CellViT: Vision Transformers for precise cell segmentation and classification.

Nuclei detection and segmentation in hematoxylin and eosin-stained (H&E) tissue images are important clinical tasks and crucial for a wide range of applications. However, it is a challenging task due to nuclei variances in staining and size, overlapping boundaries, and nuclei clustering. While convolutional neural networks have been extensively used for this task, we explore the potential of Transformer-based networks in combination with large scale pre-training in this domain. Therefore, we introduce a new method for automated instance segmentation of cell nuclei in digitized tissue samples using a deep learning architecture based on Vision Transformer called CellViT. CellViT is trained and evaluated on the PanNuke dataset, which is one of the most challenging nuclei instance segmentation datasets, consisting of nearly 200,000 annotated nuclei into 5 clinically important classes in 19 tissue types. We demonstrate the superiority of large-scale in-domain and out-of-domain pre-trained Vision Transformers by leveraging the recently published Segment Anything Model and a ViT-encoder pre-trained on 104 million histological image patches - achieving state-of-the-art nuclei detection and instance segmentation performance on the PanNuke dataset with a mean panoptic quality of 0.50 and an F1-detection score of 0.83. The code is publicly available at https://github.com/TIO-IKIM/CellViT.

Diagnostic Accuracy of 68Ga-FAPI Versus 18F-FDG PET in Patients with Various Malignancies.

To assess the diagnostic accuracy of 68Ga-labeled fibroblast activation protein inhibitor (FAPI) and 18F-labeled FDG PET for the detection of various tumors, we performed a head-to-head comparison of both imaging modalities across a range of tumor entities as part of our ongoing 68Ga-FAPI PET observational trial. Methods: The study included 115 patients with 8 tumor entities who received imaging with 68Ga-FAPI for tumor staging or restaging between October 2018 and March 2022. Of those, 103 patients received concomitant imaging with 68Ga-FAPI and 18F-FDG PET and had adequate lesion validation for accuracy analysis. Each scan was evaluated for the detection of primary tumor, lymph nodes, and visceral and bone metastases. True or false positivity and negativity to detected lesions was assigned on the basis of histopathology from biopsies or surgical excision, as well as imaging validation. Results: 68Ga-FAPI PET revealed higher accuracy than 18F-FDG PET in the detection of colorectal cancer (n = 14; per-patient, 85.7% vs. 78.6%; per-region, 95.6% vs. 91.1%) and prostate cancer (n = 22; per-patient, 100% vs. 90.9%; per-region, 96.4% vs. 92.7%). 68Ga-FAPI PET and 18F-FDG PET had comparable per-patient accuracy in detecting breast cancer (n = 16, 100% for both) and head and neck cancers (n = 10, 90% for both modalities). 68Ga-FAPI PET had lower per-patient accuracy than 18F-FDG PET in cancers of the bladder (n = 12, 75% vs. 100%) and kidney (n = 10, 80% vs. 90%), as well as lymphoma (n = 9, 88.9% vs. 100%) and myeloma (n = 10, 80% vs. 90%). Conclusion: 68Ga-FAPI PET demonstrated higher diagnostic accuracy than 18F-FDG PET in the diagnosis of colorectal cancer and prostate cancer, as well as comparable diagnostic performance for cancers of the breast and head and neck. Accuracy and impact on management will be further assessed in an ongoing prospective interventional trial (NCT05160051).

Fibroblast Activation Protein α-Directed Imaging and Therapy of Solitary Fibrous Tumor.

Fibroblast activation protein α (FAPα) is expressed at high levels in several types of tumors. Here, we report the expression pattern of FAPα in solitary fibrous tumor (SFT) and its potential use as a radiotheranostic target. Methods: We analyzed FAPα messenger RNA and protein expression in biopsy samples from SFT patients using immunohistochemistry and multiplexed immunofluorescence. Tracer uptake and detection efficacy were assessed in patients undergoing clinical 68Ga-FAPα inhibitor (FAPI)-46 PET,18F-FDG PET, and contrast-enhanced CT. 90Y-FAPI-46 radioligand therapy was offered to eligible patients with progressive SFT. Results: Among 813 patients and 126 tumor entities analyzed from the prospective observational MASTER program of the German Cancer Consortium, SFT (n = 34) had the highest median FAPα messenger RNA expression. Protein expression was confirmed in tumor biopsies from 29 of 38 SFT patients (76%) in an independent cohort. Most cases showed intermediate to high FAPα expression by immunohistochemistry (24/38 samples, 63%), which was located primarily on the tumor cell surface. Nineteen patients who underwent 68Ga-FAPI-46 PET imaging demonstrated significantly increased tumor uptake, with an SUVmax of 13.2 (interquartile range [IQR], 10.2), and an improved mean detection efficacy of 94.5% (SEM, 4.2%), as compared with 18F-FDG PET (SUVmax, 3.2 [IQR, 3.1]; detection efficacy, 77.3% [SEM, 5.5%]). Eleven patients received a total of 34 cycles (median, 3 cycles [IQR, 2 cycles]) of 90Y-FAPI-46 radioligand therapy, which resulted in disease control in 9 patients (82%). Median progression-free survival was 227 d (IQR, 220 d). Conclusion: FAPα is highly expressed by SFT and may serve as a target for imaging and therapy. Further studies are warranted to define the role of FAPα-directed theranostics in the care of SFT patients.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. RESULTS: We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. CONCLUSION: Overall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

Development of Potent Dual BET/HDAC Inhibitors via Pharmacophore Merging and Structure-Guided Optimization.

Bromodomain and extra-terminal domain (BET) proteins and histone deacetylases (HDACs) are prime targets in cancer therapy. Recent research has particularly focused on the development of dual BET/HDAC inhibitors for hard-to-treat tumors, such as pancreatic cancer. Here, we developed a new series of potent dual BET/HDAC inhibitors by choosing starting scaffolds that enabled us to optimally merge the two functionalities into a single compound. Systematic structure-guided modification of both warheads then led to optimized binders that were superior in potency to both parent compounds, with the best molecules of this series binding to both BRD4 bromodomains as well as HDAC1/2 with EC50 values in the 100 nM range in cellular NanoBRET target engagement assays. For one of our lead molecules, we could also show the selective inhibition of HDAC1/2 over all other zinc-dependent HDACs. Importantly, this on-target activity translated into promising efficacy in pancreatic cancer and NUT midline carcinoma cells. Our lead molecules effectively blocked histone H3 deacetylation in pancreatic cancer cells and upregulated the tumor suppressor HEXIM1 and proapoptotic p57, both markers of BET inhibition. In addition, they have the potential to downregulate the oncogenic drivers of NUT midline carcinoma, as demonstrated for MYC and TP63 mRNA levels. Overall, this study expands the portfolio of available dual BET/class I HDAC inhibitors for future translational studies in different cancer models.

68Ga-Labeled Fibroblast Activation Protein Inhibitor (68Ga-FAPI) PET for Pancreatic Adenocarcinoma: Data from the 68Ga-FAPI PET Observational Trial.

The fibroblast activation protein (FAP) is highly expressed on carcinoma-associated fibroblasts in the stroma of pancreatic cancer and thus is a promising target for imaging and therapy. Preliminary data on PET imaging with radiolabeled FAP inhibitors (FAPIs) demonstrate superior tumor detection. Here we assess the accuracy of FAP-directed PET in patients with pancreatic cancer. Methods: Of 64 patients with suspected or proven pancreatic cancer, 62 (97%) were included in the data analysis of the 68Ga-FAPI PET observational trial (NCT04571086). All of these patients underwent contrast-enhanced CT, and 38 patients additionally underwent 18F-FDG PET. The primary study endpoint was the association of 68Ga-FAPI PET uptake intensity and histopathologic FAP expression. Secondary endpoints were detection rate, diagnostic performance, interreader reproducibility, and change in management. Datasets were interpreted by 2 masked readers. Results: The primary endpoint was met: The association between 68Ga-FAPI SUVmax and histopathologic FAP expression was significant (Spearman r, 0.48; P = 0.04). For histopathology-validated lesions, 68Ga-FAPI PET showed high sensitivity and positive predictive values (PPVs) on per-patient (sensitivity, 100%; PPV, 96.3%) and per-region (sensitivity, 100%; PPV, 97.0%) bases. In a head-to-head comparison versus 18F-FDG or contrast-enhanced CT, 68Ga-FAPI detected more tumor on a per-lesion (84.7% vs. 46.5% vs. 52.9%), per-patient (97.4% vs. 73.7% vs. 92.1%), or per-region (32.6% vs. 18.8% vs. 23.7%) basis, respectively. 68Ga-FAPI PET readers showed substantial overall agreement on the basis of the Fleiss κ: primary κ, 0.77 (range, 0.66-0.88). Minor and major changes in clinical management occurred in 5 patients (8.4%) after 68Ga-FAPI PET. Conclusion: We confirmed an association of 68Ga-FAPI PET SUVmax and histopathologic FAP expression in pancreatic cancer patients. Additionally, we found high detection rate and diagnostic accuracy, superior to those of 18F-FDG PET/CT. 68Ga-FAPI might become a powerful diagnostic tool for pancreatic cancer work-up.

Protocol of the IntenSify-Trial: An open-label phase I trial of the CYP3A inhibitor cobicistat and the cytostatics gemcitabine and nab-paclitaxel in patients with advanced stage or metastatic pancreatic ductal adenocarcinoma to evaluate the combination's pharmacokinetics, safety, and efficacy.

Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2 ) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2 ) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.

Three-month life expectancy as inclusion criterion for clinical trials in advanced pancreatic cancer: is it really a valid tool for patient selection?

PURPOSE: To analyze the 3-month life expectancy rate in pancreatic cancer (PC) patients treated within prospective trials from the German AIO study group. PATIENTS AND METHODS: A pooled analysis was conducted for patients with advanced PC that were treated within five phase II/III studies conducted between 1997 and 2017 (Gem/Cis, Ro96, RC57, ACCEPT, RASH). The primary goal for the current report was to identify the actual 3-month survival rate, a standard inclusion criterion in oncology trials. RESULTS: Overall, 912 patients were included, 83% had metastatic and 17% locally advanced PC; the estimated median overall survival (OS) was 7.1 months. Twenty-one percent of the participants survived < 3 months, with a range from 26% in RC57 to 15% in RASH. Significant predictors for not reaching 3-month OS were > 1 previous treatment line (p < 0.001) and performance status (p < 0.001). CONCLUSIONS: Despite the definition of a life expectancy of > 3 months as a standard inclusion criterion in clinical trials for advanced PC, a significant proportion of study patients does not survive > 3 months. TRIAL REGISTRATION NUMBERS: NCT00440167 (AIO-PK0104), NCT01729481 (RASH), NCT01728818 (ACCEPT).

Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Oncologic Staging with 68Ga-FAPI PET/CT Demonstrates a Lower Rate of Nonspecific Lymph Node Findings Than 18F-FDG PET/CT.

Nonspecific lymph node uptake on 18F-FDG PET/CT imaging is a significant pitfall for tumor staging. Fibroblast activation protein α expression on cancer-associated fibroblasts and some tumor cells is less sensitive to acute inflammatory stimuli, and fibroblast activation protein-directed PET may overcome this limitation. Methods: Eighteen patients from our prospective observational study underwent 18F-FDG and 68Ga fibroblast activation protein inhibitor (FAPI) PET/CT scans within a median of 2 d (range, 0-22 d). Lymph nodes were assessed on histopathology and compared with SUV measurements. Results: On a per-patient basis, lymph nodes were rated malignant in 10 (56%) versus 7 (39%) patients by 18F-FDG PET/CT versus 68Ga-FAPI PET/CT scans, respectively, with a respective accuracy of 55% versus 94% for true lymph node metastases. Five of 6 (83%) false-positive nodes on the 18F-FDG PET/CT scans were rated true negative by the 68Ga-FAPI PET/CT scans. On a per-lesion basis, tumor detection rates were similar (85/89 lesions, 96%). Conclusion: 68Ga-FAPI PET/CT imaging demonstrated higher accuracy for true nodal involvement and therefore has the potential to replace 18F-FDG PET/CT imaging for cancer staging.

Practical guidelines for the management of adverse events of the T cell engager bispecific tebentafusp.

Tebentafusp is a new T cell receptor bispecific fusion protein and the first approved treatment option for human leucocyte antigen-A*02:01 (HLA-A*02:01) metastatic uveal melanoma, with a proven benefit in overall survival versus the investigator's choice. As a first-in-class therapeutic option, this Immune mobilising monoclonal T cell receptor Against Cancer (ImmTAC) is associated with a new adverse event (AE) profile. Based on clinical experience, a national expert group discussed recommendations for tebentafusp treatment, focusing on AE management. Further topics included prerequisites for initiating tebentafusp treatment, appropriate treatment setting, and patient selection criteria. To provide guidance for treating physicians, the resulting recommendations are summarised including a model standard operating procedure for AE management. Patients in good clinical condition and with a low tumour burden are good candidates for tebentafusp treatment, particularly if treated as early as possible after the diagnosis of metastatic disease. The safety profile of tebentafusp is manageable and includes two major pathologies: cytokine release syndrome (CRS) and skin-related events. Postdose monitoring should thus focus on pyrexia and hypotension as the first symptoms of cytokine release. To minimise the risk of hypotension associated with CRS, patients should receive intravenous fluids before starting treatment. The monitoring of liver values is crucial, as patients may experience an increase in transaminases, which can even manifest as tumour lysis syndrome.

Histology-Based Prediction of Therapy Response to Neoadjuvant Chemotherapy for Esophageal and Esophagogastric Junction Adenocarcinomas Using Deep Learning.

PURPOSE: Quantifying treatment response to gastroesophageal junction (GEJ) adenocarcinomas is crucial to provide an optimal therapeutic strategy. Routinely taken tissue samples provide an opportunity to enhance existing positron emission tomography-computed tomography (PET/CT)-based therapy response evaluation. Our objective was to investigate if deep learning (DL) algorithms are capable of predicting the therapy response of patients with GEJ adenocarcinoma to neoadjuvant chemotherapy on the basis of histologic tissue samples. METHODS: This diagnostic study recruited 67 patients with I-III GEJ adenocarcinoma from the multicentric nonrandomized MEMORI trial including three German university hospitals TUM (University Hospital Rechts der Isar, Munich), LMU (Hospital of the Ludwig-Maximilians-University, Munich), and UME (University Hospital Essen, Essen). All patients underwent baseline PET/CT scans and esophageal biopsy before and 14-21 days after treatment initiation. Treatment response was defined as a ≥35% decrease in SUVmax from baseline. Several DL algorithms were developed to predict PET/CT-based responders and nonresponders to neoadjuvant chemotherapy using digitized histopathologic whole slide images (WSIs). RESULTS: The resulting models were trained on TUM (n = 25 pretherapy, n = 47 on-therapy) patients and evaluated on our internal validation cohort from LMU and UME (n = 17 pretherapy, n = 15 on-therapy). Compared with multiple architectures, the best pretherapy network achieves an area under the receiver operating characteristic curve (AUROC) of 0.81 (95% CI, 0.61 to 1.00), an area under the precision-recall curve (AUPRC) of 0.82 (95% CI, 0.61 to 1.00), a balanced accuracy of 0.78 (95% CI, 0.60 to 0.94), and a Matthews correlation coefficient (MCC) of 0.55 (95% CI, 0.18 to 0.88). The best on-therapy network achieves an AUROC of 0.84 (95% CI, 0.64 to 1.00), an AUPRC of 0.82 (95% CI, 0.56 to 1.00), a balanced accuracy of 0.80 (95% CI, 0.65 to 1.00), and a MCC of 0.71 (95% CI, 0.38 to 1.00). CONCLUSION: Our results show that DL algorithms can predict treatment response to neoadjuvant chemotherapy using WSI with high accuracy even before therapy initiation, suggesting the presence of predictive morphologic tissue biomarkers.

Dose-dependent expression of GFI1 alters metabolism in the haematopoietic progenitors and MLL::AF9-induced leukaemic cells.

Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose-dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose-dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in-vitro and ex-vivo murine models of MLL::AF9-induced human AML and extra-cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1- MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1-low-expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism.

Tebentafusp in Patients with Metastatic Uveal Melanoma: A Real-Life Retrospective Multicenter Study.

BACKGROUND: Tebentafusp has recently been approved for the treatment of metastatic uveal melanoma (mUM) after proving to have survival benefits in a first-line setting. PATIENTS AND METHODS: This retrospective, multicenter study analyzed the outcomes and safety of tebentafusp therapy in 78 patients with mUM. RESULTS: Patients treated with tebentafusp had a median PFS of 3 months (95% CI 2.7 to 3.3) and a median OS of 22 months (95% CI 10.6 to 33.4). In contrast to a published Phase 3 study, our cohort had a higher rate of patients with elevated LDH (65.4% vs. 35.7%) and included patients with prior systemic and local ablative therapies. In patients treated with tebentafusp following ICI, there was a trend for a longer median OS (28 months, 95% CI 26.9 to 29.1) compared to the inverse treatment sequence (24 months, 95% CI 13.0 to 35.0, p = 0.257). The most common treatment-related adverse events were cytokine release syndrome in 71.2% and skin toxicity in 53.8% of patients. Tumor lysis syndrome occurred in one patient. CONCLUSIONS: Data from this real-life cohort showed a median PFS/OS similar to published Phase 3 trial data. Treatment with ICI followed by tebentafusp may result in longer PFS/OS compared to the inverse treatment sequence.

Fibroblast Activation Protein-Targeted Photodynamic Therapy of Cancer-Associated Fibroblasts in Murine Models for Pancreatic Ductal Adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.

Fibroblast Activation Protein-Targeting Minibody-IRDye700DX for Ablation of the Cancer-Associated Fibroblast with Photodynamic Therapy.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein's dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. IMPLICATIONS: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

Integrated Analysis of the RASH Study with the Use of the "Burden of Therapy" (BOTh®TM) Methodology-A Novel Tool for Assessing Adverse Events in Metastatic Pancreatic Cancer.

This analysis of the RASH trial (NCT01729481) aimed at gaining a better understanding of the "Burden of Therapy" (BOTh®TM) in pancreatic ductal adenocarcinoma (PDAC). In the RASH study, 150 patients with newly diagnosed metastatic PDAC were treated with gemcitabine plus erlotinib (gem/erlotinib) for four weeks. Patients who developed a skin rash during this four-week run-in phase continued with the gem/erlotinib treatment, while rash-negative patients were switched to FOLFIRINOX. The study demonstrated a 1-year survival rate of rash-positive patients who received gem/erlotinib as first-line treatment that was comparable to previous reports of patients receiving FOLFIRINOX. To understand whether these comparable survival rates may be accompanied by better tolerability of the gem/erlotinib treatment compared to FOLFIRINOX, the BOTh®TM methodology was used to continuously quantify and depict the burden of therapy generated by treatment emergent events (TEAEs). Sensory neuropathy was significantly more common in the FOLFIRINOX arm, and prevalence as well as severity increased over time. In both arms, the BOTh®TM associated with diarrhea decreased over the course of treatment. The BOTh®TM caused by neutropenia was comparable in both arms but decreased in the FOLFIRINOX arm over time, possibly due to chemotherapy dose reductions. Overall, gem/erlotinib was associated with a slightly higher overall BOTh®TM, but the difference was not statistically significant (p = 0.6735). In summary, the BOTh®TM analysis facilitates the evaluation of TEAEs. In patients fit for intense chemotherapeutic regimens, FOLFIRINOX is associated with a lower BOTh®TM than gem/erlotinib.

Superior Tumor Detection for 68Ga-FAPI-46 Versus 18F-FDG PET/CT and Conventional CT in Patients with Cholangiocarcinoma.

Management of cholangiocarcinoma is among other factors critically determined by accurate staging. Here, we aimed to assess the accuracy of PET/CT with the novel cancer fibroblast-directed 68Ga-fibroblast activation protein (FAP) inhibitor (FAPI)-46 tracer for cholangiocarcinoma staging and management guidance. Methods: Patients with cholangiocarcinoma from a prospective observational trial were analyzed. 68Ga-FAPI-46 PET/CT detection efficacy was compared with 18F-FDG PET/CT and conventional CT. SUVmax/tumor-to-background ratio (Wilcoxon test) and separately uptake for tumor grade and location (Mann-Whitney U test) were compared. Immunohistochemical FAP and glucose transporter 1 (GLUT1) expression of stromal and cancer cells was analyzed. The impact on therapy management was investigated by pre- and post-PET/CT questionnaires sent to the treating physicians. Results: In total, 10 patients (6 with intrahepatic cholangiocarcinoma and 4 with extrahepatic cholangiocarcinoma; 6 with grade 2 tumor and 4 with grade 3 tumor) underwent 68Ga-FAPI-46 PET/CT and conventional CT; 9 patients underwent additional 18F-FDG PET/CT. Immunohistochemical analysis was performed on the entire central tumor plain in 6 patients. Completed questionnaires were returned in 8 cases. Detection rates for 68Ga-FAPI-46 PET/CT, 18F-FDG PET/CT, and CT were 5, 5, and 5, respectively, for primary tumor; 11, 10, and 3, respectively, for lymph nodes; and 6, 4, and 2, respectively, for distant metastases. 68Ga-FAPI-46 versus 18F-FDG PET/CT SUVmax for primary tumor, lymph nodes, and distant metastases was 14.5 versus 5.2 (P = 0.043), 4.7 versus 6.7 (P = 0.05), and 9.5 versus 5.3 (P = 0.046), respectively, and tumor-to-background ratio (liver) was 12.1 versus 1.9 (P = 0.043) for primary tumor. Grade 3 tumors demonstrated a significantly higher 68Ga-FAPI-46 uptake than grade 2 tumors (SUVmax, 12.6 vs. 6.4; P = 0.009). Immunohistochemical FAP expression was high on tumor stroma (∼90% of cells positive), whereas GLUT1 expression was high on tumor cells (∼80% of cells positive). Overall, average expression intensity was estimated as grade 3 for FAP and grade 2 for GLUT1. Positive 68Ga-FAPI-46 PET findings led to a consequent biopsy workup and diagnosis of cholangiocarcinoma in 1 patient. However, patient treatment was not adjusted on the basis of 68Ga-FAPI-46 PET. Conclusion: 68Ga-FAPI-46 demonstrated superior radiotracer uptake, especially in grade 3 tumors, and lesion detection in patients with cholangiocarcinoma. In line with this result, immunohistochemistry demonstrated high FAP expression on tumor stroma. Accuracy is under investigation in an ongoing investigator-initiated trial.

Evolutionary and immune microenvironment dynamics during neoadjuvant treatment of oesophagael adenocarcinoma.

Locally advanced oesophageal adenocarcinoma (EAC) remains difficult to treat because of common resistance to neoadjuvant therapy and high recurrence rates. The ecological and evolutionary dynamics responsible for treatment failure are incompletely understood. Here, we performed a comprehensive multi-omic analysis of samples collected from EAC patients in the MEMORI clinical trial, revealing major changes in gene expression profiles and immune microenvironment composition that did not appear to be driven by changes in clonal composition. Multi-region multi-timepoint whole exome (300x depth) and paired transcriptome sequencing was performed on 27 patients pre-, during and after neoadjuvant treatment. EAC showed major transcriptomic changes during treatment with upregulation of immune and stromal pathways and oncogenic pathways such as KRAS, Hedgehog and WNT. However, genetic data revealed that clonal sweeps were rare, suggesting that gene expression changes were not clonally driven. Additional longitudinal image mass cytometry was performed in a subset of 15 patients and T-cell receptor sequencing in 10 patients, revealing remodelling of the T-cell compartment during treatment and other shifts in microenvironment composition. The presence of immune escape mechanisms and a lack of clonal T-cell expansions were linked to poor clinical treatment response. This study identifies profound transcriptional changes during treatment with limited evidence that clonal replacement is the cause, suggesting phenotypic plasticity and immune dynamics as mechanisms for therapy resistance with pharmacological relevance.