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Background: Recent advances in the personalized treatment of non-small cell lung cancer (NSCLC) require representative in vitro model systems that reflect tumor heterogeneity and maintain the characteristic genetic aberrations. We therefore aimed to establish patient-derived NSCLC organoids that offer a reliable platform for further investigations. Methods: NSCLC organoids were cultured between May 2020 and February 2022 from surgically resected NSCLC tissue specimens. After histological and immunohistochemical validation, genetic validation was performed by targeted next-generation sequencing of tissue and organoid specimens using the Oncomine Focus Assay (ThermoFisher Scientific). Results: From 37 resected NSCLC samples, 18 primary organoid cultures were successfully established and expanded during early passages. Upon histomorphological validation, organoids showed complementary characteristics when compared to the resected parental tumor, including adenocarcinoma, squamous cell carcinoma, mucoepidermoid carcinoma, and lung carcinoid differentiation. Among nine parental tumors, traceable genetic alterations were detected, and three corresponding organoids lines retained this mutational profile, including a KRAS p.Gly12Val mutation, KRAS p.Gly12Cys mutation, and RET-fusion. Conclusions: The establishment of primary NSCLC organoids from surgically resected tissue is feasible. Histological, immunohistochemical, and genetic validation is essential to identify representative NSCLC organoids that maintain the characteristics of the parental tumor. Overall, low establishment rates remain a challenge for broad clinical applications.

Abstract Immune checkpoint inhibitor treatment has the potential to prolong survival in non-small cell lung cancer (NSCLC), however, some of the patients develop resistance following initial response. Here, we analyze the immune phenotype of matching tumor samples from a cohort of NSCLC patients showing good initial response to immune checkpoint inhibitors, followed by acquired resistance at later time points. By using imaging mass cytometry and whole exome and RNA sequencing, we detect two patterns of resistance¨: One group of patients is characterized by reduced numbers of tumor-infiltrating CD8+ T cells and reduced expression of PD-L1 after development of resistance, whereas the other group shows high CD8+ T cell infiltration and high expression of PD-L1 in addition to markedly elevated expression of other immune-inhibitory molecules. In two cases, we detect downregulation of type I and II IFN pathways following progression to resistance, which could lead to an impaired anti-tumor immune response. This study thus captures the development of immune checkpoint inhibitor resistance as it progresses and deepens our mechanistic understanding of immunotherapy response in NSCLC.

BackgroundAnaplastic lymphoma kinase (ALK) rearrangements are known oncogenic drivers in non-small cell lung cancer (NSCLC). Few case reports described the occurrence of such rearrangements in large cell neuroendocrine carcinomas (LCNECs) of the lung without information on clinical responses to ALK tyrosine kinase inhibitors (TKIs) in these cases. Currently, neuroendocrine tumors of the lungs are not screened for ALK rearrangements.MethodsTo illustrate the clinical impact of molecular characterization in LCNECs, we report the disease course in three patients with ALK-rearranged metastatic LCNEC from our clinical routine, as well as their treatment response to ALK TKIs (index cases). To gain insight into the prevalence of ALK rearrangements in neuroendocrine tumors of the lung, we analyzed a retrospective cohort of 436 tumor biopsies including LCNEC (n = 61), small cell lung cancer (SCLC) (n = 206), typical (n = 91) and atypical (n = 69) carcinoids, and mixed histology (n = 9) for the presence of ALK rearrangements using a sequential diagnostic algorithm. ALK immunohistochemistry (IHC) was evaluable in 362 cases; fluorescence in situ hybridization (FISH) was evaluable in 28 out of the 35 IHC-positive cases, followed by next-generation sequencing (NGS) that was available in 12 cases.ResultsWithin the retrospective cohort, ALK IHC was positive in 35 out of 362 (9.7%) evaluable samples. FISH was positive in 3 out of the 28 (10.7%) evaluable cases: 2 with atypical carcinoids and 1 with LCNEC. Additionally, the 3 index cases showed positive ALK IHC, which was confirmed by NGS. Within the retrospective cohort, NGS confirmed the presence of an ALK genomic rearrangement in one FISH-positive atypical carcinoid where material was sufficient for sequencing. Two out of three patients with metastatic ALK-rearranged LCNEC received up-front treatment with the ALK TKI alectinib and showed rapid tumor response at all metastatic sites, including multiple brain metastases.ConclusionsALK rearrangements represent rare but targetable oncogenic driver alterations in LCNEC. Contrarily to NSCLC, the detection of ALK rearrangements in neuroendocrine tumors of the lung is challenging, since ALK IHC can lead to false-positive results and therefore needs confirmation by FISH or NGS. Up-front comprehensive molecular profiling with NGS should be performed in metastatic LCNEC in order not to miss actionable genomic alterations.

IntroductionThere is no proven therapy for chronic-active antibody-mediated rejection (caABMR), the major cause of late kidney allograft failure. Histological and molecular patterns associated with possible therapy responsiveness are not known.MethodsBased on rigorous selection criteria this single center, retrospective study identified 16 out of 1027 consecutive kidney transplant biopsies taken between 2008 and 2016 with pure, unquestionable caABMR, without other pathologic features. The change in estimated GFR pre- and post-biopsy/treatment were utilized to differentiate subjects into responders and non-responders. Gene sets reflecting active immune processes of caABMR were defined a priori, including endothelial, inflammatory, cellular, interferon gamma (IFNg) and calcineurin inhibitor (CNI) related-genes based on the literature. Transcript measurements were performed in RNA extracted from stored, formalin-fixed, paraffin-embedded (FFPE) samples using NanoString™ technology. Histology and gene expression patterns of responders and non-responders were compared.ResultsA reductionist approach applying very tight criteria to identify caABMR and treatment response excluded the vast majority of clinical ABMR cases. Only 16 out of 139 cases with a written diagnosis of chronic rejection fulfilled the caABMR criteria. Histological associations with therapy response included a lower peritubular capillaritis score (p = 0.028) along with less glomerulitis. In contrast, no single gene discriminated responders from non-responders. Activated genes associated with NK cells and endothelial cells suggested lack of treatment response.ConclusionIn caABMR active microvascular injury, in particular peritubular capillaritis, differentiates treatment responders from non-responders. Transcriptome changes in NK cell and endothelial cell associated genes may further help to identify treatment response. Future prospective studies will be needed which include more subjects, who receive standardized treatment protocols to identify biomarkers for treatment response.Clinical Trial Registration[ClinicalTrials.gov], identifier [NCT03430414].

Pleomorphic lobular carcinoma (PLC) of the breast with osteoclast like giant cells (OGC) is a very rare variant of lobular carcinoma associated with tumour-associated macrophages (TAM) in the microenvironment. High content of TAM in breast cancer is associated with poor survival rates. Using genomic testing for the individual risk assessment in breast cancers with special features of their microenvironment might be skewed by the fact, that most recommended tests investigate proliferation and oestrogen receptor associated genes of tumour cells in their risk stratification but do not take into consideration the microenvironment. So far, no disease-specific molecular alteration could be identified in NST and lobular invasive carcinoma with OGC.

Introduction: Survival of ALK-rearranged NSCLC patients has dramatically improved by the use of multiple ALK-tyrosine kinase inhibitors (ALK-TKI). However, still little is known about the impact of drug sequencing and clinical features on survival in a real-world setting.Methods: Patients with stage IV ALK-rearranged NSCLC treated at six centers in Switzerland and Italy were identified and standard clinical variables collected. OS curves were constructed using the Kaplan–Meier method and compared with the log-rank test. Multivariate Cox proportional hazard analysis was applied to determine the correlations between clinical features and OS. In four patients, biopsies were subjected to NGS.Results: One-hundred and twenty-one patients with stage IV ALK-rearranged NSCLC diagnosed between 2011 and 2016 were included. With a median follow-up time of 39.5 months, the median OS from diagnosis of stage IV disease was 48.0 months. First-line treatment consisted of an ALK-TKI in 24% of patients, with crizotinib in 83% of them. Chemotherapy as first-line treatment did not influence OS (p = 0.955). The use of more than one ALK-TKI line positively correlated with OS (p = 0.016), as well as the use of alectinib or lorlatinib in any treatment line, as compared to the use of crizotinib ± ceritinib (p = 0.022). A never smoking history was an independent prognostic factor for OS (p = 0.032). Moreover, treatment with alectinib significantly improved OS.Conclusions: Targeted treatment for ALK-positive NSCLC patients lead to prolonged OS. Smoking status was a negative independent prognostic factor in a multi-variate analysis. The use of alectinib or lorlatinib in any treatment line improved overall outcome.

Abstract Background Molecular testing of lung adenocarcinoma for oncogenic driver mutations has become standard in pathology practice. The aim of the study was to analyze the EGFR, KRAS, ALK, RET, ROS1, BRAF, ERBB2, MET and PIK3CA mutational status in a representative cohort of Swiss patients with lung adenocarcinoma and to correlate the mutational status with clinicopathological patient characteristics. Methods All patients who underwent molecular testing of newly diagnosed lung adenocarcinoma during a 4-year period (2014–2018) were included. Molecular analyses were performed with Sanger sequencing (n = 158) and next generation sequencing (n = 311). ALK, ROS1 and RET fusion gene analyses were also performed with fluorescence in situ hybridization and immunohistochemistry/immunocytochemistry. Demographic and clinical data were obtained from the medical records. Results Of 469 patients with informative EGFR mutation analyses, 90 (19.2%) had EGFR mutations. KRAS mutations were present in 33.9% of the patients, while 6.0% of patients showed ALK rearrangement. BRAF, ERBB2, MET and PIK3CA mutations and ROS1 and RET rearrangements were found in 2.6%, 1.9%, 1.9%, 1.5%, 1.7% and 0.8% of the patients, respectively. EGFR mutation was significantly associated with female gender and never smoking status. ALK translocations were more frequent in never smokers, while KRAS mutations were more commonly found in ever smokers. The association between KRAS mutational status and female gender was statistically significant only on multivariate analysis after adjusting for smoking. Conclusion The EGFR mutation rate in the current study is among the higher previously reported mutation rates, while the frequencies of KRAS, BRAF, ERBB2 and PIK3CA mutations and ALK, ROS1 and RET rearrangements are similar to the results of previous reports. EGFR and KRAS mutations were significantly associated with gender and smoking. ALK rearrangements showed a significant association with smoking status alone.

PurposeLung cancer in never smokers is recognized as a distinct molecular, clinicopathologic and epidemiologic entity. The aim of the study was to investigate the molecular profile in Swiss never smokers with lung adenocarcinoma and to correlate the mutation status with clinicopathologic and demographic patient characteristics and outcome.MethodsOne hundred thirty-eight never smokers diagnosed with lung adenocarcinoma at the University Hospital Zurich between 2011-2018 were included in the study. Data from the electronic medical records were reviewed to characterize clinicopathologic and demographic features, molecular profile, treatment and outcome.ResultsThe majority of patients were female (58.7%) with a median age at diagnosis of 64.5 years (range, 27.1-94.2 years). The most common mutations were EGFR (58.7%) followed by ALK (12.3%), TP53 (5.8%), MET (5.8%), KRAS (4.3%), ERBB2 (4.3%), PIK3CA (2.9%), BRAF (2.2%), ROS1 (1.4%), RET (1.4%), CTNNB1 (0.7%), PARP1 (0.7%), TET1 (0.7%) and PIK3CG (0.7%). Median overall survival (mOS) was 51.0 months (mo). Early clinical stage (p = 0.002) and treatment with targeted therapy (HR 2.53, 95% CI 1.35-4.74, p = 0.004) were independently associated with longer mOS. Patients with oncogenic driver mutations had significantly longer mOS (52.2 mo) compared to patients without mutations (16.9 mo) (HR 3.38, 95% CI 1.52-7.55, p = 0.003). Besides, patients with EGFR mutated (57.8 mo) or ALK rearranged (59.9 mo) tumors had significantly longer mOS compared to the EGFR wildtype (35.0 mo), ALK wildtype (46.5 mo) and pan-negative (16.9 mo) cohorts (HR 2.35, 95% CI 1.37-4.04, p = 0.002; HR 7.80, 95% CI 3.28-18.55, p < 0.001; HR 3.96, 95% CI 1.21-12.95, p = 0.023 and HR 34.78, 95% CI 3.48-34.65, p = 0.003).ConclusionNever smokers with lung adenocarcinoma display distinct clinicopathologic and molecular features and are characterized by a high incidence of targetable mutations. Never smokers with targetable mutations have significantly longer survival compared to patients without mutations.

Abstract Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium‐specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours.

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78-13.2) compared to 8.26 months (95% CI 6.02-19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn't reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.

Background: Intratumoral hypoxia plays an important role with regard to tumor biology and susceptibility to radio. and chemotherapy. For further investigation of hypoxia.related changes, areas of certain hypoxia must be reliably detected within cancer tissues. Pimonidazole, a 2.nitroimindazole, accumulates in hypoxic tissue and can be easily visualized using immunohistochemistry. Materials and Methods: To improve detection of highly hypoxic versus normoxic areas in prostate cancer, immunoreactivity of pimonidazole and a combination of known hypoxia.related proteins was used to create computational oxygen supply maps of prostate cancer. Pimonidazole was intravenously administered before radical prostatectomy in n = 15 patients, using the da Vinci robot.assisted surgical system. Prostatectomy specimens were immediately transferred into buffered formaldehyde, fixed overnight, and completely embedded in paraffin. Pimonidazole accumulation and hypoxia.related protein expression were visualized by immunohistochemistry. Oxygen supply maps were created using the normalized information from pimonidazole and hypoxia.related proteins. Results: Based on pimonidazole staining and other hypoxia.related proteins (osteopontin, hypoxia.inducible factor 1.alpha, and glucose transporter member 1) oxygen supply maps in prostate cancer were created. Overall, oxygen supply maps consisting of information from all hypoxia.related proteins showed high correlation and mutual information to the golden standard of pimonidazole. Here, we describe an improved computer.based ex vivo model for an accurate detection of oxygen supply in human prostate cancer tissue. Conclusions: This platform can be used for precise colocalization of novel candidate hypoxia.related proteins in a representative number of prostate cancer cases, and improve issues of single marker correlations. Furthermore, this study provides a source for further in situ tests and biochemical investigations