Expression of Checkpoint Molecules in the Tumor Microenvironment of Intrahepatic Cholangiocarcinoma: Implications for Immune Checkpoint Blockade Therapy.

Background: The tumor microenvironment (TME) in cholangiocarcinoma (CCA) influences the immune environment. Checkpoint blockade is promising, but reliable biomarkers to predict response to treatment are still lacking. Materials and Methods: The levels of checkpoint molecules (PD-1, PD-L1, PD-L2, LAG-3, ICOS, TIGIT, TIM-3, CTLA-4), macrophages (CD68), and T cells (CD4 and CD8 cells) were assessed by multiplexed immunofluorescence in 50 intrahepatic cases. Associations between marker expression, immune cells, and region of expression were studied in the annotated regions of tumor, interface, sclerotic tumor, and tumor-free tissue. Results: ICCA demonstrated CD4_TIM-3 high densities in the tumor region of interest (ROI) compared to the interface (p = 0.014). CD8_PD-L1 and CD8_ICOS densities were elevated in the sclerotic tumor compared to the interface (p = 0.011 and p = 0.031, respectively). In a multivariate model, high expression of CD8_PD-L2 (p = 0.048) and CD4_ICOS_TIGIT (p = 0.011) was associated with nodal metastases. Conclusions: High densities of PD-L1 were more abundant in the sclerotic tumor region; this is meaningful for the stratification of immunotherapy. Lymph node metastasis correlates with CD4_ICOS_TIGIT co-expression and CD8_PD-L2 expression, indicating the checkpoint expression profile of patients with a poor prognosis. Also, multiple co-expressions occur, and this potentially suggests a role for combination therapy with different immune checkpoint targets than just PD-1 blockade monotherapy.

PD-1+ T-Cells Correlate with Nerve Fiber Density as a Prognostic Biomarker in Patients with Resected Perihilar Cholangiocarcinoma.

BACKGROUND AND AIMS: Perihilar cholangiocarcinoma (pCCA) is a hepatobiliary malignancy, with a dismal prognosis. Nerve fiber density (NFD)-a novel prognostic biomarker-describes the density of small nerve fibers without cancer invasion and is categorized into high numbers and low numbers of small nerve fibers (high vs low NFD). NFD is different than perineural invasion (PNI), defined as nerve fiber trunks invaded by cancer cells. Here, we aim to explore differences in immune cell populations and survival between high and low NFD patients. APPROACH AND RESULTS: We applied multiplex immunofluorescence (mIF) on 47 pCCA patients and investigated immune cell composition in the tumor microenvironment (TME) of high and low NFD. Group comparison and oncological outcome analysis was performed. CD8+PD-1 expression was higher in the high NFD than in the low NFD group (12.24 × 10-6 vs. 1.38 × 10-6 positive cells by overall cell count, p = 0.017). High CD8+PD-1 expression was further identified as an independent predictor of overall (OS; Hazard ratio (HR) = 0.41; p = 0.031) and recurrence-free survival (RFS; HR = 0.40; p = 0.039). Correspondingly, the median OS was 83 months (95% confidence interval (CI): 18-48) in patients with high CD8+PD-1+ expression compared to 19 months (95% CI: 5-93) in patients with low CD8+PD-1+ expression (p = 0.018 log rank). Furthermore, RFS was significantly lower in patients with low CD8+PD-1+ expression (14 months (95% CI: 6-22)) compared to patients with high CD8+PD-1+ expression (83 months (95% CI: 17-149), p = 0.018 log rank). CONCLUSIONS: PD-1+ T-cells correlate with high NFD as a prognostic biomarker and predict good survival; the biological pathway needs to be investigated.

Limitations of Nerve Fiber Density as a Prognostic Marker in Predicting Oncological Outcomes in Hepatocellular Carcinoma.

It has been shown that the presence and density of nerve fibers (NFs; NFD) in the tumor microenvironment (TME) may play an important prognostic role in predicting long-term oncological outcomes in various malignancies. However, the role of NFD in the prognosis of hepatocellular carcinoma (HCC) is yet to be explored. To this end, we aimed to investigate the impact of NFs on oncological outcomes in a large European single-center cohort of HCC patients. In total, 153 HCC patients who underwent partial hepatectomy in a curative-intent setting between 2010 and 2021 at our university hospital were included in this study. Group comparisons between patients with and without NFs were conducted and the association of recurrence-free survival (RFS) and overall survival (OS) with the presence of NFs and other clinico-pathological variables were determined by univariate and multivariable Cox regression models. Patients with NFs in the TME presented with a median OS of 66 months (95% CI: 30−102) compared to 42 months (95% CI: 20−63) for patients without NFs (p = 0.804 log-rank). Further, RFS was 26 months (95% CI: 12−40) for patients with NFs compared to 18 months (95% CI: 9−27) for patients without NFs (p = 0.666 log-rank). In a subgroup analysis, patients with NFD ≤ 5 showed a median OS of 54 months (95% CI: 11−97) compared to 48 months (95% CI: 0−106) for the group of patients with NFD > 5 (p = 0.787 log-rank). Correspondingly, the RFS was 26 months (95% CI: 10−42) in patients with NFD ≤ 5 and 29 months (95% CI: 14−44) for the subcohort with NFD > 5 (p = 0.421 log-rank). Further, group comparisons showed no clinico-pathological differences between patients with NFs (n = 76) and without NFs (n = 77) and NFs were not associated with OS (p = 0.806) and RFS (p = 0.322) in our Cox regression models. In contrast to observations in various malignancies, NFs in the TME and NFD are not associated with long-term oncological outcomes in HCC patients undergoing surgery.

The Presence of Small Nerve Fibers in the Tumor Microenvironment as Predictive Biomarker of Oncological Outcome Following Partial Hepatectomy for Intrahepatic Cholangiocarcinoma.

The oncological role of the density of nerve fibers (NFs) in the tumor microenvironment (TME) in intrahepatic cholangiocarcinoma (iCCA) remains to be determined. Therefore, data of 95 iCCA patients who underwent hepatectomy between 2010 and 2019 was analyzed regarding NFs and long-term outcome. Extensive group comparisons were carried out and the association of cancer-specific survival (CSS) and recurrence-free survival (RFS) with NFs were assessed using Cox regression models. Patients with iCCA and NFs showed a median CSS of 51 months (5-year-CSS = 47%) compared to 27 months (5-year-CSS = 21%) in patients without NFs (p = 0.043 log rank). Further, NFs (hazard ratio (HR) = 0.39, p = 0.002) and N-category (HR = 2.36, p = 0.010) were identified as independent predictors of CSS. Patients with NFs and without nodal metastases displayed a mean CSS of 89 months (5-year-CSS = 62%), while patients without NFs or with nodal metastases but not both showed a median CCS of 27 months (5-year-CSS = 25%) and patients with both positive lymph nodes and without NFs showed a median CCS of 10 months (5-year-CSS = 0%, p = 0.001 log rank). NFs in the TME are, therefore, a novel and important prognostic biomarker in iCCA patients. NFs alone and in combination with nodal status is suitable to identify iCCA patients at risk of poor oncological outcomes following curative-intent surgery.

Development and validation of deep learning classifiers to detect Epstein-Barr virus and microsatellite instability status in gastric cancer: a retrospective multicentre cohort study.

BACKGROUND: Response to immunotherapy in gastric cancer is associated with microsatellite instability (or mismatch repair deficiency) and Epstein-Barr virus (EBV) positivity. We therefore aimed to develop and validate deep learning-based classifiers to detect microsatellite instability and EBV status from routine histology slides. METHODS: In this retrospective, multicentre study, we collected tissue samples from ten cohorts of patients with gastric cancer from seven countries (South Korea, Switzerland, Japan, Italy, Germany, the UK and the USA). We trained a deep learning-based classifier to detect microsatellite instability and EBV positivity from digitised, haematoxylin and eosin stained resection slides without annotating tumour containing regions. The performance of the classifier was assessed by within-cohort cross-validation in all ten cohorts and by external validation, for which we split the cohorts into a five-cohort training dataset and a five-cohort test dataset. We measured the area under the receiver operating curve (AUROC) for detection of microsatellite instability and EBV status. Microsatellite instability and EBV status were determined to be detectable if the lower bound of the 95% CI for the AUROC was above 0·5. FINDINGS: Across the ten cohorts, our analysis included 2823 patients with known microsatellite instability status and 2685 patients with known EBV status. In the within-cohort cross-validation, the deep learning-based classifier could detect microsatellite instability status in nine of ten cohorts, with AUROCs ranging from 0·597 (95% CI 0·522-0·737) to 0·836 (0·795-0·880) and EBV status in five of eight cohorts, with AUROCs ranging from 0·819 (0·752-0·841) to 0·897 (0·513-0·966). Training a classifier on the pooled training dataset and testing it on the five remaining cohorts resulted in high classification performance with AUROCs ranging from 0·723 (95% CI 0·676-0·794) to 0·863 (0·747-0·969) for detection of microsatellite instability and from 0·672 (0·403-0·989) to 0·859 (0·823-0·919) for detection of EBV status. INTERPRETATION: Classifiers became increasingly robust when trained on pooled cohorts. After prospective validation, this deep learning-based tissue classification system could be used as an inexpensive predictive biomarker for immunotherapy in gastric cancer. FUNDING: German Cancer Aid and German Federal Ministry of Health.

Nerve Fibers in the Tumor Microenvironment as a Novel Biomarker for Oncological Outcome in Patients Undergoing Surgery for Perihilar Cholangiocarcinoma.

INTRODUCTION: Perihilar cholangiocarcinoma (pCCA) is a biliary tract cancer with a dismal prognosis, with surgery being the only chance of cure. A characteristic aggressive biological feature of pCCA is perineural growth which is defined by the invasion of cancer cells to nerves and nerve fibers. Recently, nerve fiber density (NFD) was linked to oncological outcomes in various malignancies; however, its prognostic role in pCCA remains to be elucidated. MATERIALS AND METHODS: Data of 101 pCCA patients who underwent curative-intent surgery between 2010 and 2019 were included in this study. Extensive group comparisons between patients with high and low NFD were carried out, and the association of cancer-specific survival (CSS) and recurrence-free survival with NFD and other clinicopathological characteristics was assessed using univariate and multivariable cox regression models. RESULTS: Patients with high NFD showed a median CSS of 90 months (95% CI: 48-132, 3-year CSS = 77%, 5-year CSS = 72%) compared to 33 months (95% CI: 19-47, 3-year CSS = 46%, 5-year CSS = 32%) in patients with low NFD (p = 0.006 log rank). Further, N1 category (HR = 2.84, p = 0.001) and high NFD (HR = 0.41, p = 0.024) were identified as independent predictors of CSS in multivariable analysis. Patients with high NFD and negative lymph nodes showed a median CSS of 90 months (3-year CSS = 88%, 5-year CSS = 80%), while patients with either positive lymph nodes or low NFD displayed a median CSS of 51 months (3-year CSS = 59%, 5-year CSS = 45%) and patients with both positive lymph nodes and low NFD a median CSS of 24 months (3-year CSS = 26%, 5-year CSS = 16%, p = 0.001 log rank). CONCLUSION: NFD has been identified as an important novel prognostic biomarker in pCCA patients. NFD alone and in combination with nodal status in particular allows to stratify pCCA patients based on their risk for inferior oncological outcomes after curative-intent surgery.

Nerve Fibers in the Tumor Microenvironment Are Co-Localized with Lymphoid Aggregates in Pancreatic Cancer.

B cells and tertiary lymphoid structures (TLS) are reported to be important in survival in cancer. Pancreatic Cancer (PDAC) is one of the most lethal cancer types, and currently, it is the seventh leading cause of cancer-related death worldwide. A better understanding of tumor biology is pivotal to improve clinical outcome. The desmoplastic stroma is a complex system in which crosstalk takes place between cancer-associated fibroblasts, immune cells and cancer cells. Indirect and direct cellular interactions within the tumor microenvironment (TME) drive key processes such as tumor progression, metastasis formation and treatment resistance. In order to understand the aggressiveness of PDAC and its resistance to therapeutics, the TME needs to be further unraveled. There are some limited data about the influence of nerve fibers on cancer progression. Here we show that small nerve fibers are located at lymphoid aggregates in PDAC. This unravels future pathways and has potential to improve clinical outcome by a rational development of new therapeutic strategies.

Nerve fibers in the tumor microenvironment in neurotropic cancer-pancreatic cancer and cholangiocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) are both deadly cancers and they share many biological features besides their close anatomical location. One of the main histological features is neurotropism, which results in frequent perineural invasion. The underlying mechanism of cancer cells favoring growth by and through the nerve fibers is not fully understood. In this review, we provide knowledge of these cancers with frequent perineural invasion. We discuss nerve fiber crosstalk with the main different components of the tumor microenvironment (TME), the immune cells, and the fibroblasts. Also, we discuss the crosstalk between the nerve fibers and the cancer. We highlight the shared signaling pathways of the mechanisms behind perineural invasion in PDAC and CCA. Hereby we have focussed on signaling neurotransmitters and neuropeptides which may be a target for future therapies. Furthermore, we have summarized retrospective results of the previous literature about nerve fibers in PDAC and CCA patients. We provide our point of view in the potential for nerve fibers to be used as powerful biomarker for prognosis, as a tool to stratify patients for therapy or as a target in a (combination) therapy. Taking the presence of nerves into account can potentially change the field of personalized care in these neurotropic cancers.

Clinical-Grade Detection of Microsatellite Instability in Colorectal Tumors by Deep Learning.

BACKGROUND & AIMS: Microsatellite instability (MSI) and mismatch-repair deficiency (dMMR) in colorectal tumors are used to select treatment for patients. Deep learning can detect MSI and dMMR in tumor samples on routine histology slides faster and less expensively than molecular assays. However, clinical application of this technology requires high performance and multisite validation, which have not yet been performed. METHODS: We collected H&E-stained slides and findings from molecular analyses for MSI and dMMR from 8836 colorectal tumors (of all stages) included in the MSIDETECT consortium study, from Germany, the Netherlands, the United Kingdom, and the United States. Specimens with dMMR were identified by immunohistochemistry analyses of tissue microarrays for loss of MLH1, MSH2, MSH6, and/or PMS2. Specimens with MSI were identified by genetic analyses. We trained a deep-learning detector to identify samples with MSI from these slides; performance was assessed by cross-validation (N = 6406 specimens) and validated in an external cohort (n = 771 specimens). Prespecified endpoints were area under the receiver operating characteristic (AUROC) curve and area under the precision-recall curve (AUPRC). RESULTS: The deep-learning detector identified specimens with dMMR or MSI with a mean AUROC curve of 0.92 (lower bound, 0.91; upper bound, 0.93) and an AUPRC of 0.63 (range, 0.59-0.65), or 67% specificity and 95% sensitivity, in the cross-validation development cohort. In the validation cohort, the classifier identified samples with dMMR with an AUROC of 0.95 (range, 0.92-0.96) without image preprocessing and an AUROC of 0.96 (range, 0.93-0.98) after color normalization. CONCLUSIONS: We developed a deep-learning system that detects colorectal cancer specimens with dMMR or MSI using H&E-stained slides; it detected tissues with dMMR with an AUROC of 0.96 in a large, international validation cohort. This system might be used for high-throughput, low-cost evaluation of colorectal tissue specimens.