3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

High NANOG expression correlates with worse patients' survival in esophageal adenocarcinoma.

BACKGROUND: Patients diagnosed with esophageal cancer demonstrate a low overall survival even despite the established multimodal therapy as the current standard of care. Therefore, further biomarkers for patients with high-risk and additional therapy options are needed. NANOG is a transcription factor, which can be found in stem cells and is known to support tumorigenesis. METHODS: Six hundred sixty patients with esophageal adenocarcinoma, who were operated at the University of Cologne with a curative intent, were included. Immunohistochemical stainings for NANOG were performed. The study population was divided into NANOG-positive and -negative subgroups. RESULTS: Positive NANOG expression correlates significantly with worse overall survival (p = 0.002) and could be confirmed as an independent risk factor for worse patient survival in multivariate analysis (HR = 1.40, 95%CI = 1.09-1.80, p = 0.006). This effect could be detected in the subgroup of primarily operated patients, but not in patients after neoadjuvant therapy. CONCLUSIONS: We describe a NANOG-positive subgroup of patients with esophageal cancer, who exhibit worse overall survival in a large patient cohort. This discovery suggests the potential use of NANOG as a biomarker for both intensified therapy and stricter follow-up regimes. Additionally, NANOG-positive stem cell-like cancer cells could be used as a new antitumoral treatment target if validated in mechanistic and clinical studies.

Influence of patient sex on outcomes after pancreatic surgery: multicentre study.

BACKGROUND: Recent findings support the hypothesis of sex-related differences in inflammatory and immunological responses to trauma. The aim of this study was to address sex-specific aspects in patients who underwent pancreatic surgery. METHODS: This retrospective study used data from the German StuDoQ registry. Patients who underwent pancreatic surgery between 2010 and 2020 were stratified according to procedure (pancreatic head resection, distal pancreatectomy (DP), total pancreatectomy (TP)). Each cohort underwent propensity score matching (PSM) with the co-variables BMI, ASA, age, coronary heart disease (CHD), diabetes, hypertension with medication, and histology to level the distribution of co-morbidities between men and women. The main outcomes were morbidity and mortality. RESULTS: The total cohort consisted of 10 224 patients (45.3 per cent women). Men had higher ASA grades, and more often had CHD, diabetes, and hypertension with medication. Women had fewer overall complications (57.3 versus 60.1 per cent; P = 0.005) and a lower mortality rate (3.4 versus 4.9 per cent; P < 0.001). Rates of pancreatic surgery-specific complications, such as clinically relevant postoperative pancreatic fistula (POPF) (grade B/C: 14 versus 17 per cent; P < 0.001), delayed gastric emptying (grade B/C: 7.8 versus 9.2 per cent; P = 0.014), and postpancreatectomy haemorrhage (grade B/C: 7.1 versus 9.0 per cent; P < 0.001), were also lower in women. After PSM, 8358 patients were analysed. In the pancreatic head resection cohort (5318 patients), women had fewer complications (58.6 versus 61.4 per cent; P = 0.044), a lower in-hospital mortality rate (3.6 versus 6.1 per cent; P < 0.001), and less often had clinically relevant POPF (11.6 versus 16.2 per cent; P < 0.001). After DP, the clinically relevant POPF rate was lower in women (22.5 versus 27.3 per cent; P = 0.012). In the TP cohort, men more often developed intra-abdominal abscess requiring drainage (5.0 versus 2.3 per cent; P = 0.050). CONCLUSION: Women had favourable outcomes after pancreatic surgery.

Proposal for a definition of "Oligometastatic disease in pancreatic cancer".

BACKGROUND: To date, patients with metastasized pancreatic ductal adenocarcinoma (PDAC M1) are regarded as a uniform collective. We hypothesize the existence of oligometastatic disease (OMD): a state of PDAC M1 disease with better tumor biology, limited metastasis, and increased survival. METHODS: Data of 128 PDAC M1 patients treated at the University of Cologne between 2008 and 2018 was reviewed. Interdependence between clinical parameter was calculated using the Mann-Whitney U-Test. Survival curves were generated using the Kaplan-Meier method and analyzed using the log-rank test. RESULTS: Eighty-one (63%) patients had metastases confined to one organ (single organ metastasis, SOG) whereas the remaining 47 (37%) showed multiple metastatic sites (multi-organ metastasis, MOG). Survival analysis revealed a median overall survival (OS) of 12.2 months for SOG vs 4.5 months for MOG (95% CI 5.7-9.8; p < 0.001). We defined limited disease by the presence of ≤4 metastases in liver or lung. Limited disease together with CA 19-9 baseline < 1000 U/ml and response or stable disease after first-line chemotherapy defined OMD. We identified 8 patients with hepatic metastases and 2 with pulmonary metastases matching all OMD criteria. This group of 10 (7.8%) had a median overall survival of 19.4 vs 7.2 months compared to the remaining patients (95% CI 5.7-9.8; p = 0.009). CONCLUSION: We propose a definition of oligometastatic disease in PDAC including anatomical criteria and biological criteria reflecting better tumor biology. The 10 OMD patients (7.8%) survived significantly longer and might even benefit from surgical resection in the future.

Upregulation of the histone γ-H2AX correlates with worse patient survival and basal-like subtype in pancreatic ductal adenocarcinoma.

PURPOSE: Patients with pancreatic ductal adenocarcinoma (PDAC) have yet to experience significant benefits from targeted therapy. Olaparib is currently the only active substance in BRCA-mutated PDACs that successfully influences the DNA repair of carcinoma cells. H2AX belongs to the histone family and is known as a part of the DNA repair system. The inhibition of γ-H2AX could lead to the inhibition of mitotically active tumor cells. Therefore, we aimed to evaluate the predictive value of the γ-H2AX in patients with PDAC. METHODS: All included patients (n = 311) received a pancreatic resection with curative intention in one of our PANCALYZE study centers. Subsequently, they were enrolled in a standardized follow-up protocol. Immunohistochemical stainings for γ-H2AX were conducted on tissue microarrays. RESULTS: Patients exhibiting high levels of γ-H2AX expression experience more frequent R1 resections, indicating advanced tumor stages in this subgroup. Additionally, patients with high γ-H2AX expression demonstrated significantly poorer survival compared to those with low expression (median OS: 15 vs. 25 months, p < 0.001). In multivariate analyses, high γ-H2AX expression could be identified as an independent risk factor for worse patient survival. Moreover, high γ-H2AX expression could be more frequently observed in the more aggressive basal-like subtype. CONCLUSION: γ-H2AX can be characterized as a predictive biomarker for poorer patient survival. Consequently, upcoming clinical trials focused on the efficacy of targeted therapies influencing the DNA repair system and radiotherapy should evaluate γ-H2AX as a potential biomarker for therapy response. Furthermore, γ-H2AX may serve as a viable target for treatment in the future.

Extent and clinical significance of the therapy-relevant tight junction protein Claudin 18.2 in pancreatic ductal adenocarcinoma - real-world evidence.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis, wherefore targeted therapies have experienced increasing interest. Zolbetuximab is a novel targeted therapy under investigation in patients with PDAC and targets Claudin 18.2 (CLDN18.2), which is a component of tight junctions and is of significance in various solid tumors. As its role in PDAC is not definitively elucidated, this study aims to clarify the significance of CLDN18.2 expression in PDAC in a real-world setting. METHODS: All patients (n = 309) were recruited at one of the PANCALYZE study centers and received pancreatic resection with curative intention. Paraffin samples were analyzed using an antibody against CLDN18.2, which is known to be comparable to the antibody used by the SPOTLIGHT and GLOW studies. RESULTS: 94 PDACs are positive for CLDN18.2 (30.4 %). Positive CLDN 18.2 expression was associated with significantly better cancer differentiation (p < 0.001). Patients with positive CLDN18.2 expression showed significantly better overall survival when compared to patients with negative expression (median OS: 30 versus 18 months, p = 0.003). Additionally, in multivariable analyses, CLDN18.2 expression was identified as an independent factor for better survival in patients with PDAC (HR = 0.686, 95 %CI = 0.492-0.956, p = 0.026). CONCLUSION: Significant improvement in survival could be demonstrated by adding Zolbetuximab to known chemotherapy regimes in patients with gastro-esophageal junction adenocarcinoma with at least 75 % CLDN18.2 positive cancer cells. Our findings demonstrate, that 30.4 % of the included patients with PDAC would potentially be eligible for therapy with Zolbetuximab in a real-world patient cohort. Results of trials targeting Claudin 18.2 are pending in patients with PDAC.

Specific Subtypes of Carcinoma-Associated Fibroblasts Are Correlated with Worse Survival in Resectable Pancreatic Ductal Adenocarcinoma.

PURPOSE: The pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer entities. Effective therapy options are still lacking. The tumor microenvironment possibly bears further treatment possibilities. This study aimed to describe the expression patterns of four established carcinoma-associated fibroblast (CAFs) markers and their correlation in PDAC tissue samples. METHODS: This project included 321 patients with PDAC who underwent surgery with a curative intent in one of the PANCALYZE study centers. Immunohistochemical stainings for FAP, PDGFR, periostin, and SMA were performed. The expression patterns of each marker were divided into low- and high-expressing CAFs and correlated with patients' survival. RESULTS: Tumors showing SMAhigh-, PeriostinhighSMAhigh-, or PeriostinhighSMAlowPDGFRlowFAPhigh-positive CAFs demonstrated significantly worse survival. Additionally, a high expression of SMA in PDAC tissue samples was shown to be an independent risk factor for worse survival. CONCLUSION: This project identified three subgroups of PDAC with different expression patterns of CAF markers which showed significantly worse survival. This could be the base for the further characterization of the fibroblast subgroups in PDAC and contribute to the development of new targeted therapy options against CAFs.

Cytokeratin 6 identifies basal-like subtypes of pancreatic ductal adenocarcinoma with decreased survival.

PURPOSE: Rising incidence of pancreatic ductal adenocarcinoma (PDAC) bind with insufficient therapy options showcases a great medical challenge. Further biomarkers are required to identify patients, who will benefit from more aggressive therapy. METHODS: 320 patients were included by the PANCALYZE study group. Cytokeratin 6 (CK6) immunohistochemical staining as a putative marker for the basal-like subtype of PDAC was performed. The correlation between CK6 expression patterns and survival data, as well as various markers of the (inflammatory) tumor microenvironment, were analyzed. RESULTS: We divided the study population based on the expression pattern of CK6. Patients with a high CK6 tumor expression had a significantly shorter survival (p = 0.013), confirmed in a multivariate cox regression model. CK6-expression is an independent marker for a decreased overall survival (HR = 1.655, 95% CI 1.158-2.365, p = 0.006). In addition, the CK6-positive tumors showed significantly less plasma cell infiltration and more cancer-associated fibroblasts (CAFs) expressing Periostin and SMA. CONCLUSIONS: CK6 could be considered as an independent biomarker for a shorter overall survival. CK6 is a clinically easily accessible biomarker for the identification of the basal-like subtype of PDAC. Therefore, it could be taken into consideration in deciding for the more aggressive therapy regimes. Prospectively, studies addressing the chemosensitive characteristics of this subtype are required.

X-linked inhibitor of apoptosis protein is a prognostic marker for a favorable outcome in three identified subsets in resectable adenocarcinoma of the pancreas.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is currently one of the leading causes of cancer death worldwide. Therefore, building further subgroups as well as enabling individual patient therapy and diagnostics are needed. X-linked inhibitor of apoptosis protein (XIAP) is known to modulate apoptotic and inflammatory pathways. Its expression was found to correlate with patients' survival in other tumor entities. This study aims to examine the role of XIAP in patients with PDAC in relation to the inflammatory microenvironment. METHODS: The PANCALYZE multicenter study group included 257 patients with PDAC. Paraffin-embedded tumor samples were stained immunohistochemically for CD3, CD20, CD38, CD56, CD66b, CD117, and CD163 and XIAP. These stainings were further analyzed digitally with QuPath and survival analyses were done. RESULTS: XIAP-positive patients with T-cell, respectively, neutrophil enriched tumors survived significantly longer compared to XIAP-negative patients (CD3: 37.6 vs. 24.6 months, p = 0.028; CD66b: 34.1 vs. 14.9 months, p = 0.027). Additionally, XIAP-positive patients showed better survival in the lymph node-negative population (48.4 vs. 24.2 months, p = 0.019). Regarding the total population, our findings did not show a correlation between XIAP expression and survival. In multivariate cox regression analyzes XIAP proves to be an independent factor for better survival in the identified subgroups (CD3: p = 0.043; CD66b: p = 0.012, N0: p = 0.040). CONCLUSION: We found XIAP-positive subgroups with significantly better survival in patients with PDAC in T-cell-rich, neutrophil-rich, or lymph node-negative cohorts. This could lead to further individualized cancer treatment with less aggressive therapy protocols for XIAP-positive tumors or more intensive follow-up for XIAP-negative tumors.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Integrin beta1 (ITGB1) as a prognostic marker in esophageal adenocarcinoma.

Today, individual prognosis in patients with adenocarcinoma of the esophagus (EAC) is based on post-surgical TNM staging and valid biomarkers are still not implemented. Integrin beta1 (ITGB1) is widely expressed in epithelial cells and promotes cell adhesion and growth. Its impact on tumor progression was described for different tumor entities before, data on its function as a potential biomarker in EAC is not available. Aim of the study is to evaluate the expression level of ITGB1 in a large collective of EAC and its impact on patients´ prognosis. 640 patients with esophageal adenocarcinoma were analyzed immunohistochemically for ITGB1. The data was correlated with long term outcome, clinical, pathological and molecular data (TP53, HER2/neu, c-myc, GATA6, PIK3CA and KRAS). Of 640 patients to be analyzed, 127 (19.8%) showed expression of ITGB1. ITGB1 expression was associated with lymph node metastasis, expression of integrin alphaV and KRAS mutation status. Patients with high ITGB1 expression showed impaired overall survival (22.5 months (95% CI 15.3-29.7 months), vs. 34.1 months (95% CI 25.3-42.4 months), P = 0.024). This effect was particularly evident in the group of patients undergoing primary surgery without prior neoadjuvant therapy (10.2 months (95% CI 1.9-41.7 months) vs. 31.4 months (95% CI 21.1-144.2 months, P = 0.008). ITGB1 was also an independent prognostic marker in multivariable analysis (HR 1.696 (95% CI 1.084-2.653, P = 0.021) in patients that underwent primary surgery. We demonstrate for the first time the prognostic significance of ITGB1 expression in a large EAC patient population.

SARS-CoV-2-specific immune responses in boosted vaccine recipients with breakthrough infections during the Omicron variant surge.

BackgroundBreakthrough SARS-CoV-2 infections in vaccinated individuals have been previously associated with suboptimal humoral immunity. However, less is known about breakthrough infections with the Omicron variant.MethodsWe analyzed SARS-CoV-2-specific antibody and cellular responses in healthy vaccine recipients who experienced breakthrough infections a median of 50 days after receiving a booster mRNA vaccine with an ACE2 binding inhibition assay and an ELISpot assay, respectively.ResultsWe found that high levels of antibodies inhibited vaccine strain spike protein binding to ACE2 but that lower levels inhibited Omicron variant spike protein binding to ACE2 in 4 boosted vaccine recipients prior to infection. The levels of antibodies that inhibited vaccine strain and Omicron spike protein binding after breakthrough in 18 boosted vaccine recipients were similar to levels seen in COVID-19-negative boosted vaccine recipients. In contrast, boosted vaccine recipients had significantly stronger T cell responses to both vaccine strain and Omicron variant spike proteins at the time of breakthrough.ConclusionOur data suggest that breakthrough infections with the Omicron variant can occur despite robust immune responses to the vaccine strain spike protein.FundingThis work was supported by the Johns Hopkins COVID-19 Vaccine-related Research Fund and by funds from the National Institute of Allergy and Infectious Disease intramural program as well as awards from the National Cancer Institute (U54CA260491) and the National Institutes of Allergy and Infectious Disease (K08AI156021 and U01AI138897).

Epigenetic Modifications in Thyroid Cancer Cells Restore NIS and Radio-Iodine Uptake and Promote Cell Death.

Epigenetic modifications have been identified as being responsible for the de-differentiation of thyroid tissue and its malignant transformation. Cell proliferation inhibitory effects of the pan-deacetylase inhibitors panobinostat, SAHA and Trichostatin A (TSA), the modulation of the sodium iodide symporter (NIS; SLC5A5), thyroid transcription factor 1 (TTF1), high mobility group A2 (HMGA2), and H19 and their putative targeting miRNAs have been evaluated in vitro. The cell viability was measured in five thyroid cancer cell lines (FTC133, TPC1, BCPAP, 8505C, C643) by real time cell analyzer xCELLigence. Expression of the above mentioned markers was performed by RT-qPCR and Western Blot. Radioiodine up-take was detected by Gamma Counter with I131. Cell viability decreased after treatment in all five cell lines. 10 nM panobinostat; 1 µM TSA or 10 µM SAHA caused a significant over-expression of NIS transcript in all five cell lines, whereas NIS protein was up-regulated in FTC133, BCPAP, and C643 cell lines only. Radioiodine up-take increased in FTC133 and C643 cells after 48 h of treatment with 10 nM panobinostat and 1 µM TSA. A significant down-regulation of the oncogene HMGA2 was detected in all five cell lines; except for TPC1 cells that were treated with 1 µM TSA. In accordance, hsa-let-7b-5p and hsa-let-7f-5p were stable or significantly over-expressed in all of the cell lines, except for TPC1 cells that were treated with 10 µM SAHA. TTF1 was significantly down-regulated in FTC133, BCPAP, and 8505C cells; whereas, TPC1 and C643 showed an up-regulated or stable expression. TTF1 was over-expressed in samples of human anaplastic thyroid cancer; whereas, it was down-regulated in follicular and undetectable in papillary thyroid cancer. H19 was over-expressed after 48 h treatment, except for BCPAP cells that were treated with panobinostat and SAHA. H19 was differently expressed in human anaplastic, follicular and papillary thyroid tumor samples. Deacetylase inhibitors reduced cell viability, restored NIS and H19, and suppressed the oncogenes HMGA2 and TTF1 in thyroid cancer cells.

MicroRNAs let7 expression in thyroid cancer: correlation with their deputed targets HMGA2 and SLC5A5.

PURPOSE: Thyroid cancer (TC), the most common endocrine malignancy, increases its incidence worldwide. MicroRNAs have been shown to be abnormally expressed in tumors and could represent valid diagnostic markers for patients affected by TC. Our aim was to analyze the expression of tumorsuppressor hsa-let7b-5p and hsa-let7f-5p, together with their predicted targets SLC5A5 (NIS) and HMGA2, in papillary (PTC), follicular (FTC) and anaplastic (ATC). METHODS: 8 FTC, 14 PTC, 12 ATC and three normal thyroid tissue samples were analyzed for the expression of pre-let7b, hsa-let7b-5p and hsa-let7f-5p as SLC5A5 and HMGA2 by RT-qPCR. Data were analyzed by REST 2008. RESULTS: FTC patients showed a significant down-regulation of hsa-let7b-5p and its precursor. hsa-let7f-5p was overexpressed, and SLC5A5 was strongly suppressed. HMGA2 was overexpressed, reflecting no correlation with its regulatory let7 miRNAs. PTC samples were characterized by up-regulation of hsa-let7b-5p, its precursor and hsa-let7f-5p. SLC5A5 was strongly suppressed in comparison with normal thyroid tissue. HMGA2 was overexpressed, as shown in FTC, also. ATC samples showed a similar miRNAs profile as PTC. In contrast with FTC and PTC, these patients showed a stable or up-regulated SLC5A5 and HMGA2. CONCLUSIONS: Expression of HMGA2 is not correlated with the regulatory let7 miRNAs. Interestingly, SLC5A5 was down-regulated in FTC and PTC. Its expression could be modulated by hsa-let-7f-5p. ATC showed a loss of SLC5A5/hsa-let7f-5p correlation. SLC5A5, in ATC, needs further investigation to clarify the genetic/epigenetic mechanism altering its expression.