Time to Deliver on Promises: The Role of ERBB2 Alterations as Treatment Options for Colorectal Cancer Patients in the Era of Precision Oncology.

Receptor tyrosine kinase erythroblastic oncogene B2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2), represents an oncogenic driver and has been effectively targeted in breast and gastric cancer. Recently, next-generation sequencing (NGS) discovered ERBB2 as a promising therapeutic target in metastatic colorectal cancer (mCRC), where it is altered in 3-5% of patients, but no therapies are currently approved for this use. Herein, we present the experience of a single center in diagnosing actionable genetic ERBB2 alterations using NGS and utilizing the latest therapeutic options. Between October 2019 and December 2022, a total of 107 patients with advanced CRC underwent molecular analysis, revealing actionable ERBB2 mutations in two patients and ERBB2 amplifications in two other patients. These findings correlated with immunohistochemical (IHC) staining. Of these four patients, two were treated with trastuzumab-deruxtecan (T-DXd). We present two exemplary cases of patients with actionable ERBB2 alterations to demonstrate the effectiveness of T-DXd in heavily pretreated ERBB2-positive mCRC patients and the need for early molecular profiling. To fully exploit the potential of this promising treatment, earlier molecular profiling and the initiation of targeted therapies are essential.

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.

NTRK Gene Fusions in Non-Small-Cell Lung Cancer: Real-World Screening Data of 1068 Unselected Patients.

(1) Background: The main objectives of our study are (i) to determine the prevalence of NTRK (neurotrophic tyrosine kinase) fusions in a routine diagnostic setting in NSCLC (non-small cell lung cancer) and (ii) to investigate the feasibility of screening approaches including immunohistochemistry (IHC) as a first-line test accompanied by fluorescence in situ hybridization (FISH) and RNA-(ribonucleic acid-)based next-generation sequencing (RNA-NGS). (2) Methods: A total of 1068 unselected consecutive patients with NSCLC were screened in two scenarios, either with initial IHC followed by RNA-NGS (n = 973) or direct FISH testing (n = 95). (3) Results: One hundred and thirty-three patients (14.8%) were IHC positive; consecutive RNA-NGS testing revealed two patients (0.2%) with NTRK fusions (NTRK1-EPS15 (epidermal growth factor receptor pathway substrate 15) and NTRK1-SQSTM1 (sequestosome 1)). Positive RNA-NGS was confirmed by FISH, and NTRK-positive patients benefited from targeted treatment. All patients with direct FISH testing were negative. RNA-NGS- or FISH-positive results were mutually exclusive with alterations in EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (proto-oncogene B-Raf), RET (rearranged during transfection) or KRAS (kirsten rat sarcoma viral oncogene). Excluding patients with one of these alterations raised the prevalence of NTRK-fusion positivity among panTrk-(tropomyosin receptor kinase-) IHC positive samples to 30.5%. (4) Conclusions: NTRK fusion-positive lung cancers are exceedingly rare and account for less than 1% of patients in unselected all-comer populations. Both RNA-NGS and FISH are suitable to determine clinically relevant NTRK fusions in a real-world setting. We suggest including panTrk-IHC in a diagnostic workflow followed by RNA-NGS. Excluding patients with concurrent molecular alterations to EGFR/ALK/ROS1/BRAF/RET or KRAS might narrow the target population.

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.

KRAS mutation status concordance between the primary tumor and the corresponding metastasis in patients with rectal cancer.

INTRODUCTION: Oncogenic mutation within the KRAS gene represents a negative predictor for treatment response to anti-epidermal growth factor receptor (EGFR) in patients with colorectal cancer. Recently, we have shown no relevant heterogeneity for KRAS mutation status within and between pre- and posttherapeutic samples from the primary tumor in patients with locally advanced rectal cancer. The aim of this study was to evaluate the intertumoral heterogeneity of KRAS mutation status between the primary tumor and the corresponding metastasis or local recurrence in the similar cohort and to evaluate the ideal representative tissue for KRAS mutation testing. MATERIALS AND METHODS: KRAS mutation status was analyzed from 47 patients with locally advanced rectal cancer, which were enrolled in the CAO/ARO/AIO-94 or CAO/ARO/AIO-04 trial. Mutations in KRAS codons 12, 13, and 61 were analyzed by using the KRAS RGQ PCR Kit (therascreen® KRAS test). Six patients needed to be excluded due to incomplete follow up data. 11 patients showed a relapse of the disease during the follow up presented by distant metastases or local recurrence. DNA from representative areas of metastatic tissue was obtained from formalin-fixed paraffin-embedded specimens. RESULTS: The mean patient age was 64.13 ± 10.64 years. In total, 19 patients showed a KRAS mutation (46.34%) in the primary tumor. Of the eleven patients with a metastatic disease or local recurrence, five patients showed a KRAS mutation whereas six patients had a KRAS wildtype status. Metastatic localizations included the liver (n = 2), lung (n = 4), local recurrence (n = 1), liver + lung (n = 3), lung + local recurrence (n = 1). For these eleven patients with paired data available for the primary tumor and metastatic tissue, a significant KRAS mutation status concordance was detected in 81.18% (9/11) of the patients (p = 0.03271). Only two patients showed intertumoral heterogeneity, which harbored in one patient a KRAS G12C mutation status in the primary tumor, but a G12V KRAS mutation status in the corresponding lung lesion, and in the other patient a G12A mutation in the primary lesion and a WT in the lung metastasis. CONCLUSIONS: We show a significant concordance of the KRAS mutation status between tumor samples obtained from the primary tumor and the corresponding metastasis and/ or local recurrence in patients with rectal cancer indicating no relevant intertumoral heterogeneity. Our data suggest that sampling either the primary (pre- or posttherapeutical tumor tissue) or metastatic lesion may be valid for the initial evaluation of KRAS mutation status predicting the response to anti-EGFR treatment and guiding clinical decisions.

Top-level MET gene copy number gain defines a subtype of poorly differentiated pulmonary adenocarcinomas with poor prognosis.

BACKGROUND: MET amplifications occur in human tumors, including non-small cell lung cancer (NSCLC). MET inhibitors have demonstrated some clinical activity in MET amplified NSCLC, presumably with a gene dose effect. However, the definition of MET positivity or MET amplification as a potential oncogenic driver is still under debate. In this study, we aimed to establish the molecular subgroup of NSCLC with the highest unequivocal MET amplification level and to describe the prevalence, and histologic and clinical phenotype of this subgroup. METHODS: A total of 373 unselected patients with NSCLC were consecutively tested for MET gene copy number (GCN) by FISH. Mean GCN, MET/CEN7 ratio and other FISH parameters were identified and correlated with morphological and molecular pathological characteristics of the tumors as well as with clinical data. RESULTS: Based on the variability of obtained data a top-level category of MET amplification was newly defined (>90th percentile of average GCN; ≥10 MET gene copies per tumor cell). This criterion was fulfilled in 2% of analyzed tumors. These tumors were exclusively poorly differentiated adenocarcinomas with a predominant solid subtype and pleomorphic features. Rarely, co-alterations were detected (KRAS mutation or MET exon 14 skipping mutation). In this top-level group, there were no EGFR mutations or ALK or ROS1 alterations. The most important clinical feature was a significantly shortened overall survival (HR 3.61; median OS 8.2 vs. 23.6 months). Worse prognosis did not depend on initial stage or treatment. CONCLUSIONS: The newly defined top-level category of MET amplification in NSCLC defines a specific subgroup of pulmonary adenocarcinoma with adverse prognosis and characteristic morphological features. Lower levels of MET gene copy number seem to have probably no specific value as a prognostic or predictive biomarker.

Fibroblast growth factor receptor 1 gene amplification and protein expression in human lung cancer.

BACKGROUND: Targeting fibroblast growth factor receptor 1 (FGFR1) is a potential treatment for squamous cell lung cancer (SQCLC). So far, treatment decision in clinical studies is based on gene amplification. However, only a minority of patients have shown durable response. Furthermore, former studies have revealed contrasting results regarding the impact of FGFR1 amplification and expression on patient's prognosis. AIMS: Here, we analyzed prevalence and correlation of FGFR1 gene amplification and protein expression in human lung cancer and their impact on overall survival. MATERIALS & METHODS: FGFR1 gene amplification and protein expression were analyzed by fluorescence in situ hybridization and immunohistochemistry (IHC) in 208 SQCLC and 45 small cell lung cancers (SCLC). Furthermore, FGFR1 protein expression was analyzed in 121 pulmonary adenocarcinomas (ACs). Amplification and expression were correlated to each other, clinicopathological characteristics, and overall survival. RESULTS: FGFR1 was amplified in 23% of SQCLC and 8% of SCLC. Amplification was correlated to males (P = .027) but not to overall survival. Specificity of immunostaining was verified by cellular CRISPR/Cas9 FGFR1 knockout. FGFR1 was strongly expressed in 9% of SQCLC, 35% of AC, and 4% of SCLC. Expression was correlated to females (P = .0187) and to the absence of lymph node metastasis in SQCLC (P = .018) with no significant correlation to overall survival. Interestingly, no significant correlation between amplification and expression was detected. DISCUSSION: FGFR1 gene amplification does not seem to correlate to protein expression. CONCLUSION: We believe that patient selection for FGFR1 inhibitors in clinical studies should be reconsidered. Neither FGFR1 amplification nor expression influences patient's prognosis.

FGFR3 mRNA overexpression defines a subset of oligometastatic colorectal cancers with worse prognosis.

OBJECTIVES: Metastatic colorectal cancer (CRC) remains a leading cause of cancer related deaths. Patients with oligometastatic liver disease represent a clinical subgroup with heterogeneous course. Until now, biomarkers to characterize outcome and therapeutic options have not been fully established. METHODS: We investigated the prevalence of FGFR alterations in a total of 140 primary colorectal tumors and 63 liver metastases of 55 oligometastatic CRC patients. FGF receptors (FGFR1-4) and their ligands (FGF3, 4 and 19) were analyzed for gene amplifications and rearrangements as well as for RNA overexpression in situ. Results were correlated with clinico-pathologic data and molecular subtypes. RESULTS: Primary tumors showed FGFR1 (6.3%) and FGF3,4,19 (2.2%) amplifications as well as FGFR1 (10.1%), FGFR2 (5.5%) and FGFR3 (16.2%) overexpression. In metastases, we observed FGFR1 amplifications (4.8%) as well as FGFR1 (8.5%) and FGFR3 (14.9%) overexpression. Neither FGFR2-4 amplifications nor gene rearrangements were observed. FGFR3 overexpression was significantly associated with shorter overall survival in metastases (mOS 19.9 vs. 47.4 months, HR=3.14, p=0.0152), but not in primary CRC (HR=1.01, p=0.985). Although rare, also FGFR1 amplification was indicative of worse outcome (mOS 12.6 vs. 47.4 months, HR=8.83, p=0.00111). CONCLUSIONS: We provide the so far most comprehensive analysis of FGFR alterations in primary and metastatic CRC. We describe FGFR3 overexpression in 15% of CRC patients with oligometastatic liver disease as a prognosticator for poor outcome. Recently FGFR3 overexpression has been shown to be a potential therapeutic target. Therefore, we suggest focusing on this subgroup in upcoming clinical trials with FGFR-targeted therapies.

Comparative proteomics reveals a diagnostic signature for pulmonary head-and-neck cancer metastasis.

Patients with head-and-neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head-and-neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross- and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC.

ABCA3 Phenotype in Non-Small Cell Lung Cancer Indicates Poor Outcome.

BACKGROUND: ATP-binding cassette transport protein A3 (ABCA3) is expressed in non-small cell lung cancer (NSCLC). We hypothesize that high-level ABCA3 expression may have a negative prognostic impact in patients with NSCLC. METHODS: In 89 patients with NSCLC and curative intended surgery, we analyzed postoperative immunohistochemistry staining of primary tumors (anti-ABCA3) and clinicopathological parameters. We used a unidimensional four point score (FPS) system for intensity assessment and, furthermore, a combined bidimensional scoring of intensity and quantity resulting in the positive index (PI). RESULTS: Former or never-smokers were more likely to have intermediate or strong ABCA3 unidimensional expression (FPS) compared with current smokers (p < 0.01). Patients >65 years of age had a higher probability of intermediate/strong ABCA3 expression (FPS) than younger patients (p < 0.05). In PI measurement, there were no significant correlations between ABCA3 and clinicopathological parameters. Patients with high-level PI had a significantly worse disease-free survival as well as overall survival than patients with low-level PI (p < 0.05). CONCLUSIONS: High-level PI of ABCA3 in NSCLC showed poor disease-free and overall survival in this patient cohort, potentially indicating the relevance of ABCA3 in lung cancer. This observation needs to be validated in larger series.

Context-Dependent Epigenetic Regulation of Nuclear Factor of Activated T Cells 1 in Pancreatic Plasticity.

BACKGROUND & AIMS: The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation. METHODS: We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1 expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic approaches of EZH2 and NFATC1 inhibition to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays. RESULTS: NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on the NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1. CONCLUSIONS: In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity as an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.

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.

A new probe for super-resolution imaging of membranes elucidates trafficking pathways.

The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.