B-Cos Aligned Transformers Learn Human-Interpretable Features

Vision Transformers (ViTs) and Swin Transformers (Swin) are currently state-of-the-art in computational pathology. However, domain experts are still reluctant to use these models due to their lack of interpretability. This is not surprising, as critical decisions need to be transparent and understandable. The most common approach to understanding transformers is to visualize their attention. However, attention maps of ViTs are often fragmented, leading to unsatisfactory explanations. Here, we introduce a novel architecture called the B-cos Vision Transformer (BvT) that is designed to be more interpretable. It replaces all linear transformations with the B-cos transform to promote weight-input alignment. In a blinded study, medical experts clearly ranked BvTs above ViTs, suggesting that our network is better at capturing biomedically relevant structures. This is also true for the B-cos Swin Transformer (Bwin). Compared to the Swin Transformer, it even improves the F1-score by up to 4.7% on two public datasets.Comment: Accepted at MICCAI 2023 (oral). Camera-ready available at https://doi.org/10.1007/978-3-031-43993-3_5

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine

The immunologic tumor microenvironment in endometrioid endometrial cancer in the morphomolecular context: mutual correlations and prognostic impact depending on molecular alterations

OBJECTIVE POLE-mutant, microsatellite-instable (MSI), p53-mutant and non-specific molecular profile (NSMP) are TCGA-defined molecular subgroups of endometrial cancer (EC). Hypothesizing that morphology and tumor immunology might differ depending on molecular background concerning composition and prognostic impact, we aimed to comprehensively interconnect morphologic, immunologic and molecular data. METHODS TCGA-defined molecular groups were determined by immunohistochemistry and sequencing in n = 142 endometrioid EC. WHO-defined histopathological grading was performed. The immunologic microenvironment (iTME) was characterised by the quantification of intraepithelial and stromal populations of tumor-infiltrating lymphocytes (TIL: overall T-cells; T-Killer cells; regulatory T-cells (Treg)). Immunologic parameters were correlated with WHO-grading, TCGA-subgroups and prognosis. RESULTS High density TIL were significantly more frequent in high-grade (G3) compared to low-grade (G1/2) EC in the whole cohort and in the subgroup of POLE-wildtype-/microsatellite-stable-EC. MSI was associated with high-level TIL-infiltration when taking into account the type of mismatch repair defect (MLH1/PMS2; MSH2/MSH6). Prognostic impact of biomarkers depended on molecular subgroups: In p53-mutant EC, Treg were independently prognostic, in NSMP, the unique independently prognostic biomarker was WHO-grading. CONCLUSIONS EC morphology and immunology differ depending on genetics. Our study delineated two molecularly distinct subgroups of immunogenic EC characterized by high-density TIL-infiltration: MSI EC and high-grade POLE-wildtype/microsatellite-stable-EC. Prognostic impact of TIL-populations relied on TCGA-subgroups indicating specific roles for TIL depending on molecular background. In NSMP, histopathological grading was the only prognostic biomarker demonstrating the relevance of WHO-grading in an era of molecular subtyping

NOTCH1 Intracellular Domain and the Tumor Microenvironment as Prognostic Markers in HNSCC

(1) Background: NOTCH1 is the second most common mutated gene in whole-exome sequencing of HNSCC. The aim of this project was to gain further insight into the relevance of NOTCH1 in HNSCC, potentially establishing NOTCH1 as a prognostic marker or therapeutic target; (2) Methods: NOTCH1 was silenced via RNA interference in six HNSCC cell lines and the impact was evaluated in migration and proliferation assays. Subsequently, the protein expression of NOTCH1 intracellular domain (NICD) and NOTCH1 mRNA expression were examined in 70 oropharyngeal squamous cell cancer tissue samples. Lastly, the NICD expression was compared with the local infiltration of lymphocytes, measured with the immunoscore; (3) Results: Knockdown of NOTCH1 decreased migration and proliferation. A high NICD expression was associated with lower OS. A high immunoscore resulted in significantly better OS. NICD expression was independent of the immunoscore and as a whole differentiated three distinct prognostic groups; (4) Conclusions: These data suggest that NOTCH1 is involved in migration and proliferation of HNSCC cell lines. In vivo, NICD expression was associated with overall survival and could, therefore, be used as a prognostic marker. NICD expression differs from NOTCH1 mRNA levels, potentially explaining the previously suggested bimodal role as an oncogene and tumor suppressor in HNSCC

Loss of CDX2 in colorectal cancer is associated with histopathologic subtypes and microsatellite instability but is prognostically inferior to hematoxylin-eosin-based morphologic parameters from the WHO classification

BACKGROUND Immunohistochemical loss of CDX2 has been proposed as a biomarker of dismal survival in colorectal carcinoma (CRC), especially in UICC Stage II/III. However, it remains unclear, how CDX2 expression is related to central hematoxylin-eosin (HE)-based morphologic parameters defined by 2019 WHO classification and how its prognostic relevance is compared to these parameters. METHODS We evaluated CDX2 expression in 1003 CRCs and explored its prognostic relevance compared to CRC subtypes, tumour budding and WHO grade in the overall cohort and in specific subgroups. RESULTS CDX2-low/absent CRCs were enriched in specific morphologic subtypes, right-sided and microsatellite-instable (MSI-H) CRCs (P \textless 0.001) and showed worse survival characteristics in the overall cohort/UICC Stage II/III (e.g. DFS: P = 0.005) and in microsatellite stable and left-sided CRCs, but not in MSI-H or right-sided CRCs. Compared with CDX2, all HE-based markers showed a significantly better prognostic discrimination in all scenarios. In multivariate analyses including all morphologic parameters, CDX2 was not an independent prognostic factor. CONCLUSION CDX2 loss has some prognostic impact in univariate analyses, but its prognostic relevance is considerably lower compared to central HE-based morphologic parameters defined by the WHO classification and vanishes in multivariate analyses incorporating these factors

TP53 germline mutations in the context of families with hereditary breast and ovarian cancer: a clinical challenge

Purpose!#!TP53germline (g) mutations, associated with the Li-Fraumeni syndrome (LFS), have rarely been reported in the context of hereditary breast and ovarian cancer (HBOC). The prevalence and cancer risks in this target group are unknown and counseling remains challenging. Notably an extensive high-risk surveillance program is implemented, which evokes substantial psychological discomfort. Emphasizing the lack of consensus about clinical implications, we aim to further characterize TP53g mutations in HBOC families.!##!Methods!#!Next-generation sequencing was conducted on 1876 breast cancer (BC) patients who fulfilled the inclusion criteria for HBOC.!##!Results!#!(Likely) pathogenic variants in TP53 gene were present in 0.6% of the BC cohort with higher occurrence in early onset BC < 36 years. (1.1%) and bilateral vs. unilateral BC (1.1% vs. 0.3%). Two out of eleven patients with a (likely) pathogenic TP53g variant (c.542G > A; c.375G > A) did not comply with classic LFS/Chompret criteria. Albeit located in the DNA-binding domain of the p53-protein and therefore revealing no difference to LFS-related variants, they only displayed a medium transactivity reduction constituting a retainment of wildtype-like anti-proliferative functionality.!##!Conclusion!#!Among our cohort of HBOC families, we were able to describe a clinical subgroup, which is distinct from the classic LFS-families. Strikingly, two families did not adhere to the LFS criteria, and functional analysis revealed a reduced impact on TP53 activity, which may suit to the attenuated phenotype. This is an approach that could be useful in developing individualized screening efforts for TP53g mutation carrier in HBOC families. Due to the low incidence, national/international cooperation is necessary to further explore clinical implications. This might allow providing directions for clinical recommendations in the future

A humanized bone microenvironment uncovers HIF2 alpha as a latent marker for osteosarcoma

The quest for predictive tumor markers for osteosarcoma (OS) has not well progressed over the last two decades due to a lack of preclinical models. The aim of this study was to investigate if microenvironmental modifications in an original humanized in vivo model alter the expression of OS tumor markers. Human bone micro-chips and bone marrow, harvested during hip arthroplasty, were implanted at the flanks of NOD/scid mice. We administered recombinant human bone morphogenetic protein 7 (rhBMP-7) in human bone micro-chips/bone marrow group I in order to modulate bone matrix and bone marrow humanization. Ten weeks post-implantation, human Luc-SAOS-2 OS cells were injected into the humanized tissue-engineered bone organs (hTEBOs). Tumors were harvested 5 weeks post-implantation to determine the expression of the previously described OS markers ezrin, periostin, VEGF, HIF1 alpha and HIF2 alpha. Representation of these proteins was analyzed in two different OS patient cohorts. Ezrin was downregulated in OS in hTEBOs with rhBMP-7, whereas HIF2 alpha was significantly upregulated in comparison to hTEBOs without rhBMP-7. The expression of periostin, VEGF and HIF1 alpha did not differ significantly between both groups. HIF2 alpha was consistently present in OS patients and dependent on tumor site and clinical stage. OS patients post-chemotherapy had suppressed levels of HIF2 alpha. In conclusion, we demonstrated the overall expression of OS-related factors in a preclinical model, which is based on a humanized bone organ. Our preclinical research results and analysis of two comprehensive patient cohorts imply that HIF2 alpha is a potential prognostic marker and/or therapeutic target. Statement of Significance This study demonstrates the clinical relevance of the humanized organ bone microenvironment in osteosarcoma research and validates the expression of tumor markers, especially HIF2 alpha. The convergence of clinically proven bone engineering concepts for the development of humanized mice models is a new starting point for investigations of OS-related marker expression. The validation and first data set in such a model let one conclude that further clinical studies on the role of HIF2 alpha as a prognostic marker and its potential as therapeutic target is a condition sine qua non. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options

Increased intraepithelial CD3+ T-lymphocytes and high PD-L1 expression on tumor cells are associated with a favorable prognosis in esophageal squamous cell carcinoma and allow prognostic immunogenic subgrouping.

Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer associated with poor prognosis and additional therapeutic strategies must be implemented to optimize ESCC treatment. Meanwhile, the important biologic role and potential prognostic and therapeutic implications of a tumors immunologic microenvironment (IM) have been recognized in various cancers.In order to investigate the contexture and the prognostic relevance of the IM in ESCC, we immunohistochemically evaluated the extent of overall/intraepithelial TILs (CD3+/CD8+) and of PD-1 / PD-L1 expression in a cohort of 125 therapy-naive ESCCs, additionally assessing PD-L1 copy number status via fluorescence in-situ hybridization.High intraepithelial CD3+ TILs (CD3ihigh) and high PD-L1 expression on tumor cells (PD-L1high) were each significantly associated with improved overall- (OS) (CD3+: p = 0.019; PD-L1: p = 0.028), disease specific- (DSS) (CD3+: p = 0.05; PD-L1: p = 0.006) and disease free survival (DFS) (CD3+: p = 0.009; PD-L1: p < 0.001). CD3ihigh- and PD-L1high cases were significantly associated with one another (p < 0.001). Subgrouping of ESCC revealed decreased OS (p = 0.031), DSS (p = 0.012) and DFS (p < 0.001) for CD3ilow/PD-L1low cancers.Our data not only associate CD3ihigh- and PD-L1high ESCC with a beneficial outcome, but also demonstrate PD-L1high- and CD3ihigh status to be closely intertwined. Furthermore, our study demarcates a prognostically unfavorable, "non-immunoreactive" CD3ilow / PD-L1low ESCC-subgroup, potentially forming the basis for an immune-based stratification of ESCC