Uterine mesenchymal neoplasms comprise a heterogeneous group of tumours with distinct morphological and molecular features. High-grade endometrial stromal sarcomas (HG-ESS) are typically defined by YWHAE::NUTM2 or BCOR alterations; however, a subset lacks these canonical rearrangements. We report a 59-year-old woman with a HG-ESS-like uterine mesenchymal neoplasm, composed of atypical oval cells with infiltrative growth, brisk mitotic activity, and tumour necrosis. Immunohistochemistry showed diffuse SMA and p16 expression with partial CD10 and cyclin D1 positivity while ER, PR, desmin, ALK, and panTRK were negative. RNA sequencing identified an AKAP9 exon 8 to BRAF exon 9 fusion, but no canonical ESS-associated rearrangements or additional alterations (including p53). This finding expands the molecular spectrum of uterine mesenchymal neoplasms and defines a novel kinase-driven subset. The BRAF fusion indicates MAPK pathway activation and raises the possibility of a distinct entity or a novel pathogenetic pathway in HG-ESS, with potential therapeutic implications.

Abstract Purpose Colorectal cancer is one of the most common and deadliest cancer types worldwide. In the last years, changes in the mitochondrial DNA (mtDNA) copy number have been described to correlate with the prognostic outcome for colorectal cancer patients by impacting different tumorigenic properties. One key regulator of mtDNA is the mitochondrial transcription factor A (TFAM) that acts as a limiting factor of mtDNA copy number. Here, we investigated the effect of TFAM deficiency on mtDNA and tumorigenic properties in the human colorectal cancer cell line SW480. Methods TFAM expression was stably downregulated in the colorectal cancer cell line SW480 using the CRISPR-Cas9 approach. To dissect the molecular alterations induced by deletion of TFAM, RNA sequencing and gene set enrichment analysis was performed on TFAM-wild-type and TFAM-deficient SW480 cells. Functional consequences of TFAM downregulation were assessed in cellular assays. Results We showed that TFAM deficiency leads to decreased mtDNA copy number and reduced expression of mtDNA-encoded genes. TFAM-deficient cells also revealed higher activity of senescence-associated β-galactosidase and decreased cell growth parameters. Moreover, RNA sequencing showed that the expression of cyclin dependent kinase inhibitor 1A (CDKN1A/p21) is significantly increased in TFAM-deficient cells. Conclusion Our results suggest that TFAM-induced changes of the mitochondrial genome lead to upregulated CDKN1A/p21 expression in colorectal cancer cells identifying p21 as a new possible linker between mitochondria and nucleus.

Deleterious mutations in the BRCA1 and BRCA2 genes have significant therapeutic relevance in clinical settings regarding personalized therapy approaches. BRCA1 and BRCA2 play a pivotal role in homologous recombination (HR) and thus are sensitive for PARP inhibitors (PARPi). Beyond the narrow scope of evaluating only the BRCA mutation status, PARPi can be beneficial for HR deficient (HRD) patients, who harbor mutations in other HR-associated genes. In the present retrospective study, a novel targeted HRD gene panel was validated and implemented for use with FFPE tissue. Samples of patients with ovarian, breast, pancreatic and prostate cancer were included. Variants were robustly detected with various DNA input amounts and the use of test samples showed complete concordance between previously known mutations and HRD panel results. From all the 90 samples included in this cohort, TP53 was the most frequently altered gene (73%). Deleterious BRCA1/2 mutations were found in 20 (22%) of all samples. New pathogenic or likely pathogenic mutations in additional HR-associated genes were identified in 22 (24%) patients. Taken together, the present study proves the feasibility of a new HRD gene panel with reliable panel performance and offers the possibility to easily screen for resistance mutations acquired over treatment time. Mutations in HR-associated genes, besides BRCA1/2 , might represent promising potential targets for synthetic lethality approaches. Thus, a substantial number of patients may benefit from expanding the scope of therapeutic agents like PARPi.

Background: Treatment of CRLM with major vessel involvement is still challenging and valid data on outcomes are still rare. We analyzed our experience of hepatectomies with resection and reconstruction of major hepatic vessels with regard to operative and perioperative details, histopathological findings and oncological outcome. Methods: Data of 32 hepatectomies with major hepatic vessel resections and reconstructions were included. Results were correlated with perioperative and oncological outcome. Results: Out of 1236 surgical resections due to CRLM, we performed 35 major hepatic vessel resections and reconstructions in 32 cases (2.6%) during the study period from January 2008 to March 2023. The vena cava inferior (VCI) was resected and reconstructed in 19, the portal vein (PV) in 6 and a hepatic vein (HV) in 10 cases. Histopathological examination confirmed a vascular infiltration in 6/32 patients (VCI 3/17, HV 2/10 and PV 1/6). There were 27 R0 and 5 R1 resections. All R1 situations affected the parenchymal margin. Vascular wall margins were R0. Ninety-day mortality was 0. The median overall survival (OS) for the patient group with vascular infiltration (V1) was 21 months and for the V0 group 33.3 months. Conclusion: Liver resections with vascular resection and reconstruction are rare and histological vessel infiltration occurs seldom. In cases with presumed vascular wall infiltration, liver resection combined with major vessel resection and reconstruction can be performed with low morbidity and mortality. We prefer a parenchymal sparing liver resection with vascular resection and reconstruction to achieve negative resection margins, but in technically difficult cases with higher risk for postoperative complications, tumor detachment from vessels without resection is a most reasonable surgical alternative.

Papillary thyroid carcinoma (PTC), the most common malignancy of follicular cell derivation, is generally associated with good prognosis. Nevertheless, it is important to identify patients with aggressive PTCs and unfavorable outcome. Molecular markers such as BRAFV600E mutation and TERT promoter mutations have been proposed for risk stratification. While TERT promoter mutations have been frequently associated with aggressive PTCs, the association of BRAFV600E mutation with increased recurrence and mortality is less clear and has been controversially discussed. The aim of the present study was to analyze whether differentially expressed genes can predict BRAFV600E mutations as well as TERT promoter mutations in PTCs. RNA sequencing identified a large number of differentially expressed genes between BRAFV600E and BRAFwildtype PTCs. Of those, AHNAK2, DCSTAMP, and FN1 could be confirmed in a larger cohort (n = 91) to be significantly upregulated in BRAFV600E mutant PTCs using quantitative RT-PCR. Moreover, individual PTC expression values of DCSTAMP and FN1 were able to predict the BRAFV600E mutation status with high sensitivity and specificity. The expression of TERT was detected in all PTCs harboring TERT promoter mutations and in 19% of PTCs without TERT promoter mutations. Tumors with both TERT expression and TERT promoter mutations were particularly associated with aggressive clinicopathological features and a shorter recurrence-free survival. Altogether, it will be interesting to explore the biological function of AHNAK2, DCSTAMP, and FN1 in PTC in more detail. The analysis of their expression patterns could allow the characterization of PTC subtypes and thus enabling a more individualized surgical and medical treatment.

INTRODUCTION:Whole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis. METHODS:To assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics. Somatic and germline variants, copy-number alterations (CNAs), and complex biomarkers were investigated. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. The raw data were collected and re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability. RESULTS:The mean positive percentage agreement (PPA) of somatic variant calling was 76 % while the positive predictive value (PPV) was 89 % in relation to a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88 % for all and 97 % for the clinically relevant variants. CNA calls were concordant for 82 % of genomic regions. Homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94 %, 93 %, and 93 % of calls, respectively. Variability of CNAs and complex biomarkers did not decrease considerably after harmonization of the bioinformatic processing and was hence attributed mainly to wet-lab differences. CONCLUSION:Continuous optimization of bioinformatic workflows and participating in round robin tests are recommended.

Das zunehmende Verständnis der Onkogenese sowie die immer umfangreichere Diagnostik ermöglichen zunehmend die Detektion von Mutationen, die für die Tumorentwicklung und Progression relevant sind. Daraus resultierend lassen sich Ansatzpunkte für zielgerichtete Therapien ableiten. Die vorliegende Übersichtsarbeit soll mittels Pro- und Kontra-Abschnitten einen Überblick über die Möglichkeiten, aber auch Limitationen entitätsagnostischer Therapieansätze aufzeigen und einen Ausblick in die Zukunft geben.

Although it has long been known that the immune cell composition has a strong prognostic and predictive value in colorectal cancer (CRC), scoring systems such as the immunoscore (IS) or quantification of intraepithelial lymphocytes are only slowly being adopted into clinical routine use and have their limitations. To address this we established and evaluated a multistain deep learning model (MSDLM) utilizing artificial intelligence (AI) to determine the AImmunoscore (AIS) in more than 1,000 patients with CRC. Our model had high prognostic capabilities and outperformed other clinical, molecular and immune cell-based parameters. It could also be used to predict the response to neoadjuvant therapy in patients with rectal cancer. Using an explainable AI approach, we confirmed that the MSDLM's decisions were based on established cellular patterns of anti-tumor immunity. Hence, the AIS could provide clinicians with a valuable decision-making tool based on the tumor immune microenvironment.

BACKGROUND The aim of this study was to analyse the role of associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) for advanced colorectal liver disease. Surgery offers the best long-term survival in patients with colorectal liver metastases (CRLM). To increase the rates of resectability, two-stage procedures (TSH) and ALPPS are established methods in cases of advanced colorectal liver disease to avoid post hepatectomy liver failure (PHLF). There is still a debate of the oncological utility and the surgical ranking of ALPPS in this clinical scenario. The aim of this analysis was to share our ALPPS data of the perioperative and oncological outcome in patients with CRLM and to compare them with regard to recommendations of published data. METHODS Ten patients (1.1%) out of 881 received a classical ALPPS procedure between January 2008 and November 2021 at our institution. The median volume increase was 76% (range 55-125%) in a median time interval of 7 days. RESULTS The completion rate was 100% and all resections were R0-situations (100%). No patient developed PHLF. The median overall survival (OS) was 36.7 months and the median recurrence-free survival (RFS) 6.1 months. CONCLUSIONS The ALPPS procedure is a surgical approach to achieve a R0 situation in patients with an extensive intrahepatic tumor burden. Nevertheless, the use of ALPPS should be allocated for patients who have no other surgical options.

Recent progress in clinical development of KRAS inhibitors has raised interest in predicting the tumor dependency on frequently mutated RAS-pathway oncogenes. However, even without such activating mutations, RAS proteins represent core components in signal integration of several membrane-bound kinases. This raises the question of applications of specific inhibitors independent from the mutational status. Here, we examined CRISPR/RNAi data from over 700 cancer cell lines and identified a subset of cell lines without KRAS gain-of-function mutations (KRASwt) which are dependent on KRAS expression. Combining machine learning-based modeling and whole transcriptome data with prior variable selection through protein-protein interaction network analysis by a diffusion kernel successfully predicted KRAS dependency in the KRASwt subgroup and in all investigated cancer cell lines. In contrast, modeling by RAS activating events (RAE) or previously published RAS RNA-signatures did not provide reliable results, highlighting the heterogeneous distribution of RAE in KRASwt cell lines and the importance of methodological references for expression signature modeling. Furthermore, we show that predictors of KRASwt models contain non-substitutable information signals, indicating a KRAS dependency phenotype in the KRASwt subgroup. Our data suggest that KRAS dependent cancers harboring KRAS wild type status could be targeted by directed therapeutic approaches. RNA-based machine learning models could help in identifying responsive and non-responsive tumors.

Background: Outcome after resection of CRLM is hampered by a high rate of recurrence. There are little data about the role of cancer related genes and their mutations in this scenario. The aim of our analysis was to assess the predictive power of cancer-related genes and their mutations on risk for and distribution of recurrence and the time of occurrence after resection of colorectal liver metastases (CRLM). Methods: We included 130 patients with 167 liver resections. The work-up consisted of the analysis of a total of 720 cancer-related genes by next-generation sequencing (NGS). Results were correlated with the patterns and time of recurrence and survival. Results: At the time of analysis, 89/130 patients (68%) had developed recurrence. This included liver only recurrence in 52%, lung only recurrence in 11% and disseminated disease in 37% of cases. In univariate analysis, alterations in the RAS/RAF pathway and in the SMAD family had significant predictive power for the time of recurrence (P<0.0001) whereas single mutations did not reach statistical significance in multivariate analysis. Mutations of PIK3CA were associated with a better prognosis and a later occurrence of relapse. A recurrence risk score (r-RS) based on mutations in these cancer related genes is predictive of the time of recurrence. Conclusions: In conclusion, mutations in the RAS/RAF pathway and the SMAD family are risk factors for early recurrence. Mutations of PIK3CA are associated with a lower risk for recurrence after resection of CRLM. Cancer related genes and their mutations do not correlate with patterns of recurrence but are predictive for the timely onset of recurrence.

ZusammenfassungMutationen des hepatocyte nuclear factor 1 α-(HNF1α) Gens lösen einen MODY3-Diabetes aus und sind mit einer hepatozellulären Adenomatose assoziiert. Eine maligne Transformation von HNF1α-mutierten hepatozellulären Adenomen ist selten. Wir beschreiben hier den Fall eines 28-jährigen Mannes mit heterozygoter HNF1α-Keimbahnmutation mit MODY3-Diabetes und einer hepatozellulären Adenomatose mit multiplen, HNF1α-inaktivierten Adenomen, der ein inflammatorisches Adenom mit β-Catenin-Mutation mit maligner Transformation in ein gut differenziertes hepatozelluläres Karzinom (HCC) entwickelte.

Hepatocyte nuclear factor 1 alpha (HNF1 alpha) mutations cause maturity-onset diabetes of the young (MODY) type 3 and are associated with hepatocellular adenomatosis. Malignant transformation of HNF1 alpha-mutated hepatocellular adenomas is rare. We report a case of a 28-year-old man with HNF1-inactivated adenomatosis who developed an inflammatory adenoma with beta catenin mutation with malignant transformation into a well-differentiated hepatocellular carcinoma (HCC).

Two-stage hepatectomy (TSH) with or without portal vein ligation (PVL) or portal vein embolization (PVE) and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) are surgical strategies in the treatment of advanced colorectal liver metastases (CRLM). The role of each strategy is yet ill defined. The aim of this analysis is to share our center experience with conventional TSH with or without PVL/PVE and ALPPS in patients with advanced bilateral CRLM.

Serrated or Hyperplastic Polyposis Syndrome (SPS, HPS) is a yet poorly defined colorectal cancer (CRC) predisposition characterised by the occurrence of multiple and/or large serrated polyps throughout the colon. A serrated polyp-CRC sequence (serrated pathway) of CRC formation has been postulated, however, to date only few molecular signatures of serrated neoplasia (BRAF, KRAS, RNF43 mutations, CpG Island Methylation, MSI) have been described in a subset of SPS patients and neither the etiology of the syndrome nor the distinct genetic alterations during tumorigenesis have been identified.

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Esophageal cancer (EC) is one of the most common malignancies diagnosed in the Western world with an increasing incidence noted for esophageal adenocarcinoma (EAC). Despite improvements in staging, surgical procedures and postoperative treatments, the overall survival of patients with EC remains low. Murine double minute‑2 (MDM2) acts as an oncogene by inducing the degradation of the tumor‑suppressor protein TP53. In order to evaluate the MDM2 gene amplification status in EAC and squamous cell carcinoma (SCC), we established a quantitative PCR (qPCR) assay, screening a total of 127 esophageal carcinoma cases for MDM2 amplification. Esophageal carcinoma cases with enhanced MDM2 gene copy numbers were further analyzed by fluorescence in situ hybridisation (FISH) and MDM2 immunostaining. Among a total of 23 specimens (18%), identified by qPCR to possess elevated MDM2 gene copy numbers, one third (6.3%) showed a cluster‑like fluorescence pattern by FISH analyses and marked MDM2 protein immunostaining. MDM2 gene amplifications did not correlate with the occurrence of TP53 mutations. Due to the high therapeutic relevance of MDM2 overexpression, but the high cost of FISH, we suggest a primary screening of MDM2 copy number variations by qPCR, followed by detailed FISH analysis of the identified ECs.