Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been ...described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress.
Abbreviations: ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA
1
: bafilomycin A
1
; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.
Purpose
IDH1
mutation is a known biomarker for targeted therapy of intrahepatic cholangiocarcinoma (iCCA), while its prognostic relevance for current palliative chemotherapy is still unclear. Aim of ...this study was to analyze clinicopathological characteristics of patients with
IDH1
mutations and to outline a potential impact on the outcome after state-of-the-art palliative chemotherapy regimens.
Methods
All patients with iCCA receiving large panel molecular profiling and follow-up treatment at Frankfurt University Hospital until 04/2022 were retrospectively analyzed. Clinicopathological characteristics were assessed for
IDH1
mutated (mut) and
IDH1
wild type (wt) patients, and progression-free survival (PFS) and overall survival (OS) were determined.
Results
In total, 75 patients with iCCA received molecular profiling. Of the patients with available DNA data, pathogenic mutations in
IDH1
were found in 14.5% (
n
= 10).
IDH1
mut status was associated with lower serum CA-19/9 (
p
= 0.023), lower serum lactate dehydrogenase (
p
= 0.006), and a higher proportion of primary resectability (
p
= 0.028) as well as response to chemotherapy after recurrence (
p
= 0.009). Median PFS was 5.9 months (95% CI 4.4–7.3 months) for
IDH1
wt in comparison to 9.8 months (95% CI 7.7–12 months) for patients with
IDH1
mut (
p
= 0.031).
IDH1
wt was a significant risk factor for shortened PFS in univariate (
p
= 0.043), but not in multivariate analysis (
p
= 0.061). There was no difference in OS between both groups.
Conclusion
Patients with
IDH1
mutated iCCA seem to have a favorable tumor biology including a longer PFS for palliative chemotherapy regimens compared to
IDH1
wild type.
We aimed to identify hepatocellular carcinoma (HCC) patients who will respond to repetitive transarterial chemoembolization (TACE) to improve the treatment algorithm. Retrospectively, 61 patients ...(mean age, 65.3 years ± 10.0 SD; 49 men) with 94 HCC mRECIST target-lesions who had three consecutive TACE between 01/2012 and 01/2020 were included. Robust and non-redundant radiomics features were extracted from the 24 h post-embolization CT. Five different clinical TACE-scores were assessed. Seven different feature selection methods and machine learning models were used. Radiomics, clinical and combined models were built to predict response to TACE on a lesion-wise and patient-wise level as well as its impact on overall-survival prognostication. 29 target-lesions of 19 patients were evaluated in the test set. Response rates were 37.9% (11/29) on the lesion-level and 42.1% (8/19) on the patient-level. Radiomics top lesion-wise response prognostications was AUC 0.55-0.67. Clinical scores revealed top AUCs of 0.65-0.69. The best working model combined the radiomic feature LargeDependenceHighGrayLevelEmphasis and the clinical score mHAP_II_score_group with AUC = 0.70, accuracy = 0.72. We transferred this model on a patient-level to achieve AUC = 0.62, CI = 0.41-0.83. The two radiomics-clinical features revealed overall-survival prognostication of C-index = 0.67. In conclusion, a random forest model using the radiomic feature LargeDependenceHighGrayLevelEmphasis and the clinical mHAP-II-score-group seems promising for TACE response prognostication.
Data on the impact of autophagy in primary cholangiocarcinoma (CCA) remain scarce. Here, we therefore investigated the role of active autophagy and its impact on survival in CCA patients. All CCA ...patients who underwent surgical resection with curative intent between 08/2005 and 12/2021 at University Hospital Frankfurt were evaluated. Autophagic key proteins were studied by immunohistochemistry. iCCA processed for gene expression profiling of immune-exhaustion gene sets was used for an autophagy approach in silico. Active autophagy was present in 23.3% of the 172 CCA patients. Kaplan–Meier curves revealed median OS of 68.4 months (95% CI = 46.9–89.9 months) and 32.7 months (95% CI = 23.6–41.8 months) for active and non-active autophagy, respectively (p ≤ 0.001). In multivariate analysis, absence of active autophagy (HR = 2, 95% CI = 1.1–3.5, p = 0.015) was an independent risk factor for OS. Differential-expression profiling revealed significantly upregulated histone deacetylases (HDAC) mRNA in patients showing non-active autophagy. In line with this, pan-acetylated lysine was significantly more prominent in CCA patients with ongoing autophagy (p = 0.005). Our findings strengthen the role of active autophagy as a prognostically relevant marker and a potential therapeutic target.
Fluorescence confocal microscopy (FCM) is a rapidly evolving tool that provides real-time virtual HE images of native tissue. Data about the potential of FCM as an alternative to frozen sections for ...the evaluation of donor liver specimens are lacking so far. The aim of the current study was to determine the value of FCM in liver specimens according to the criteria of the German Society for Organ Procurement. In this prospective study, conventional histology and FCM scans of 50 liver specimens (60% liver biopsies, 26% surgical specimens, and 14% donor samples) were evaluated according to the German Society for Organ Procurement. A comparison of FCM scans and conventional frozen sections revealed almost perfect levels of agreement for cholangitis (κ = 0.877), fibrosis (κ = 0.843), and malignancy (κ = 0.815). Substantial levels of agreement could be obtained for macrovesicular steatosis (κ = 0.775), inflammation (κ = 0.763), necrosis (κ = 0.643), and steatohepatitis (κ = 0.643). Levels of agreement were moderate for microvesicular steatosis (κ = 0.563). The strength of agreement between frozen sections and FCM was superior to the comparison of conventional HE and FCM imaging. We introduce FCM as a potential alternative to the frozen section that may represent a novel approach to liver transplant pathology where timely feedback is crucial and the deployment of human resources is becoming increasingly difficult.
MUC16/CA125 is associated with cancer proliferation in several tumor entities. The data on MUC16 expression in cholangiocarcinoma (CCA) tissue are very limited. The aim of this study was to assess ...the MUC16 status and its impact on survival in CCA patients. All the patients with surgically resected CCA that were diagnosed between August 2005 and December 2021 at the University Hospital Frankfurt were retrospectively analyzed. A 7-Mucin biomarker panel was assessed by immunohistochemistry. For overall survival (OS), Kaplan−Meier curves and Cox-regression analyses were performed. Randomly selected intrahepatic cholangiocarcinoma (iCCA) were further processed for differential expression profiling. A total of 168 patients with CCA were classified as MUC16 (−) (66%, n = 111) and MUC16 (+) (34%, n = 57). Subgroup analyses revealed a median OS of 56.1 months (95% CI = 42.4−69.9 months) and 27.4 months (95% CI = 15.8−39.1 months) for MUC16 (−) and MUC16 (+), respectively (p < 0.001). In multivariate analysis, MUC16 (+) (HR = 1.6, 95% CI = 1−2.6, p = 0.032) was an independent risk factor for poor prognosis. Prominently deregulated pathways have been identified following MUC16 expression, overrepresented in cell cycle and immune system exhaustion processes. These findings suggest including MUC16 in clinical routine diagnostics as well as studying its molecular pathways to identify further mechanistic key players.
Aims
The histological subtype of intrahepatic cholangiocarcinoma (iCCA) is associated with different mutational characteristics that impact clinical management. So far, data are lacking on the ...presence of small duct iCCA (SD‐iCCA) and large duct iCCA (LD‐iCCA) in a single patient. The aim of the current study was to determine the presence and degree of intratumoural heterogeneity of SD‐ and LD‐iCCA features in different tumour regions.
Methods and results
All patients treated with surgically resected iCCA at Frankfurt University Hospital between December 2005 and March 2023 were retrospectively analysed. Histomorphological features of SD‐ and LD‐iCCA were evaluated by an expert hepatobiliary pathologist. Tissue samples suspicious for subtype heterogeneity were further investigated. Immunohistochemistry for N‐cadherin, S100P, MUC5AC, MUC6, TFF1 and AGR2 and mutational profiling with the Illumina TruSight Oncology 500 (TSO500) assay were performed separately for the SD‐ and LD‐iCCA regions. Of 129 patients with surgically resected iCCA, features of either SD‐ or LD‐iCCA were present in 67.4% (n = 87) and 24.8% of the patients (n = 32), respectively; 7.8% (n = 10) had histomorphological features of both SD‐ and LD‐iCCA, seven patients (5.4%) of which had sufficient formalin‐fixed, paraffin‐embedded tissue for further analysis. Heterogeneity of both subtypes could be confirmed with immunohistochemistry. In five of seven (71.4%) patients, molecular profiling revealed intratumoural differences in genetic alterations between the SD‐ and LD‐iCCA region. In one patient, a BRAF mutation (p.V600E) was found in the SD‐iCCA but not in the LD‐iCCA region of the tumour.
Conclusions
A marked portion of patients with iCCA exhibits both SD‐ and LD‐iCCA in different tumour regions. In case of the presence of histopathological heterogeneity, mutational profiling should be considered to avoid missing therapeutically relevant genetic alterations.
•Comparison of DECT biomarkers revealed significant differences between iCCA and HCC.•ID at the arterial phase yielded the best diagnostic performance.•An ID threshold of 2.33 mg/dl was determined to ...differentiate between both entities.
To assess the potential of material decomposition in dual-energy CT (DECT) to differentiate intrahepatic cholangiocarcinoma (iCCA) from hepatocellular carcinoma (HCC).
In this retrospective study, we included 94 patients (26 female (27.7 %), median age 64.5 (interquartile range 55.5–74.5) years) with either iCCA or HCC who underwent abdominal contrast-enhanced DECT in arterial phase. To test for differences between iCCA (n = 47) and HCC (n = 47), we evaluated mean attenuation and DECT material density values including iodine density (ID), normalized iodine uptake (NIU), fat fraction, and lesion-to-liver parenchyma ratio. Histopathology served as reference standard for all lesions. We used univariate logistic regression models for the outcome iCCA versus HCC. ROC curve analysis was applied to assess discriminative ability of the model. Model accuracy was evaluated by calculating the Brier score. Youden index was applied to establish thresholds to differentiate between iCCA and HCC.
Comparison of quantitative image parameters revealed significant differences between iCCA and HCC for ID (1.6 ± 0.5 mg/ml vs 2.8 ± 0.8 mg/ml, p < 0.001), NIU (14.5 ± 4.8 vs 24.8 ± 10.3, p < 0.001), attenuation (41.9 ± 10.1 HU vs 47.9 ± 8.9 HU, p = 0.003), and fat fraction (12.0 ± 7.8 % vs 9.0 ± 6.4 %, p = 0.045). ROC curve analysis revealed highest ability to differentiate iCCA from HCC for ID (AUC = 0.93, 95 % CI 0.89–0.98). For ID, an optimal threshold of 2.33 mg/dl was determined to discriminate between iCCA and HCC (sensitivity 89.4 %, specificity 76.6 %).
DECT-based iodine quantification can serve as a tool for the differentiation of iCCA and HCC in contrast-enhanced CT. ID yielded the highest diagnostic performance and may assist in clinical routine CT diagnostics.
Distinct immune patterns of hepatocellular carcinoma (HCC) may have prognostic implications in the response to transarterial chemoembolization (TACE). Thus, we aimed to exploratively analyze tumor ...tissue of HCC patients who do or do not respond to TACE, and to identify novel prognostic biomarkers predictive of response to TACE. We retrospectively included 15 HCC patients who had three consecutive TACE between January 2019 and November 2019. Eight patients had a response while seven patients had no response to TACE. All patients had measurable disease according to mRECIST. Corresponding tumor tissue samples were processed for differential expression profiling using NanoString nCounter
PanCancer immune profiling panel. Immune-related pathways were broadly upregulated in TACE responders. The top differentially regulated genes were the upregulated CXCL1 (log2fc 4.98, Benjamini-Hochberg (BH)-
< 0.001), CXCL6 (log2fc 4.43, BH-
= 0.016) and the downregulated MME (log2fc -4.33, BH-
0.001). CD8/T-regs was highly increased in responders, whereas the relative number of T-regs to tumor-infiltrating lymphocytes (TIL) was highly decreased. We preliminary identified CXCL1 and CXCL6 as candidate genes that might have the potential to serve as therapeutically relevant biomarkers in HCC patients. This might pave the way to improve patient selection for TACE in HCC patients beyond expert consensus.
In recent years, histopathological characterization of intrahepatic cholangiocarcinoma revealed small duct type (SD-iCCA) and large duct type (LD-iCCA). Data on the prevalence of the subtypes are ...limited and highly varying. The aim of this study was to assess the prevalence of SD-iCCA and LD-iCCA and their impact on survival for the first time in a European cohort.
All patients with surgically resected iCCA diagnosed between December 2005 and December 2021 at the University Hospital Frankfurt were analyzed by an expert hepatobiliary pathologist. For overall survival (OS) and progression-free survival (PFS), Kaplan-Meier curves and Cox-regression analyses were performed.
In total, 116 patients with surgically resected iCCA treated in our tertiary hospital were classified as SD-iCCA (73.3%, n = 85) and LD-iCCA (26.7%, n = 31). Subgroup analyses revealed median OS of 54.4 months (95% CI = 38.3 – 70.4 months) and 25.4 months (95% CI = 15.1 – 35.7 months) for SD-iCCA and LD-iCCA, respectively (p = 0.027). The median PFS for patients receiving gemcitabine-based chemotherapy with SD- and LD-iCCA was 8.4 months (95% CI = 4.7 – 12 months) and 3.3 months (95% CI = 1.8 – 4.7 months), respectively (p = 0.011). While LD-iCCA was as a significant risk factor of OS (HR = 1.7, 95% CI = 1 – 2.8, p = 0.031) in univariate analysis, it was not significant in multivariate analysis.
In contrast to data from Asia, SD-iCCA is more prevalent than LD-iCCA in our cohort. LD-iCCA is associated with impaired OS after surgical resection and decreased PFS for patients receiving chemotherapy. These findings may suggest including the histological subtype in clinical routine diagnostics.
•SD-iCCA is more prevalent than LD-iCCA in this European cohort.•LD-iCCA is associated with poor overall survival.•PFS is impaired for LD-iCCA receiving gemcitabine-based chemotherapy.
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