Checkpoint inhibition enhances cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells of classic Hodgkin lymphoma.

Although checkpoint molecules like CTLA-4 and PD1 have been described several years ago, checkpoint inhibitors such as Nivolumab (an anti-PD-1 antibody) have only recently been used to treat classic Hodgkin lymphoma (cHL). Several studies have shown convincing therapeutic effects of Nivolumab in cHL. However, the mechanism of action of Nivolumab in cHL is not fully understood. The aim of this study was to monitor changes in cell motility and cell contacts after administration of Nivolumab to an in vitro model of cHL as well as to native hyperplastic lymphoid tissue and native human tissue from cHL. In both tissue and in vitro, CD4+, CD8+, CD30+ and CD20+ cell velocities were unchanged after Nivolumab incubation. In contrast, in primary cHL tissue, the duration of cell contacts between CD4+ T cells and HRS cells was significantly increased after 5 h Nivolumab treatment, and the number of contacts with HRS cells was also slightly increased for CD4+ T cells (not significant), suggesting that CD4+ T cells in particular contribute to the cytotoxicity observed as a result of Nivolumab therapy. There was no change in the duration of cell contacts in the hyperplastic lymphoid tissue after Nivolumab incubation. In conclusion, we show here for the first time by imaging of native lymphoma tissue an enhanced interaction of CD4+ T cells and HRS cells in cHL after Nivolumab administration.

[Fluorescence confocal microscopy-complete digitization of pathology]. / Fluoreszenzbasierte Konfokalmikroskopie ­ vollständige Digitalisierung der Pathologie.

BACKGROUND: Fluorescence-based confocal microscopy (FCM) can be used to create virtual H&E sections in real time. So far, FCM has been used in dermato-, uro-, and gynecopathology. FCM allows the creation of a completely digitized frozen section, which could potentially replace conventional frozen sections in the future. OBJECTIVE: The aim of the current work is to implement FCM technology as a component of fully digitized processes in the pathological workflow. For this purpose, the current use of FCM in liver transplant pathology will be extended to other disciplines such as urology and otorhinolaryngology. MATERIALS AND METHODS: The FCM technique continues to be used prospectively on native tissue samples from potential donor livers. Conventional frozen sections are used comparatively to virtual FCM scans. RESULTS: The data show a nearly perfect agreement for the detection of cholangitis, fibrosis, and malignancy, and a high level of agreement for, e.g., macrovesicular steatosis, inflammation, steatohepatitis, and necrosis between virtual FCM scans and conventional routine diagnostic frozen sections. CONCLUSION: Since the availability of time- and cost-intensive frozen section diagnostics in the context of transplant pathology in continuous operation (24/7) is now only established at very few university centers in Germany due to an increasing shortage of specialists, the use of FCM could be an important building block in the current process leading towards a fully digitized pathology workflow and should thus be extended to various disciplines.

Heterogeneity of small duct- and large duct-type intrahepatic cholangiocarcinoma.

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.

[The model transferability of AI in digital pathology : Potential and reality]. / Die Modelltransferierbarkeit von KI in der digitalen Pathologie : Potenzial und Realität.

OBJECTIVE: Artificial intelligence (AI) holds the potential to make significant advancements in pathology. However, its actual implementation and certification for practical use are currently limited, often due to challenges related to model transferability. In this context, we investigate the factors influencing transferability and present methods aimed at enhancing the utilization of AI algorithms in pathology. MATERIALS AND METHODS: Various convolutional neural networks (CNNs) and vision transformers (ViTs) were trained using datasets from two institutions, along with the publicly available TCGA-MIBC dataset. These networks conducted predictions in urothelial tissue and intrahepatic cholangiocarcinoma (iCCA). The objective was to illustrate the impact of stain normalization, the influence of various artifacts during both training and testing, as well as the effects of the NoisyEnsemble method. RESULTS: We were able to demonstrate that stain normalization of slides from different institutions has a significant positive effect on the inter-institutional transferability of CNNs and ViTs (respectively +13% and +10%). In addition, ViTs usually achieve a higher accuracy in the external test (here +1.5%). Similarly, we showcased how artifacts in test data can negatively affect CNN predictions and how incorporating these artifacts during training leads to improvements. Lastly, NoisyEnsembles of CNNs (better than ViTs) were shown to enhance transferability across different tissues and research questions (+7% Bladder, +15% iCCA). DISCUSSION: It is crucial to be aware of the transferability challenge: achieving good performance during development does not necessarily translate to good performance in real-world applications. The inclusion of existing methods to enhance transferability, such as stain normalization and NoisyEnsemble, and their ongoing refinement, is of importance.

[Challenges of automation in quantitative evaluation of liver biopsies : Automatic quantification of liver steatosis]. / Herausforderungen der Automation bei der quantitativen Auswertung von Leberbiopsien : Automatische Quantifizierung von Leberverfettung.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD), or non-alcoholic fatty liver disease (NAFLD), is a common disease that is diagnosed through manual evaluation of liver biopsies, an assessment that is subject to high interobserver variability (IBV). IBV can be reduced using automated methods. OBJECTIVES: Many existing computer-based methods do not accurately reflect what pathologists evaluate in practice. The goal is to demonstrate how these differences impact the prediction of hepatic steatosis. Additionally, IBV complicates algorithm validation. MATERIALS AND METHODS: Forty tissue sections were analyzed to detect steatosis, nuclei, and fibrosis. Data generated from automated image processing were used to predict steatosis grades. To investigate IBV, 18 liver biopsies were evaluated by multiple observers. RESULTS: Area-based approaches yielded more strongly correlated results than nucleus-based methods (⌀ Spearman rho [ρ] = 0.92 vs. 0.79). The inclusion of information regarding tissue composition reduced the average absolute error for both area- and nucleus-based predictions by 0.5% and 2.2%, respectively. Our final area-based algorithm, incorporating tissue structure information, achieved a high accuracy (80%) and strong correlation (⌀ Spearman ρ = 0.94) with manual evaluation. CONCLUSION: The automatic and deterministic evaluation of steatosis can be improved by integrating information about tissue composition and can serve to reduce the influence of IBV.

Active Autophagy Is Associated with Favorable Outcome in Patients with Surgically Resected Cholangiocarcinoma.

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.

Increased MCL1 dependency leads to new applications of BH3-mimetics in drug-resistant neuroblastoma.

BACKGROUND: Neuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells. METHODS: We utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance. RESULTS: In a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells. CONCLUSIONS: These data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.

Fluorescence confocal microscopy on liver specimens for full digitization of transplant pathology.

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.

Tissue Shrinkage of Resected Specimens in Hirschsprung's Disease: Why Pediatric Surgeons Think the Bowel Specimen was Longer Than Indicated in the Pathology Report.

BACKGROUND: Hirschsprung disease (HD) is an aganglionosis of variable length starting at the rectosigmoid colon with surgery as sole therapeutic option. The length of the resected bowel segment is a crucial information for the treating surgeons and influences the prognosis of the patient. It is often artificially altered due to post operative tissue shrinkage. The objective of this study is to quantify the extent tissue shrinkage of HD specimens. MATERIAL AND METHODS: Colorectal HD specimens were measured at the time of surgery and at the time of cut-up, either fresh or after formalin fixation and statistically analyzed. RESULTS: Sixteen colorectal specimens were included. Following formalin fixation the specimen length decreased by 22.7% (P < .001). Without formalin fixation the specimens shrank by an average of 24.9% (P = .05). There was no significant difference in the extent of tissue shrinkage with or without formalin fixation (P = .76). CONCLUSION: This study showed that there is significant tissue shrinkage in HD specimens. The 2 different cohorts revealed that tissue shrinkage is mostly caused by tissue retraction/alteration after organ removal but also to a lesser extent by fixation with formalin. Surgeons and (neuro-)pathologists should be aware of the sizeable shrinking artifact to avoid unnecessary confusion.

Expression of MUC16/CA125 Is Associated with Impaired Survival in Patients with Surgically Resected Cholangiocarcinoma.

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.

How to learn with intentional mistakes: NoisyEnsembles to overcome poor tissue quality for deep learning in computational pathology.

There is a lot of recent interest in the field of computational pathology, as many algorithms are introduced to detect, for example, cancer lesions or molecular features. However, there is a large gap between artificial intelligence (AI) technology and practice, since only a small fraction of the applications is used in routine diagnostics. The main problems are the transferability of convolutional neural network (CNN) models to data from other sources and the identification of uncertain predictions. The role of tissue quality itself is also largely unknown. Here, we demonstrated that samples of the TCGA ovarian cancer (TCGA-OV) dataset from different tissue sources have different quality characteristics and that CNN performance is linked to this property. CNNs performed best on high-quality data. Quality control tools were partially able to identify low-quality tiles, but their use did not increase the performance of the trained CNNs. Furthermore, we trained NoisyEnsembles by introducing label noise during training. These NoisyEnsembles could improve CNN performance for low-quality, unknown datasets. Moreover, the performance increases as the ensemble become more consistent, suggesting that incorrect predictions could be discarded efficiently to avoid wrong diagnostic decisions.

Impact of small duct- and large duct type on survival in patients with intrahepatic cholangiocarcinoma: Results from a German tertiary center.

BACKGROUND & AIMS: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Deep learning based on hematoxylin-eosin staining outperforms immunohistochemistry in predicting molecular subtypes of gastric adenocarcinoma.

In gastric cancer (GC), there are four molecular subclasses that indicate whether patients respond to chemotherapy or immunotherapy, according to the TCGA. In clinical practice, however, not every patient undergoes molecular testing. Many laboratories have used well-implemented in situ techniques (IHC and EBER-ISH) to determine the subclasses in their cohorts. Although multiple stains are used, we show that a staining approach is unable to correctly discriminate all subclasses. As an alternative, we trained an ensemble convolutional neuronal network using bagging that can predict the molecular subclass directly from hematoxylin-eosin histology. We also identified patients with predicted intra-tumoral heterogeneity or with features from multiple subclasses, which challenges the postulated TCGA-based decision tree for GC subtyping. In the future, deep learning may enable targeted testing for molecular subtypes and targeted therapy for a broader group of GC patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy.

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

Leydig Cell Tumor in a Patient with 46,XX Disorder of Sex Development (DSD), Ovotesticular: A Case Report and a Review of the Literature.

Disorder of sex development (DSD) is a rare condition with atypical development of chromosomal, gonadal, or anatomical sex. It is classified in different subgroups based on the patient's karyotype, gonadal dysgenesis, and the appearance of the internal and external genitalia. Within the subgroups, the risk for developing neoplasms varies a lot. Here, we report the case of a 41-year-old patient with disorder of sex development, showing a 46,XX karyotype with an ovotestis and the simultaneous manifestation of a Leydig cell tumor in the ovotestis. The patient initially presented with infertility, and a suspicious lesion of the left testicle was noted on MRI-Scan. Upon resection, a Leydig cell tumor and an ovotestis were diagnosed. Nongerm call tumors are rare in patients with DSD. We report a nongerm cell tumor in a patient with 46,XX DSD, ovotesticular. This shows that although 46,XX DSD, ovotesticular is known to have a low potential for germ cell neoplasia, nongerm cell tumors can develop and should be into account for the management of those patients.