Clinicopathological characterization of next-generation sequencing detected mutations in lung cancers-a single-center experience.

Background: Despite many advances in molecular procedures many lung cancer patients do not receive full panel testing. This can limit the comprehensive understanding of their disease and potentially hinder personalized treatment options. Methods: In this retrospective analysis, we used results from next-generation sequencing (NGS) testing of 154 patients with adenocarcinoma (AC) or squamous cell carcinoma (SCC) of the lung treated at the University Hospital, Ludwig-Maximilians Universität (LMU) Munich between 2018 and 2021. We compared different clinicopathological features and patients' baseline characteristics with results of NGS testing. We used t-test and analysis of variance (ANOVA) to compare metric- and χ2-test and Fisher's exact test to compare categorical variables. Results: NGS testing found mutations in 107 (69.5%) patients; 44 patients (28.6%) had more than one mutation. The majority (79.2%) of patients had AC and 64.9% were metastasized at diagnosis. Patients with detected mutations had significantly higher PD-L1 expression than those without mutations (36.4% vs. 19.2%, P=0.005). Mean PD-L1 expression also differed between different mutations ranging from 24.0% in EGFR to 56.8% in patients with MET alterations, and increased with the number of different mutations (P=0.07). EGFR mutations were significantly more common in females compared to males (22.9% vs. 9.5%, P=0.04) and PIK3CA mutations significantly more common in SCC (21.9% vs. 2.5%, P=0.004). We found 23 different mutations in AC and 13 different gene mutations in SCC. Conclusions: Mutation profiles differed by histological type and metastases status and were significantly associated with PD-L1 expression. In the context of limited resources, our results may help prioritize patient for testing when tissue material and funding is limited.

Identification of a gene expression signature associated with brain metastasis in colorectal cancer.

PURPOSE: Brain metastasis (BM) in colorectal cancer (CRC) is a rare event with poor prognosis. Apart from (K)RAS status and lung and bone metastasis no biomarkers exist to identify patients at risk. This study aimed to identify a gene expression signature associated with colorectal BM. METHODS: Three patient groups were formed: 1. CRC with brain metastasis (BRA), 2. exclusive liver metastasis (HEP) and, 3. non-metastatic disease (M0). RNA was extracted from primary tumors and mRNA expression was measured using a NanoString Panel (770 genes). Expression was confirmed by qPCR in a validation cohort. Statistical analyses including multivariate logistic regression followed by receiver operating characteristic (ROC) analysis were performed. RESULTS: EMILIN3, MTA1, SV2B, TMPRSS6, ACVR1C, NFAT5 and SMC3 were differentially expressed in BRA and HEP/M0 groups. In the validation cohort, differential NFAT5, ACVR1C and SMC3 expressions were confirmed. BRA patients showed highest NFAT5 levels compared to HEP/M0 groups (global p = 0.02). High ACVR1C expression was observed more frequently in the BRA group (42.9%) than in HEP (0%) and M0 (7.1%) groups (global p = 0.01). High SMC3 expressions were only detectable in the BRA group (global p = 0.003). Only patients with BM showed a combined high expression of NFAT5, ACVR1C or SMC3 as well as of all three genes. ROC analysis revealed a good prediction of brain metastasis by the three genes (area under the curve (AUC) = 0.78). CONCLUSIONS: The NFAT5, ACVR1C and SMC3 gene expression signature is associated with colorectal BM. Future studies should further investigate the importance of this biomarker signature.

Unresectable biliary tract cancer: Current and future systemic therapy.

For decades, treatment of advanced biliary tract cancer (BTC) was confined to the use of chemotherapy. In recent years however, the number of therapeutic options available for patients with unresectable BTC have drastically increased, with immunotherapy and targeted treatment gradually joining the ranks of guideline-recommended treatment regimens. The aim of the present review is to summarise the current knowledge on unresectable BTC focusing on epidemiology, anatomical distribution and current strategies for systemic treatment. We further outline ongoing clinical trials and provide an outlook on future therapeutic interventions. In the realm of gastrointestinal malignancies, the increasing number of systemic treatment options for BTC is finally delivering on the longstanding commitment to personalised oncology. This emphasises the need for considering a comprehensive genomic-based pathology assessment right from the initial diagnosis to fully leverage the expanding array of therapeutic options that have recently become accessible.

Risk factors for intracerebral hemorrhage in small-vessel disease and non-small-vessel disease etiologies-an observational proof-of-concept study.

Background: Sporadic cerebral small-vessel disease (CSVD), i.e., hypertensive arteriopathy (HA) and cerebral amyloid angiopathy (CAA), is the main cause of spontaneous intracerebral hemorrhage (ICH). Nevertheless, a substantial portion of ICH cases arises from non-CSVD etiologies, such as trauma, vascular malformations, and brain tumors. While studies compared HA- and CAA-related ICH, non-CSVD etiologies were excluded from these comparisons and are consequently underexamined with regard to additional factors contributing to increased bleeding risk beyond their main pathology. Methods: As a proof of concept, we conducted a retrospective observational study in 922 patients to compare HA, CAA, and non-CSVD-related ICH with regard to factors that are known to contribute to spontaneous ICH onset. Medical records (available for n = 861) were screened for demographics, antithrombotic medication, and vascular risk profile, and CSVD pathology was rated on magnetic resonance imaging (MRI) in a subgroup of 185 patients. The severity of CSVD was assessed with a sum score ranging from 0 to 6, where a score of ≥2 was defined as advanced pathology. Results: In 922 patients with ICH (median age of 71 years), HA and CAA caused the majority of cases (n = 670, 73%); non-CSVD etiologies made up the remaining quarter (n = 252, 27%). Individuals with HA- and CAA-related ICH exhibited a higher prevalence of predisposing factors than those with non-CSVD etiologies. This includes advanced age (median age: 71 vs. 75 vs. 63 years, p < 0.001), antithrombotic medication usage (33 vs. 37 vs. 19%, p < 0.001), prevalence of vascular risk factors (70 vs. 67 vs. 50%, p < 0.001), and advanced CSVD pathology on MRI (80 vs. 89 vs. 51%, p > 0.001). However, in particular, half of non-CSVD ICH patients were either aged over 60 years, presented with vascular risk factors, or had advanced CSVD on MRI. Conclusion: Risk factors for spontaneous ICH are less common in non-CSVD ICH etiologies than in HA- and CAA-related ICH, but are still frequent. Future studies should incorporate these factors, in addition to the main pathology, to stratify an individual's risk of bleeding.

TGFß signalling pathway impacts brain metastases profiles in locally advanced colorectal cancer.

RATIONALE: Colorectal Cancer (CRC) represents the third most common type of cancer in Germany and the second most common cancer-related cause of death worldwide. Distant metastases are still the main limit for patient survival. While liver metastases as well as peritoneal carcinomatosis can often either be resected or treated with systemic therapy, little options remain for brain metastases. Additionally, a number of studies has already investigated hepatic, peritoneal, pulmonary as well as continuing distant metastases in colorectal cancer. Yet, with respect to tumor biology and brain metastases, little is known so far. MATERIAL AND METHODS: Two cohorts, M0 without distant spread and BRA with brain metastases were build. RNA was isolated from paraffin embedded specimen. Gene expression was performed by an RNA NanoString-Analysis using the nCounter® PanCancer Progression Panel by NanoString-Technologies (Hamburg, Germany). Results were analysed by principal component analysis, gene expression and pathway analysis using commonly available databases such as KEGG as benchmark for comparison. RESULTS: We were able to determine a gene signature that provides a sophisticated group separation between M0 and BRA using principal component analysis. All genes with strong loading characteristics on principal component 1 were cross-referenced with the subsequently performed accurate gene set enrichment analysis (GSEA). The GSEA revealed a clear dysregulation of the TGFß pathway in compared cohorts M0 and BRA. Interestingly, the targeted pathways analysis of the identified genes confirmed that in fact almost all strong loading genes of PC1 play a role in the TGFß pathway. CONCLUSION: Our results suggest the TGFß pathway as a crucial player in the development of brain metastases in primary CRC. In some types of colorectal cancer, downregulation of the TGFß pathway might hinder primary colorectal cancer to metastasize to the nervous system. While the paradoxical functioning of the TGFß pathway is still not fully understood, these shed light on yet another clinical implication of this complex pathway.

PPARG activation promotes the proliferation of colorectal cancer cell lines and enhances the antiproliferative effect of 5-fluorouracil.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARG) is a member of the nuclear receptor family. It is involved in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, vascular homeostasis and inflammation. In addition, PPARG agonists, known as thiazolidinediones, are well established in the treatment of type 2 diabetes mellitus. PPARGs role in cancer is a matter of debate, as pro- and anti-tumour properties have been described in various tumour entities. Currently, the specific role of PPARG in patients with colorectal cancer (CRC) is not fully understood. MATERIAL AND METHODS: The prognostic impact of PPARG expression was investigated by immunohistochemistry in a case-control study using a matched pair selection of CRC tumours (n = 246) with either distant metastases to the liver (n = 82), lung (n = 82) or without distant metastases (n = 82). Its effect on proliferation as well as the sensitivity to the chemotherapeutic drug 5-fluorouracil (5-FU) was examined after activation, inhibition, and transient gene knockdown of PPARG in the CRC cell lines SW403 and HT29. RESULTS: High PPARG expression was significantly associated with pulmonary metastasis (p = 0.019). Patients without distant metastases had a significantly longer overall survival with low PPARG expression in their tumours compared to patients with high PPARG expression (p = 0.045). In the pulmonary metastasis cohort instead, a trend towards longer survival was observed for patients with high PPARG expression in their tumour (p = 0.059). Activation of PPARG by pioglitazone and rosiglitazone resulted in a significant dose-dependent increase in proliferation of CRC cell lines. Inhibition of PPARG by its specific inhibitor GW9662 and siRNA-mediated knockdown of PPARG significantly decreased proliferation. Activating PPARG significantly increased the CRC cell lines sensitivity to 5-FU while its inhibition decreased it. CONCLUSION: The prognostic effect of PPARG expression depends on the metastasis localization in advanced CRC patients. Activation of PPARG increased malignancy associated traits such as proliferation in CRC cell lines but also increases sensitivity towards the chemotherapeutic agent 5-FU. Based on this finding, a combination therapy of PPARG agonists and 5-FU-based chemotherapy constitutes a promising strategy which should be further investigated.

Comparative transcriptomic analyses reveal activation of the epithelial-mesenchymal transition program in non-metastasizing low grade pseudomyxoma peritonei.

Epithelial-mesenchymal transition (EMT), angiogenesis, cell adhesion and extracellular matrix (ECM) interaction are essential for colorectal cancer (CRC) metastasis. Low grade mucinous neoplasia of the appendix (LAMN) and its advanced state low grade pseudomyxoma peritonei (lgPMP) show local aggressiveness with very limited metastatic potential as opposed to CRC. To better understand the underlying processes that foster or impede metastatic spread, we compared LAMN, lgPMP, and CRC with respect to their molecular profile with subsequent pathway analysis. LAMN, lgPMP and (mucinous) CRC cases were subjected to transcriptomic analysis utilizing Poly(A) RNA sequencing. Successfully sequenced cases (LAMN n = 10, 77%, lgPMP n = 13, 100% and CRC n = 8, 100%) were investigated using bioinformatic and statistical tests (differential expression analysis, hierarchical clustering, principal component analysis and gene set enrichment analysis). We identified a gene signature of 28 genes distinguishing LAMN, lgPMP and CRC neoplasias. Ontology analyses revealed that multiple pathways including EMT, ECM interaction and angiogenesis are differentially regulated. Fifty-three significantly differentially regulated gene sets were identified between lgPMP and CRC followed by CRC vs. LAMN (n = 21) and lgPMP vs. LAMN (n = 16). Unexpectedly, a substantial enrichment of the EMT gene set was observed in lgPMP vs. LAMN (FDR=0.011) and CRC (FDR=0.004). Typical EMT markers were significantly upregulated (Vimentin, TWIST1, N-Cadherin) or downregulated (E-Cadherin) in lgPMP. However, MMP1 and MMP3 levels, associated with EMT, ECM and metastasis, were considerably higher in CRC. We show that the different tumor biological behaviour and metastatic spread pattern of midgut malignancies is reflected in a different gene expression profile. We revealed a strong activation of the EMT program in non-metastasizing lgPMP vs. CRC. Hence, although EMT is considered a key step in hematogenous spread, successful EMT does not necessarily lead to hematogenous dissemination. This emphasizes the need for further pathway analyses and forms the basis for mechanistic and therapy-targeting research.

Differential Immune Infiltration Profiles in Colitis-Associated Colorectal Cancer versus Sporadic Colorectal Cancer.

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.

Development and Validation of a 15-gene Expression Signature with Superior Prognostic Ability in Stage II Colorectal Cancer.

Currently, there is no consensus about the use of adjuvant chemotherapy for patients with stage II colorectal cancer. Here, we aimed to identify and validate a prognostic mRNA expression signature for the stratification of patients with stage II colorectal cancer according to their risk for relapse. First, publicly available mRNA expression profiling datasets from 792 primary, stage II colorectal cancers from six different training cohorts were analyzed to identify genes that are consistently associated with patient relapse-free survival (RFS). Second, the identified gene expression signature was experimentally validated using NanoString technology and computationally refined on primary colorectal cancer samples from 205 patients with stage II colorectal cancer. Third, the refined signature was validated in two independent publicly available cohorts of 166 patients with stage II colorectal cancer. Bioinformatics analysis of training cohorts identified a 61-gene signature that was highly significantly associated with RFS (HR = 37.08, P = 2.68*10-106, sensitivity = 89.29%, specificity = 89.61%, and AUC = 0.937). The experimental validation and refinement revealed a 15-gene signature that robustly predicted relapse in three independent cohorts: an in-house cohort (HR = 20.4, P = 8.73*10-23, sensitivity = 90.32%, specificity = 80.99%, AUC = 0.812), GSE161158 (HR = 5.81, P = 3.57*10-4, sensitivity = 64.29%, specificity = 81.67%, AUC = 0.796), and GSE26906 (HR = 7.698, P = 7.26*10-8, sensitivity = 61.54%, specificity = 78.33%, AUC = 0.752). In the pooled training cohort, the 15-gene signature (HR = 4.72, P = 7.76*10-25, sensitivity = 75%, specificity = 67.44%, AUC = 0.784) was superior to the Oncotype DX colon 7-gene signature (HR = 2.698, P = 6.3*10-8, sensitivity = 62.16%, specificity = 55.5%, AUC = 0.633). We report the identification and validation of a novel mRNA expression signature for robust prognostication and stratification of patients with stage II colorectal cancer, with superior performance in the analyzed validation cohorts when compared with clinicopathologic biomarkers and signatures currently used for stage II colorectal cancer prognostication. Significance: We identified and validated a 15-gene expression signature for robust prognostication and stratification of patients with stage II colorectal cancer, with superior performance when compared with currently used biomarkers. Therefore, the 15-gene expression signature has the potential to improve the prognostication and treatment decisions for patients with stage II colorectal cancer.

Molecular characteristics of microsatellite stable early-onset colorectal cancer as predictors of prognosis and immunotherapeutic response.

The incidence of early-onset colorectal cancer (EO-CRC, in patients younger than 50) is increasing worldwide. The specific gene signatures in EO-CRC patients are largely unknown. Since EO-CRC with microsatellite instability is frequently associated with Lynch syndrome, we aimed to comprehensively characterize the tumor microenvironment (TME) and gene expression profiles of EO-CRC with microsatellite stable (MSS-EO-CRC). Here, we demonstrated that MSS-EO-CRC has a similar pattern of tumor-infiltrating immune cells, immunotherapeutic responses, consensus molecular subtypes, and prognosis as late-onset CRC with MSS (MSS-LO-CRC). 133 differential expressed genes were identified as unique gene signatures of MSS-EO-CRC. Moreover, we established a risk score, which was positively associated with PD-L1 expression and could reflect both the level of tumor-infiltrating immune cells and the prognosis of MSS-EO-CRC patients. Application of this score on the anti-PD-L1 treatment cohort demonstrated that the low-risk score group has significant therapeutic advantages and clinical benefits. In addition, candidate driver genes were identified in the different-sidedness of MSS-EO-CRC patients. Altogether, MSS-EO-CRC exhibits distinct molecular profiles that differ from MSS-LO-CRC even though they have a similar TME characterization and survival pattern. Our risk score appears to be robust enough to predict prognosis and immunotherapeutic response and therefore could help to optimize the treatment of MSS-EO-CRC.

Guideline adherence of tumor board recommendations in lung cancer and transfer into clinical practice.

PURPOSE: Evaluating patients and treatment decisions in a multidisciplinary tumor board has led to better quality of care and longer survival in cancer patients. The aim of this study was to evaluate tumor board recommendations for thoracic oncology patients regarding guideline adherence and transferal of recommendations into clinical practice. METHODS: We evaluated tumor board recommendations of the thoracic oncology tumor board at Ludwig-Maximilians University (LMU) Hospital Munich between 2014 and 2016. We compared patient characteristics between guideline-adherent and non-guideline-adherent recommendations, as well as between transferred and non-transferred recommendations. We used multivariate logistic regression models to evaluate factors associated with guideline adherence. RESULTS: Over 90% of recommendations by the tumor board were either adherent to the guidelines (75.5%) or over fulfilling guidelines (15.6%). Almost 90% of recommendations were transferred to clinical practice. If a recommendation was not according to the guidelines, the reason was mostly associated with the general condition (age, Charlson comorbidity index, ECOG) of the patient or due to the patients' request. Surprisingly, sex also had a significant influence on the guideline adherence of recommendations, with females being more likely to get recommendations not according to the guidelines. CONCLUSION: In conclusion, the results of this study are promising, as the guideline adherence of recommendations as well as the transferal of recommendations into clinical practice were high. In the future, a special focus should be put on fragile patients as well as female patients.

[Molecular pathology of colorectal cancer]. / Molekularpathologie kolorektaler Karzinome.

In recent years, the treatment of colorectal carcinoma has experienced increasing individualization. In addition to RAS and BRAF mutational status that is firmly established in routine diagnostics, new therapeutic options evolved based on MSI and HER2 status as well as primary tumour localization. Offering the best targeted options in therapy requires new evidence-based decision-making algorithms regarding timing and scope of molecular pathological diagnostics in order for patients to receive an optimized therapy according to current treatment guidelines. New targeted therapies, some of which are about to be approved and for which pathology has to provide new molecular pathological biomarkers, will also play an increasingly important role in the future.

Treatment of Mediastinal Endometrial Carcinoma Developed from Extragenital Endometriosis and Simultaneous Rectal Adenocarcinoma in a 55-year-old Woman.

BACKGROUND/AIM: Endometriosis is a common disorder in reproductive-age women leading to a broad range of symptoms and is associated with a higher risk for endometrioid ovarian carcinoma. CASE REPORT: We report the case of a 55 year-old woman with previously undiagnosed endometriosis presenting with a large mediastinal cancer of unknown primary (CUP) and synchronous Union Internationale Contre le Cancer (UICC) stage II rectal adenocarcinoma. Histopathologically the mediastinal tumor resembled endometrial carcinoma and laparoscopically endometriotic lesions on the patient's peritoneum were detected. The patient was treated with neoadjuvant carboplatin and paclitaxel, followed by resection of the mediastinal tumor. After recovery, the patient received neoadjuvant short-course radiation to the rectal adenocarcinoma, which was resected afterwards. No primary endometrial carcinoma was found in the uterus, leading to the most likely conclusion that the mediastinal tumor derived from an extragenital endometriotic lesion. CONCLUSION: Although rare, cases of degeneration of endometriosis have been described. In this case not only the localization of endometriosis was uncommon, but also its malignant transformation and synchronous diagnosis of a rectal adenocarcinoma, complicating diagnosis, and treatment of the patient. This rare case highlights the importance of diagnosing and treating patients with CUP or multiple malignancies at large interdisciplinary centers to reach the best possible outcome.

Olfactomedin 4 associates with expression of differentiation markers but not with properties of cancer stemness, EMT nor metastatic spread in colorectal cancer.

Tumor stem cells play a pivotal role in carcinogenesis and metastatic spread in colorectal cancer (CRC). Olfactomedin 4 (OLFM4) is co-expressed with the established stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 at the bottom of intestinal crypts and has been suggested as a surrogate for cancer stemness and a biomarker in gastrointestinal tumors associated with prognosis. Therefore, it was the aim of the present study to clarify whether OLFM4 is involved in carcinogenesis and metastatic spread in CRC. We used a combined approach of functional assays using forced OLFM4 overexpression in human CRC cell lines, xenograft mice, and an immunohistochemical approach using patient tissues to investigate the impact of OLFM4 on stemness, canonical Wnt signaling, properties of metastasis and differentiation as well as prognosis. OLFM4 expression correlated weakly with tumor grade in one patient cohort (metastasis collection: p = 0.05; pooled analysis of metastasis collection and survival collection: p = 0.19) and paralleled the expression of differentiation markers (FABP2, MUC2, and CK20) (p = 0.002) but did not correlate with stemness-associated markers. Further analyses in CRC cells lines as well as xenograft mice including forced overexpression of OLFM4 revealed that OLFM4 neither altered the expression of markers of stemness nor epithelial-mesenchymal transition, nor did OLFM4 itself drive proliferation, migration, or colony formation, which are all prerequisites of carcinogenesis and tumor progression. In line with this, we found no significant correlation between OLFM4 expression, metastasis, and patient survival. In summary, expression of OLFM4 in human CRC seems to be characteristic of differentiation marker expression in CRC but is not a driver of carcinogenesis nor metastatic spread.

Analysis of Circulating Immune Subsets in Primary Colorectal Cancer.

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.

SMAD4 Loss Induces c-MYC-Mediated NLE1 Upregulation to Support Protein Biosynthesis, Colorectal Cancer Growth, and Metastasis.

Growth and metastasis of colorectal cancer is closely connected to the biosynthetic capacity of tumor cells, and colorectal cancer stem cells that reside at the top of the intratumoral hierarchy are especially dependent on this feature. By performing disease modeling on patient-derived tumor organoids, we found that elevated expression of the ribosome biogenesis factor NLE1 occurs upon SMAD4 loss in TGFß1-exposed colorectal cancer organoids. TGFß signaling-mediated downregulation of NLE1 was prevented by ectopic expression of c-MYC, which occupied an E-box-containing region within the NLE1 promoter. Elevated levels of NLE1 were found in colorectal cancer cohorts compared with normal tissues and in colorectal cancer subtypes characterized by Wnt/MYC and intestinal stem cell gene expression. In colorectal cancer cells and organoids, NLE1 was limiting for de novo protein biosynthesis. Upon NLE1 ablation, colorectal cancer cell lines activated p38/MAPK signaling, accumulated p62- and LC3-positive structures indicative of impaired autophagy, and displayed more reactive oxygen species. Phenotypically, knockout of NLE1 inhibit.ed proliferation, migration and invasion, clonogenicity, and anchorage-independent growth. NLE1 loss also increased the fraction of apoptotic tumor cells, and deletion of TP53 further sensitized NLE1-deficient colorectal cancer cells to apoptosis. In an endoscopy-guided orthotopic mouse transplantation model, ablation of NLE1 impaired tumor growth in the colon and reduced primary tumor-derived liver metastasis. In patients with colorectal cancer, NLE1 mRNA levels predicted overall and relapse-free survival. Taken together, these data reveal a critical role of NLE1 in colorectal cancer growth and progression and suggest that NLE1 represents a potential therapeutic target in colorectal cancer patients. SIGNIFICANCE: NLE1 limits de novo protein biosynthesis and the tumorigenic potential of advanced colorectal cancer cells, suggesting NLE1 could be targeted to improve the treatment of metastatic colorectal cancer.

Prospective evaluation of immunological, molecular-genetic, image-based and microbial analyses to characterize tumor response and control in patients with unresectable stage III NSCLC treated with concurrent chemoradiotherapy followed by consolidation therapy with durvalumab (PRECISION): protocol for a prospective longitudinal biomarker study.

Background: Concurrent platinum-based chemoradiotherapy (CRT) followed by durvalumab maintenance treatment represents the new standard of care in unresectable stage III non-small cell lung cancer (NSCLC). In this prospective hypothesis-generating single-center study, we aim to identify a framework of prognostic and predictive biomarkers by longitudinal characterization of tumor- and patient (host)-related parameters over all phases of multimodal treatment. Methods: This study will enroll 40 patients (≥18 years, Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2, with a diagnosis of PD-L1 positive (≥1%), inoperable stage III NSCLC) with an indication for CRT followed by maintenance treatment with durvalumab according to European Medicines Agency (EMA) approval. Comprehensive analysis will include peripheral blood cellular and humoral immunophenotyping and circulating tumor DNA as well as gut/saliva microbiota analyses. Additional morphological analysis with 18F-FDG-PET/computed tomography (CT) before, 6 weeks, 6 and 12 months after the end of CRT is included. Statistical analysis using multiple testing will be used to examine the impact of different parameters on progression-free survival (PFS) and overall survival (OS) as well as tumor response and response duration. Discussion: This protocol describes the methodology of a comprehensive biomarker study in order to identify a framework of prognostic and predictive markers for unresectable stage III NSCLC in a real-world setting. Trial Registration: ClinicalTrials.gov identifier (NCT05027165), data registered on August 2021.

First-line fluoropyrimidine plus bevacizumab followed by irinotecan-escalation versus initial fluoropyrimidine, irinotecan and bevacizumab in patients with metastatic colorectal cancer - Final survival and per-protocol analysis of the randomised XELAVIRI trial (AIO KRK 0110).

BACKGROUND: The randomised open-label phase III XELAVIRI trial failed to demonstrate non-inferiority of the sequential application of fluoropyrimidine plus bevacizumab followed by additional irinotecan at first progression (Arm A) versus initial combination of all agents (Arm B) for untreated metastatic colorectal cancer in the initial analysis of time-to-failure-of-strategy (TFS, 90% confidence boundary of 0.8). Here, we evaluate efficacy in the full analysis set (FAS), the per-protocol set, in addition to age-related and molecular subgroups. METHODS: Median TFS, overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier method and log-rank test. Cox regression models assessed hazard ratios (HRs) and confidence intervals (CIs) (TFS: 90%; OS, PFS: 95%). RESULTS: Of 421 patients, 390 (92.6%), 391 (92.9%) and 357 (84.8%) events for TFS, OS and PFS were observed in the FAS with a median follow-up of 54.2 months (Arm A) versus 52.9 months (Arm B). Non-inferiority of sequential treatment for TFS was missed in the FAS (HR 0.93; 90% CI, 0.79-1.10; P = 0.482) and not shown in the per-protocol set (HR 0.93; 90% CI, 0.75-1.13, P = 0.433). Formal non-inferiority for TFS was observed for patients older than 70 years (HR 1.06; 90% CI, 0.80-1.41; P = 0.670) and patients with RAS mutant tumours (HR 1.12; 90% CI, 0.87-1.43; P = 0.465). In RAS/BRAF wild-type tumours, combination treatment was significantly superior to sequential therapy in all end-points. CONCLUSIONS: In the overall population, XELAVIRI just missed to demonstrate the non-inferiority of sequential compared to combination therapy for TFS. However, the non-inferiority of sequential treatment was observed in elderly patients and RAS mutant tumours. TRIAL REGISTRATION: Trial registration ID (clinicaltrials.gov) NCT01249638.