Teacher-student collaborated multiple instance learning for pan-cancer PDL1 expression prediction from histopathology slides.

Programmed cell death ligand 1 (PDL1), as an important biomarker, is quantified by immunohistochemistry (IHC) with few established histopathological patterns. Deep learning aids in histopathological assessment, yet heterogeneity and lacking spatially resolved annotations challenge precise analysis. Here, we present a weakly supervised learning approach using bulk RNA sequencing for PDL1 expression prediction from hematoxylin and eosin (H&E) slides. Our method extends the multiple instance learning paradigm with the teacher-student framework, which assigns dynamic pseudo-labels for intra-slide heterogeneity and retrieves unlabeled instances using temporal ensemble model distillation. The approach, evaluated on 12,299 slides across 20 solid tumor types, achieves a weighted average area under the curve of 0.83 on fresh-frozen and 0.74 on formalin-fixed specimens for 9 tumors with PDL1 as an established biomarker. Our method predicts PDL1 expression patterns, validated by IHC on 20 slides, offering insights into histologies relevant to PDL1. This demonstrates the potential of deep learning in identifying diverse histological patterns for molecular changes from H&E images.

Reevaluating Thrombocytosis as a Risk Factor in Free Flap Surgery: Does Timing Matter?

INTRODUCTION: Thrombocytosis, defined as a platelet count >400,000, has been implicated as a risk factor in free flap failure. Despite proposed mechanisms of pedicle thrombosis, recent studies have suggested that thrombocytosis has no effect on free tissue transfer viability. Risk factors that may compromise successful free tissue transfer should be understood and elucidated, with particular attention to thrombocytosis and its conflicting evidence in the literature. We hypothesize that thrombocytosis has no bearing on free flap success or the rates of pedicle thrombosis. METHODS: Our institution performed a retrospective chart review on all patients who underwent free flap reconstruction over the past 6 years. Patient demographics, medical history, type and location of free tissue transfer, preoperative platelets, postoperative platelets, and flap outcomes and complications (wound dehiscence, infection, hematoma, seroma, and need for blood transfusion) were recorded. Independent t test, Mann-Whitney U tests, χ2 test, and Fisher exact tests were used to determine P values to compare flap outcomes in patients with thrombocytosis (platelet count >400,000) and those with platelet counts less than 400,000. RESULTS: In our 502-patient cohort, 71 were found to have a platelet count >400,000 (35 preoperatively and 36 postoperatively) and 431 patients had platelet counts <400,000. There were 42 reconstructive failures (flap success rate of 91.6%) and 111 returns to the operating room (OR). For patients with postoperative thrombocytosis, 24 flaps returned to the OR (44.4%), whereas in patients without thrombocytosis, 87 flaps returned to the OR (19.4%; P < 0.001). In patients with postoperative thrombocytosis, 10 OR returns were due to pedicle venous thrombosis (18.5%), in comparison to 10 returns for venous thrombosis in those with normal platelets (2.2%; P < 0.001). There was a small difference in free flap success rates between those with postoperative thrombocytosis and normal platelets, 88.7% versus 92.11%; however, this was not statistically significant ( P = 0.71). The thrombocytosis group had a higher incidence of overall postoperative complications ( P = 0.002). CONCLUSIONS: Thrombocytosis has historically been cited as a risk factor for free flap reconstruction failure with recent conflicting evidence in the literature. In patients with postoperative thrombocytosis, we found an increased risk of venous thrombosis; however, this did not result in increased flap failure. There was an increase in postoperative complications, which corresponds with National Surgical Quality Improvement Program data reported in the literature. We suspect that thrombocytosis is not a harbinger of free flap failure but rather a marker for overall inflammation, which may confer a higher rate of venous thrombosis requiring reoperation and postoperative complications.

Synchronous Pleural and Peritoneal Mesothelioma: a Case Report and Narrative Review.

Malignant mesotheliomas most often affect the pleura and tend to spread locally within the originating cavity. Mesotheliomas are already rare diseases, and cases with synchronous pleural and peritoneal involvement are scarce in the literature. Mesothelioma in children is a rare disease representing only 0.9% of all mesotheliomas. They exhibit similar distribution and characteristics as mesotheliomas in adults and generally, a poor prognosis. Due to the rarity, there is no standardized treatment recommendation for children with mesothelioma. Though the malignant mesothelioma tends to spread locally within the originating cavity, pleuM have been reported to metastasize into the peritoneal cavity and vice versa. As there are only few studies concerning the metastatic spread of mesothelioma, it is difficult to define a precise incidence and risk factors for patients to develop metastases of the other mesothelium. There is no standardized therapeutic recommendation for patients with synchronous pleuM and perM. Our patient proved to profit from a radical two-stage surgical approach in combination with locoregional chemotherapy; she showed no sign of tumor recurrences 9 years after tumor resection. In conclusion, clinical studies are needed to confirm the benefit of this treatment and to determine its limitations and selection criteria.

Happiness Behind the Scenes: Associations between Heuristic Happiness and Related Dimensions in Skin Diseases.

Positive health approaches, such as happiness, are largely unexplored in medicine, including dermatology. Taking into consideration the various happiness measures, the aims of this cross-sectional study were to assess this concept using a 1-item heuristic happiness question and its related dimensions (Satisfaction with Life, Positive and Negative Affect, Quality of Life, and Compound Psychological Capital) in outpatients with different skin diseases between December 2019 and June 2020, and to examine the link between these dimensions. Overall, 414 dermatological patients were included: 67 with psoriasis, 84 atopic eczema, 10 mastocytosis, 19 nummular eczema, 84 malignant melanoma and 150 keratinocyte carcinoma. Comparing the skin diseases, differences were observed for heuristic happiness, Positive Affect, and some domains of Quality of Life and Compound Psychological Capital. Analysing the relationship between heuristic happiness and other happiness measures, the data revealed moderate to strong correlations (r = 0.30-0.46, p< 0.001) and variations in the understanding of happiness between the skin diseases. Overall, the most important facet of happiness was Hope as a domain of Compound Psychological Capital. This study emphasizes the individual definition of heuristic happiness in patients with skin diseases. A 1-item heuristic approach may be a simple and practical method to assess the complexity and individuality of happiness.

Inhibition of Vascular Endothelial Growth Factor Protects against the Development of Oxaliplatin-Induced Sinusoidal Obstruction Syndrome in Wild-Type but Not in CD39-Null Mice.

(1) Background: Sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy is associated with unfavorable outcomes after partial hepatectomy for colorectal liver metastases (CLM). Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), may prevent SOS development. We investigated the impact of VEGF-inhibition on the development of SOS in a murine model. (2) Methods: Male wild-type and CD39-null mice received oxaliplatin, additional anti-VEGF (OxAV), or controls, and were sacrificed or subjected to major partial hepatectomy (MH). Specimen were used for histological analysis of SOS. Liver damage was assessed by plasma transaminases. The VEGF pathway was elucidated by quantitative PCR of liver tissue and protein analysis of plasma. (3) Results: Mice treated with oxaliplatin developed SOS. Concomitant anti-VEGF facilitated a reduced incidence of SOS, but not in CD39-null mice. SOS was associated with increased plasma VEGF-A and decreased hepatocyte growth factor (HGF). After OxAV treatment, VEGF-R2 was upregulated in wild-type but downregulated in CD39-null mice. Oxaliplatin alone was associated with higher liver damage after MH than in mice with concomitant VEGF-inhibition. (4) Conclusions: We established a murine model of oxaliplatin-induced SOS and provided novel evidence on the protective effect of VEGF-inhibition against the development of SOS that may be associated with changes in the pathway of VEGF and its receptor VEGF-R2.

R-spondin/YAP axis promotes gastric oxyntic gland regeneration and Helicobacter pylori-associated metaplasia in mice.

The stomach corpus epithelium is organized into anatomical units that consist of glands and pits. Mechanisms that control the cellular organization of corpus glands and enable their recovery upon injury are not well understood. R-spondin 3 (RSPO3) is a WNT-signaling enhancer that regulates stem cell behavior in different organs. Here, we investigated the function of RSPO3 in the corpus during homeostasis, upon chief and/or parietal cell loss, and during chronic Helicobacter pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland toward parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells - caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneration was maintained long term, promoting severe glandular hyperproliferation and the development of premalignant metaplasia.

Prognostic impact of activin subunit inhibin beta A in gastric and esophageal adenocarcinomas.

PURPOSE: Adenocarcinomas of the esophagus (AEG) and stomach (AS) are among the most common cancers worldwide. Novel markers for risk stratification and guiding treatment are strongly needed. Activin is a multi-functional cytokine with context specific pro- and anti-tumorigenic effects. We aimed to investigate the prognostic role of activin tumor protein expression in AEG/ASs. METHODS: Tissue from a retrospective cohort of 277 patients with AEG/AS treated primarily by surgery at the Charité - Universitätsmedizin Berlin was collected and analyzed by immunohistochemistry using a specific antibody to the activin homodimer inhibin beta A. Additionally, we evaluated T-cell infiltration and PD1 expression as well as expression of PD-L1 by immunohistochemistry as possible confounding factors. Clinico-pathologic data were collected and correlated with activin protein expression. RESULTS: Out of 277 tumor samples, 72 (26.0%) exhibited high activin subunit inhibin beta A protein expression. Higher expression was correlated with lower Union for International Cancer Control (UICC) stage and longer overall survival. Interestingly, activin subunit expression correlated with CD4+ T-cell infiltration, and the correlation with higher overall survival was exclusively seen in tumors with high CD4+ T-cell infiltration, pointing towards a role of activin in the tumor immune response in AEG/ASs. CONCLUSION: In our cohort of AEG/AS, higher activin subunit levels were correlated with longer overall survival, an effect exclusively seen in tumors with high CD4+ cell infiltration. Further mechanistic research is warranted discerning the exact effect of this context specific cytokine.

Laparoscopic liver resection in Caroli disease: A single-centre case series.

BACKGROUND: Liver resection is the treatment of choice for patients with localised Caroli disease. While liver resection was traditionally performed as open procedure, this case series aims to evaluate the safety and efficacy of minimally invasive, laparoscopic liver surgery in these patients. METHODS: A systematic review of electronic case files of patients seen between April 2015 and December 2017 at the Department of Surgery, Charité University Hospital Berlin, was conducted. Patients with Caroli disease in whom laparoscopic liver resection had been performed were identified and analysed in this single-centre case series. RESULTS: Seven patients who underwent laparoscopic liver surgery for Caroli syndrome were identified and presented with a median age of 49 (range = 44-66) years, of which four (57%) were female. Preoperatively, six patients were classified as the American Society of Anaesthesiologists (ASA) 2 and one patient as ASA 3. Two operations were performed as single-incision laparoscopic surgery, whereas the others were done as multi-incision laparoscopic surgery. One patient required a conversion to an open procedure. The length of operation varied between patients, ranging from 128 to 758 min (median = 355). The length of stay in the intensive care unit ranged from 0 to 2 days. Two patients presented with post-operative complications (Clavien-Dindo Grade ≥3a), whereas no patient died. In histopathological analysis, all patients demonstrated characteristic findings of Caroli disease and no cholangiocarcinoma was found. CONCLUSION: These results indicate that minimally invasive, laparoscopic liver surgery is a safe and efficacious treatment option for patients with Caroli disease who require liver resection.

Correction to: The majority of ß-catenin mutations in colorectal cancer is homozygous.

An amendment to this paper has been published and can be accessed via the original article.

The majority of ß-catenin mutations in colorectal cancer is homozygous.

BACKGROUND: ß-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of ß-catenin gene (CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous ß-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs. RESULTS: Among 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, ß-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of ß-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found. CONCLUSIONS: In conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of ß-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on ß-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active ß-catenin, but it is not directly linked to the CTNNB1 mutational status.

Malignant transformation and genetic alterations are uncoupled in early colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) development is generally accepted as a sequential process, with genetic mutations determining phenotypic tumor progression. However, matching genetic profiles with histological transition requires the analyses of temporal samples from the same patient at key stages of progression. RESULTS: Here, we compared the genetic profiles of 34 early carcinomas with their respective adenomatous precursors to assess timing and heterogeneity of driver alterations accompanying the switch from benign adenoma to malignant carcinoma. In almost half of the cases, driver mutations specific to the carcinoma stage were not observed. In samples where carcinoma-specific alterations were present, TP53 mutations and chromosome 20 copy gains commonly accompanied the switch from adenomatous tissue to carcinoma. Remarkably, 40% and 50% of high-grade adenomas shared TP53 mutations and chromosome 20 gains, respectively, with their matched carcinomas. In addition, multi-regional analyses revealed greater heterogeneity of driver mutations in adenomas compared to their matched carcinomas. CONCLUSION: Genetic alterations in TP53 and chromosome 20 occur at the earliest histological stage in colorectal carcinomas (pTis and pT1). However, high-grade adenomas can share these alterations despite their histological distinction. Based on the well-defined sequence of CRC development, we suggest that the timing of genetic changes during neoplastic progression is frequently uncoupled from histological progression.

Age-dependent performance of BRAF mutation testing in Lynch syndrome diagnostics.

BRAF V600E mutations have been reported as a marker of sporadic microsatellite instability (MSI) colorectal cancer (CRC). Current international diagnostic guidelines recommend BRAF mutation testing in MSI CRC patients to predict low risk of Lynch syndrome (LS). We evaluated the age-specific performance of BRAF testing in LS diagnostics. We systematically compared the prevalence of BRAF mutations in LS-associated CRCs and unselected MSI CRCs in different age groups as available from published studies, databases and population-based patient cohorts. Sensitivity/specificity analysis of BRAF testing for exclusion of LS and cost calculations were performed. Among 969 MSI CRCs from LS carriers in the literature and German HNPCC Consortium, 15 (1.6%) harbored BRAF mutations. Six of seven LS patients with BRAF-mutant CRC and reported age were <50 years. Among 339 of 756 (44.8%) of BRAF mutations detected in unselected MSI CRC, only 2 of 339 (0.6%) BRAF mutations were detected in patients <50 years. The inclusion of BRAF testing led to high risk of missing LS patients and increased costs at age <50 years. BRAF testing in patients <50 years carries a high risk of missing a hereditary cancer predisposition and is cost-inefficient. We suggest direct referral of MSI CRC patients <50 years to genetic counseling without BRAF testing.

The human liver matrisome - Proteomic analysis of native and fibrotic human liver extracellular matrices for organ engineering approaches.

The production of biomaterials that endow significant morphogenic and microenvironmental cues for the constitution of cell integration and regeneration remains a key challenge in the successful implementation of functional organ replacements. Despite the vast development in the production of biological and architecturally native matrices, the complex compositions and pivotal figures by which the human matrisome mediates many of its essential functions are yet to be defined. Here we present a thorough analysis of the native human liver proteomic landscape using decellularization and defatting protocols to create extracellular matrix scaffolds of natural origin that can further be used in both bottom-up and top-down approaches in tissue engineering based organ replacements. Furthermore, by analyzing human liver extracellular matrices in different stages of fibrosis and cirrhosis, we have identified distinct attributes of these tissues that could potentially be exploited therapeutically and thus require further investigation. The general experimental pipeline presented in this study is applicable to any type of tissue and can be widely used for different approaches in regenerative medicine and in the construction of novel biomaterials for organ engineering approaches.

Next generation sequencing of lung adenocarcinoma subtypes with intestinal differentiation reveals distinct molecular signatures associated with histomorphology and therapeutic options.

OBJECTIVES: We aim to provide a better understanding of the molecular landscape of primary lung adenocarcinomas with intestinal differentiation. MATERIAL AND METHODS: Five invasive mucinous adenocarcinomas (IMA) and seven pulmonary enteric adenocarcinomas (PEAD) were included in this study. Furthermore, we analyzed six pulmonary colloid adenocarcinomas (CAD), including one primary tumor, one metastasis, and two sample pairs consisting of the primary colloid lung tumor and a matching metastasis and an acinar component, respectively. All samples were characterized using immunohistochemistry (TTF-1, CK7, CK20, CDX2, Ki-67, ALK and PD-L1) and a next generation sequencing panel covering 404 cancer-related genes (FoundationOne® gene panel). RESULTS AND CONCLUSION: While Ki-67 expression was comparably low in IMA (range: 8-15%) and in primary CAD (range: 5-8%), we observed considerably higher proliferation rates in the non-colloid tumor compartment (16%) and metastases (72%) from CAD, as well as in the PEAD-group (36-71%). The overall tumor mutational burden was lowest in IMA (2.5 mutations per megabase), intermediate in CAD (5.8 mutations per megabase) and highest in PEAD (16.8 mutations per megabase). KRAS mutations were frequent in all three tumor subtypes, but TP53 mutations were mostly limited to PEAD. While chromosomal alterations were rare in IMA, we discovered MYC amplifications in three of four CAD. Comparing primary and metastatic CAD, we observed the acquisition of multiple mutations and chromosomal alterations. PEAD had a variety of chromosomal alterations, including two cases with RICTOR amplification. PD-L1 expression (20%, 50% and 80% of tumor cells) was limited to three PEAD samples, only. In conclusion, we provide a detailed insight into the molecular alterations across and within the different subtypes of pulmonary adenocarcinomas with intestinal differentiation. From a clinical perspective, we provide data on potential treatment strategies for patients with PEAD, including immunotherapy.

Machine learning analysis of DNA methylation profiles distinguishes primary lung squamous cell carcinomas from head and neck metastases.

Head and neck squamous cell carcinoma (HNSC) patients are at risk of suffering from both pulmonary metastases or a second squamous cell carcinoma of the lung (LUSC). Differentiating pulmonary metastases from primary lung cancers is of high clinical importance, but not possible in most cases with current diagnostics. To address this, we performed DNA methylation profiling of primary tumors and trained three different machine learning methods to distinguish metastatic HNSC from primary LUSC. We developed an artificial neural network that correctly classified 96.4% of the cases in a validation cohort of 279 patients with HNSC and LUSC as well as normal lung controls, outperforming support vector machines (95.7%) and random forests (87.8%). Prediction accuracies of more than 99% were achieved for 92.1% (neural network), 90% (support vector machine), and 43% (random forest) of these cases by applying thresholds to the resulting probability scores and excluding samples with low confidence. As independent clinical validation of the approach, we analyzed a series of 51 patients with a history of HNSC and a second lung tumor, demonstrating the correct classifications based on clinicopathological properties. In summary, our approach may facilitate the reliable diagnostic differentiation of pulmonary metastases of HNSC from primary LUSC to guide therapeutic decisions.

External validation of molecular subtype classifications of colorectal cancer based on microsatellite instability, CIMP, BRAF and KRAS.

BACKGROUND: Competing molecular classification systems have been proposed to complement the TNM staging system for a better prediction of survival in colorectal cancer (CRC). However, validation studies are so far lacking. The aim of this study was to validate and extend previously published molecular classifications of CRC in a large independent cohort of CRC patients. METHODS: CRC patients were recruited into a population-based cohort study (DACHS). Molecular subtypes were categorized based on three previously published classifications. Cox-proportional hazard models, based on the same set of patients and using the same confounders as reported by the original studies, were used to determine overall, cancer-specific, or relapse-free survival for each subtype. Hazard ratios and confidence intervals, as well as Kaplan-Meier plots were compared to those reported by the original studies. RESULTS: We observed similar patterns of worse survival for the microsatellite stable (MSS)/BRAF-mutated and MSS/KRAS-mutated subtypes in our validation analyses, which were included in two of the validated classifications. Of the two MSI subtypes, one defined by additional presence of CIMP-high and BRAF-mutation and the other by tumors negative for CIMP, BRAF and KRAS-mutations, we could not confirm associations with better prognosis as suggested by one of the classifications. For two of the published classifications, we were able to provide results for additional subgroups not included in the original studies (men, other disease stages, other locations). CONCLUSIONS: External validation of three previously proposed classifications confirmed findings of worse survival for CRC patients with MSS subtypes and BRAF or KRAS mutations. Regarding MSI subtypes, other patient characteristics such as stage of the tumor, may influence the potential survival benefit. Further integration of methylation, genetic, and immunological information is needed to develop and validate a comprehensive classification that will have relevance for use in clinical practice.

The efficacy of treatment options for patients with gastric cancer and peritoneal metastasis.

BACKGROUND: Patients with peritoneal metastases of gastric cancer have a poor prognosis and median survival of 7 months. This study compared treatment options and outcomes based on the Peritoneal Cancer Index (PCI). METHODS: This retrospective analysis included patients with gastric cancer treated between August 2008 and December 2017 with synchronous peritoneal metastases only diagnosed by laparoscopy. The three treatments were as follows: (1) cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) in combination with pre- and postoperative systemic chemotherapy (n = 58), (2) laparotomy/laparoscopy without CRS, but HIPEC in combination with pre- and postoperative systemic chemotherapy (n = 11), and (3) systemic chemotherapy only (n = 19). RESULTS: A total of 88 patients aged 54.6 ± 10.9 years with mean PCI of 14.3 ± 11.3 were included. The PCI was significantly lower in group 1 (8.3 ± 5.7) than in group 2 (23.9 ± 11.1, p < 0.001) and group 3 (27.3 ± 9.3, p < 0.001). Mean time from diagnosis to laparoscopy was 5.2 ± 2.9 months. The median overall survival was 9.8 ± 0.7 for group 1, 6.3 ± 3.0 for group 2 and 4.9 ± 1.9 months for group 3 (p < 0.001). Predictors for deteriorated overall patient survival included > 4 cycles of preoperative chemotherapy (HR 4.49, p < 0.001), lymph-node metastasis (HR 3.53, p = 0.005), PCI ≥ 12 (HR 2.11, p = 0.036), and incompleteness of cytoreduction (HR 4.30, p = 0.001) in patients treated with CRS and HIPEC. CONCLUSION: CRS and HIPEC showed convincing results in selected patients with PCI < 12 and complete cytoreduction. Prolonged duration (> 4 cycles) of preoperative intravenous chemotherapy reduced patient survival in patients suitable for CRS and HIPEC.