68Ga-Fibroblast Activation Protein Inhibitor PET/CT Improves Detection of Intermediate and Low-Grade Sarcomas and Identifies Candidates for Radiopharmaceutical Therapy.

Fibroblast activation protein-α (FAP) is often highly expressed by sarcoma cells and by sarcoma-associated fibroblasts in the tumor microenvironment. This makes it a promising target for imaging and therapy. The level of FAP expression and the diagnostic value of 68Ga-FAP inhibitor (FAPI) PET for sarcoma subtypes are unknown. We assessed the diagnostic performance and accuracy of 68Ga-FAPI PET in various bone and soft-tissue sarcomas. Potential eligibility for FAP-targeted radiopharmaceutical therapy (FAP-RPT) was evaluated. Methods: This prospective observational trial enrolled 200 patients with bone and soft-tissue sarcoma who underwent 68Ga-FAPI PET/CT and 18F-FDG PET/CT (186/200, or 93%) for staging or restaging. The number of lesions detected and the uptake (SUVmax) of the primary tumor, lymph nodes, and visceral and bone metastases were analyzed. The Wilcoxon test was used for semiquantitative assessment. The association of 68Ga-FAPI uptake intensity, histopathologic grade, and FAP expression in sarcoma biopsy samples was analyzed using Spearman r correlation. The impact of 68Ga-FAPI PET on clinical management was investigated using questionnaires before and after PET/CT. Eligibility for FAP-RPT was defined by an SUVmax greater than 10 for all tumor regions. Results: 68Ga-FAPI uptake was heterogeneous among sarcoma subtypes. The 3 sarcoma entities with the highest uptake (mean SUVmax ± SD) were solitary fibrous tumor (24.7 ± 11.9), undifferentiated pleomorphic sarcoma (18.8 ± 13.1), and leiomyosarcoma (15.2 ± 10.2). Uptake of 68Ga-FAPI versus 18F-FDG was significantly higher in low-grade sarcomas (10.4 ± 8.5 vs. 7.0 ± 4.5, P = 0.01) and in potentially malignant intermediate or unpredictable sarcomas without a World Health Organization grade (not applicable [NA]; 22.3 ± 12.5 vs. 8.5 ± 10.0, P = 0.0004), including solitary fibrous tumor. The accuracy, as well as the detection rates, of 68Ga-FAPI was higher than that of 18F-FDG in low-grade sarcomas (accuracy, 92.2 vs. 80.0) and NA sarcomas (accuracy, 96.9 vs. 81.9). 68Ga-FAPI uptake and the histopathologic FAP expression score (n = 89) were moderately correlated (Spearman r = 0.43, P < 0.0002). Of 138 patients, 62 (45%) with metastatic sarcoma were eligible for FAP-RPT. Conclusion: In patients with low-grade and NA sarcomas, 68Ga-FAPI PET demonstrates uptake, detection rates, and accuracy superior to those of 18F-FDG PET. 68Ga-FAPI PET criteria identified eligibility for FAP-RPT in about half of sarcoma patients.

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).

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.

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.

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).

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.

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.

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.

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.

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.

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.

Interactive enhancer hubs (iHUBs) mediate transcriptional reprogramming and adaptive resistance in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. DESIGN: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. RESULTS: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. CONCLUSION: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.

Fibroblast Activation Protein Inhibitor Theranostics: The Case for Use in Sarcoma.

The theranostic use of fibroblast activation protein inhibitors (FAPIs) is a novel approach in oncology. Sarcomas are a heterogenous group of rare malignant tumors. Prognosis remains poor in advanced/metastatic disease due to limited therapeutic options. Sarcoma frequently demonstrate high expression of fibroblast activation protein alpha on the tumor cells themselves, in contrast to other solid tumors, where it is mainly expressed on cancer-associated fibroblasts. Consequently, high in vivo uptake of FAPI in PET is observed in sarcoma. Moreover, retrospective case reports and series demonstrated feasibility of FAPI radioligand therapy with signs of tumor response.

SOX2 dosage sustains tumor-promoting inflammation to drive disease aggressiveness by modulating the FOSL2/IL6 axis.

BACKGROUND: Inflammation is undoubtedly a hallmark of cancer development. Its maintenance within tumors and the consequences on disease aggressiveness are insufficiently understood. METHODS: Data of 27 tumor entities (about 5000 samples) were downloaded from the TCGA and GEO databases. Multi-omic analyses were performed on these and in-house data to investigate molecular determinants of tumor aggressiveness. Using molecular loss-of-function data, the mechanistic underpinnings of inflammation-induced tumor aggressiveness were addressed. Patient specimens and in vivo disease models were subsequently used to validate findings. RESULTS: There was significant association between somatic copy number alterations (sCNAs) and tumor aggressiveness. SOX2 amplification was the most important feature among novel and known aggressiveness-associated alterations. Mechanistically, SOX2 regulates a group of genes, in particular the AP1 transcription factor FOSL2, to sustain pro-inflammatory signaling pathways, such as IL6-JAK-STAT3, TNFA and IL17. FOSL2 was found overexpressed in tumor sections of specifically aggressive cancers. In consequence, prolonged inflammation induces immunosuppression and activates cytidine deamination and thus DNA damage as evidenced by related mutational signatures in aggressive tumors. The DNA damage affects tumor suppressor genes such as TP53, which is the most mutated gene in aggressive tumors compared to less aggressive ones (38% vs 14%), thereby releasing cell cycle control. These results were confirmed by analyzing tissues from various tumor types and in vivo studies. CONCLUSION: Our data demonstrate the implication of SOX2 in promoting DNA damage and genome instability by sustaining inflammation via FOSL2/IL6, resulting in tumor aggressiveness.

Atezolizumab Plus PEGPH20 Versus Chemotherapy in Advanced Pancreatic Ductal Adenocarcinoma and Gastric Cancer: MORPHEUS Phase Ib/II Umbrella Randomized Study Platform.

BACKGROUND: The MORPHEUS platform comprises multiple open-label, randomized, phase Ib/II trials designed to identify early efficacy and safety signals of treatment combinations across cancers. Atezolizumab (anti-programmed cell death 1 ligand 1 [PD-L1]) was evaluated in combination with PEGylated recombinant human hyaluronidase (PEGPH20). METHODS: In 2 randomized MORPHEUS trials, eligible patients with advanced, previously treated pancreatic ductal adenocarcinoma (PDAC) or gastric cancer (GC) received atezolizumab plus PEGPH20, or control treatment (mFOLFOX6 or gemcitabine plus nab-paclitaxel [MORPHEUS-PDAC]; ramucirumab plus paclitaxel [MORPHEUS-GC]). Primary endpoints were objective response rates (ORR) per RECIST 1.1 and safety. RESULTS: In MORPHEUS-PDAC, ORRs with atezolizumab plus PEGPH20 (n = 66) were 6.1% (95% CI, 1.68%-14.80%) vs. 2.4% (95% CI, 0.06%-12.57%) with chemotherapy (n = 42). In the respective arms, 65.2% and 61.9% had grade 3/4 adverse events (AEs); 4.5% and 2.4% had grade 5 AEs. In MORPHEUS-GC, confirmed ORRs with atezolizumab plus PEGPH20 (n = 13) were 0% (95% CI, 0%-24.7%) vs. 16.7% (95% CI, 2.1%-48.4%) with control (n = 12). Grade 3/4 AEs occurred in 30.8% and 75.0% of patients, respectively; no grade 5 AEs occurred. CONCLUSION: Atezolizumab plus PEGPH20 showed limited clinical activity in patients with PDAC and none in patients with GC. The safety of atezolizumab plus PEGPH20 was consistent with each agent's known safety profile. (ClinicalTrials.gov Identifier: NCT03193190 and NCT03281369).