 in tumor and 150 in benign samples exceeding the 1% threshold. Variant heteroplasmy levels were significantly increased in cancer compared to benign tissues (p = 0.0002). Furthermore, pairwise high heteroplasmy frequency difference variants (∆HF% > 20) with potential functional impact were increased in the cancer tissues (p = 0.024). Fourteen mutations were identified in the protein-coding region, out of which thirteen were detected in cancer and only one in benign tissue. After eight years of follow-up, the risk of mortality was higher for patients who harbored at least one ∆HF% > 20 variant in mtDNA protein-coding regions relative to those with no mutations (HR = 4.6, (95%CI = 1.3–17); p = 0.019 in primary tumor carriers). Haplogroup affiliation showed an impact on survival time, which however needs confirmation in a larger study. In conclusion, we observed a significantly higher accumulation of somatic mutations in the cancer tissues associated with a worse prognosis.

Werner syndrome gene (WRN) contributes to DNA repair. In cancer, WRN mutations (WRN-mut) lead to genomic instability. Thus, WRN is a promising target in cancers with microsatellite instability (MSI). We assessed this study to investigate the molecular profile of WRN-mut in colorectal cancer (CRC). Tumor samples were analyzed using next-generation sequencing (NGS) in-situ hybridization and immunohistochemistry. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. Determination of tumor mismatch repair (MMR) or microsatellite instability (MSI) status was conducted by fragment analysis. WRN-mut were detected in 80 of 6854 samples (1.2%). WRN-mut were more prevalent in right-sided compared to left-sided CRC (2.5% vs. 0.7%, p < 0.0001). TMB, PD-L1 and MSI-H/dMMR were significantly higher in WRN-mut than in WRN wild-type (WRN-wt). WRN-mut were associated with a higher TMB in the MSI-H/dMMR and in the MSS (microsatellite stable) subgroups. Several genetic differences between WRN-mut and WRN-wt CRC were observed, i.e., TP53 (47% vs. 71%), KRAS (34% vs. 49%) and APC (56% vs. 73%). This is the largest molecular profiling study investigating the genetic landscape of WRN-mut CRCs so far. A high prevalence of MSI-H/dMMR, higher TMB and PD-L1 in WRN-mut tumors were observed. Our data might serve as an additional selection tool for trials testing immune checkpoint antibodies in WRN-mut CRC.

Evidence has accumulated asserting the importance of cullin-RING (really interesting new gene) ubiquitin ligases (CRLs) and their regulator Cullin-associated neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) dissociated protein 1 (Cand1) in various cancer entities. However, the role of Cand1 in prostate cancer (PCa) has not been intensively investigated so far. Thus, in the present study, we aimed to assess the relevance of Cand1 in the clinical and preclinical setting. Immunohistochemical analyses of radical prostatectomy specimens of PCa patients showed that Cand1 protein levels are elevated in PCa compared to benign areas. In addition, high Cand1 levels were associated with higher Gleason Scores, as well as higher tumor recurrence and decreased overall survival. In line with clinical findings, in vitro experiments in different PCa cell lines revealed that knockdown of Cand1 reduced cell viability and proliferation and increased apoptosis, therefore underlining its role in tumor progression. We also found that the cyclin-dependent kinase inhibitor p21 is significantly upregulated upon downregulation of Cand1. Using bioinformatic tools, we detected genes encoding for proteins linked to mRNA turnover, protein polyubiquitination, and proteasomal degradation to be significantly upregulated in Cand1high tumors. Next generation sequencing of PCa cell lines resistant to the anti-androgen enzalutamide revealed that Cand1 is mutated in enzalutamide-resistant cells, however, with little functional and clinically relevant impact in the process of resistance development. To summarize the present study, we found that high Cand1 levels correlate with PCa aggressiveness.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34 + /CD38 - MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34 + /CD38 - MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.