Procoagulant platelets promote immune evasion in triple-negative breast cancer.

ABSTRACT: Triple-negative breast cancer (TNBC) is an aggressive tumor entity in which immune checkpoint (IC) molecules are primarily synthesized in the tumor environment. Here, we report that procoagulant platelets bear large amounts of such immunomodulatory factors and that the presence of these cellular blood components in TNBC relates to protumorigenic immune-cell activity and impaired survival. Mechanistically, tumor-released nucleic acids attract platelets to the aberrant tumor microvasculature, where they undergo procoagulant activation, thus delivering specific stimulatory and inhibitory IC molecules. This concomitantly promotes protumorigenic myeloid leukocyte responses and compromises antitumorigenic lymphocyte activity, ultimately supporting tumor growth. Interference with platelet-leukocyte interactions prevented immune cell misguidance and suppressed tumor progression, nearly as effective as systemic IC inhibition. Hence, our data uncover a self-sustaining mechanism of TNBC by using platelets to misdirect immune-cell responses. Targeting this irregular multicellular interplay may represent a novel immunotherapeutic strategy for TNBC without the adverse effects of systemic IC inhibition.

Proteomic Characterization of Undifferentiated Small Round Cell Sarcomas With EWSR1 and CIC::DUX4 Translocations Reveals Diverging Tumor Biology and Distinct Diagnostic Markers.

Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.

Sensitive Positron Emission Tomography Imaging of PD-L1 Expression in Human Breast and Lung Carcinoma Xenografts Using the Radiometalated Peptide Ga-68-TRAP-WL12.

Noninvasive imaging of the immune checkpoint protein programmed death ligand 1 (PD-L1; synonyms: CD274, B7-H1) holds great promise to improve patient selection and, thus, response rates for immune checkpoint therapy (ICT) with monoclonal antibodies targeting the PD1/PD-L1 axis. The PD-L1 specific peptide WL12 (cyclo(AcY-(NMe)A-N-P-H-L-Hyp-W-S-W(Me)-(NMe)Nle-(NMe)Nle-O-C)-G-NH2) was functionalized with the Gallium-68 chelator TRAP by means of click chemistry (CuAAC). The resulting conjugate TRAP-WL12 was labeled with Gallium-68 within 16 min, with approximately 90% radiochemical yield and 99% radiochemical purity, affording Ga-68-TRAP-WL12 with molar activities typically exceeding 100 MBq/nmol. This radiotracer was characterized by positron emission tomography (PET) imaging and ex vivo biodistribution in murine xenografts of nontransfected PD-L1 expressing tumor cell lines, MDA-MB-231 (human breast carcinoma), and H2009 (human lung adenocarcinoma). It showed a favorable biodistribution profile with rapid renal clearance and low background (tumor-to-blood ratio = 26.6, 3 h p.i.). Conjugation of the Ga-68-TRAP moiety to WL12 successfully mitigated the nonspecific uptake of this peptide in organs, particularly the liver. This was demonstrated by comparing Ga-68-TRAP-WL12 with the archetypical Ga-68-DOTA-WL12, for which tumor-to-liver ratios of 1.4 and 0.5, respectively, were found. Although immunohistochemistry (IHC) revealed a low PD-L1 expression in MDA-MB-213 and H2009 xenografts that corresponds well to the clinical situation, PET showed high tumor uptakes (6.6 and 7.3% injected activity per gram of tissue (iA/g), respectively) for Ga-68-TRAP-WL12. Thus, this tracer has the potential for routine clinical PD-L1 PET imaging because it detects even very low PD-L1 expression densities with high sensitivity and may open an avenue to replace PD-L1 IHC of biopsies as the standard means to select potential responders for ICT.

Concordance between an FDA-approved companion diagnostic and an alternative assay kit for assessing homologous recombination deficiency in ovarian cancer.

OBJECTIVE: Authors evaluated the performance of a commercially available next-generation sequencing assay kit; this was based on genomic content from Illumina's TruSight™ Oncology 500 research assay that identifies BRCA variants and proprietary algorithms licensed from Myriad and, with additional genomic content, measures the homologous recombination deficiency (HRD) genomic instability score (GIS) in tumor tissue (TSO 500 HRD assay). METHODS: Data from the TSO 500 HRD assay were compared with data from the Myriad MyChoice®CDx PLUS assay (Myriad assay). Prevalence rates for overall HRD status and BRCA mutations (a deleterious or suspected deleterious BRCA1 or BRCA2 mutation or both) and assay agreement rates for HRD GIS and BRCA analysis were assessed in ovarian tumor samples. Pearson correlations of the continuous HRD GIS and analytic sensitivity and specificity were evaluated. RESULTS: The prevalence of overall HRD positivity was 51.2% (TSO 500 HRD assay) versus 49.2% (Myriad assay) and the prevalence of BRCA mutations was 27.6% (TSO 500 HRD assay) versus 25.5% (Myriad assay). After post-processing optimization, concordance of the HRD GIS was 0.980 in all samples and 0.976 in the non-BRCA mutation cohort; the area under the receiver operating characteristic curve was 0.995 and 0.992, respectively. CONCLUSIONS: Comparison between the Illumina and Myriad assays showed that overall HRD status, the individual components of BRCA analysis, and HRD GIS detection results were highly concordant (>93%), suggesting the TSO 500 HRD assay will approach the analytical accuracy of the FDA-approved Myriad assay.

Stroma AReactive Invasion Front Areas (SARIFA) proves prognostic relevance in gastric carcinoma and is based on a tumor-adipocyte interaction indicating an altered immune response.

BACKGROUND: Recently, we presented Stroma AReactive Invasion Front Areas (SARIFA) as a new histomorphologic negative prognostic biomarker in gastric cancer. It is defined as direct contact between tumor cells and fat cells. The aim of this study was to further elucidate the underlying genomic, transcriptional, and immunological mechanisms of the SARIFA phenomenon. METHODS: To address these questions, SARIFA was classified on H&E-stained tissue sections of three cohorts: an external cohort (n = 489, prognostic validation), the TCGA-STAD cohort (n = 194, genomic and transcriptomic analysis), and a local cohort (n = 60, digital spatial profiling (whole transcriptome) and double RNA in situ hybridization/immunostaining of cytokines). RESULTS: SARIFA status proved to be an independent negative prognostic factor for overall survival in an external cohort of gastric carcinomas. In TCGA-STAD cohort, SARIFA is not driven by distinct genomic alterations, whereas the gene expression analyses showed an upregulation of FABP4 in SARIFA-positive tumors. In addition, the transcriptional regulations of white adipocyte differentiation, triglyceride metabolism, and catabolism were upregulated in pathway analyses. In the DSP analysis of SARIFA-positive tumors, FABP4 and the transcriptional regulation of white adipocyte differentiation were upregulated in macrophages. Additionally, a significantly lower expression of the cytokines IL6 and TNFα was observed at the invasion front. CONCLUSIONS: SARIFA proves to be a strong negative prognostic biomarker in advanced gastric cancer, implicating an interaction of tumor cells with tumor-promoting adipocytes with crucial changes in tumor cell metabolism. SARIFA is not driven by tumor genetics but is very likely driven by an altered immune response as a causative mechanism.

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.

How to use AI in pathology.

AI plays an important role in pathology, both in clinical practice supporting pathologists in their daily work, and in research discovering novel biomarkers for improved patient care. Still, AI is in its starting phase, and many pathology labs still need to transition to a digital workflow to be able to enjoy the benefits of AI. In this perspective, we explain the major benefits of AI in pathology, highlight key requirements that need to be met and example how to use it in a typical workflow.

GATA4 and GATA6 loss-of-expression is associated with extinction of the classical programme and poor outcome in pancreatic ductal adenocarcinoma.

OBJECTIVE: GATA6 is a key regulator of the classical phenotype in pancreatic ductal adenocarcinoma (PDAC). Low GATA6 expression associates with poor patient outcome. GATA4 is the second most expressed GATA factor in the pancreas. We assessed whether, and how, GATA4 contributes to PDAC phenotype and analysed the association of expression with outcome and response to chemotherapy. DESIGN: We analysed PDAC transcriptomic data, stratifying cases according to GATA4 and GATA6 expression and identified differentially expressed genes and pathways. The genome-wide distribution of GATA4 was assessed, as well as the effects of GATA4 knockdown. A multicentre tissue microarray study to assess GATA4 and GATA6 expression in samples (n=745) from patients with resectable was performed. GATA4 and GATA6 levels were dichotomised into high/low categorical variables; association with outcome was assessed using univariable and multivariable Cox regression models. RESULTS: GATA4 messenger RNA is enriched in classical, compared with basal-like tumours. We classified samples in 4 groups as high/low for GATA4 and GATA6. Reduced expression of GATA4 had a minor transcriptional impact but low expression of GATA4 enhanced the effects of GATA6 low expression. GATA4 and GATA6 display a partially overlapping genome-wide distribution, mainly at promoters. Reduced expression of both proteins in tumours was associated with the worst patient survival. GATA4 and GATA6 expression significantly decreased in metastases and negatively correlated with basal markers. CONCLUSIONS: GATA4 and GATA6 cooperate to maintain the classical phenotype. Our findings provide compelling rationale to assess their expression as biomarkers of poor prognosis and therapeutic response.

Next-generation diagnostics for precision oncology: Preanalytical considerations, technical challenges, and available technologies.

Molecular diagnostics as the centrepiece of precision oncology has gone through revolutionary developments over the last decade, becoming tremendously broad, deep and precise with still ongoing advancements. In the majority of scenarios, treatment selection for cancer patients without any type of molecular characterization is no longer conceivable. Considering the impact of sample quality on the reliability of molecular analyses and the importance of the results for the fate of an individual patient, it is surprising how sparsely preanalytical and analytical requirements are addressed scientifically. Standardization and rigorous quality assessment continue to play only a marginal role in the field. Within this review, we will systematically discuss influencing preanalytic parameters and technology setups affecting molecular test results. We will shed light on the specifics of different analytes, technical modalities, and analysis pipelines. The review will have a certain focus on broad molecular genetic tumour testing with next generation sequencing but will go beyond that including other molecular diagnostic modalities and will give a glimpse into the future of molecular testing.

Tumour budding-based grading as independent prognostic biomarker in HPV-positive and HPV-negative head and neck cancer.

BACKGROUND: The prognostic significance of tumour budding (TB) and minimal cell nest size (MCNS) was shown in human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC). However, the optimisation of cutpoints, the prognostic impact in HPV-positive HNSCC, and the comparison with other histopathological grading systems are insufficiently investigated. METHODS: TB and MCNS were analysed digitally in 1 and 10 high-power fields (HPF) of 331 HPV-positive and HPV-negative cases from TCGA. Optimising the cutpoints a new cellular dissociation grading (CDG) system was defined and compared to the WHO grading and the Brandwein-Gensler (BG) risk model. RESULTS: The two-tiered CDG system based solely on TB yielded optimal prognostic stratification with shortened overall survival for CDG-high cases. Optimal cut-offs were two buds (1 HPF) and six buds (10 HPF), respectively. Analysing MCNS did not add prognostic significance to quantifying TB. CDG was a significant prognostic marker in HPV-negative and HPV-positive tumours and prognostically superior to the WHO and BG systems. High CDG was associated with clinically occult lymph-node metastases. CONCLUSIONS: The most comprehensive study of TB in HNSCC so far confirmed its prognostic impact in HPV-negative tumours and for the first time in HPV-positive tumours. Further studies are warranted to evaluate its applicability for therapy guidance in HNSCC.

Expression of Nectin-4 in Chromophobe Renal Cell Carcinoma in a multicenter cohort - early prognostic and therapeutic considerations.

INTRODUCTION: Nectin-4 is a member of the nectin family and a calcium-independent immunoglobuline-like transmembrane protein that contributes to tumor growth and angiogenesis in malignant tumors. A Nectin-4 directed antibody drug conjugate, Enfortumab vedotin-ejf, has recently been approved for treatment in urothelial cancer and is currently under investigation in other tumor entities such as breast, lung, and prostate cancer. In non-clear cell renal cell carcinoma (RCC) vascular endothelial growth factor (VEGF)-directed tyrosine kinase inhibitors and checkpoint inhibitors are currently treatments of choice. However, due to the rarity of disease treatment recommendations for chromophobe RCC (chRCC) are limited and new therapeutic agents urgently needed. In this study, we investigated the expression and prognostic impact of Nectin-4 in a large cohort of chRCC. METHODS: patients who underwent renal surgery due to chRCC were recruited. Clinical data was retrospectively evaluated. Tumor specimen were analyzed for Nectin-4 expression by immunohistochemistry. RESULTS: 81 chRCC patients were eligible for analysis. In 15 (18.5 %) samples tumors were positive for Nectin-4. No significant associations were found for Nectin-4 expression and clinical attributes in patients with chRCC. Kaplan-Meier analysis disclosed a 5 year- overall survival for Nectin-4-negative and Nectin-4-positive tumors of 91.8% versus 100.0% (p=0.316, log rank). CONCLUSIONS: In chRCC a small subset of tumors expresses Nectin-4 potentially amenable to Nectin-4 directed treatment. Expression of Nectin-4 is not associated with parameters of aggressiveness or survival. Due to the rare incidence of chRCC further studies with larger cohorts are warranted.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging: Diagnostic Pathways and Metabolites for Renal Tumor Entities.

BACKGROUND: Correct tumor subtyping of primary renal tumors is essential for treatment decision in daily routine. Most of the tumors can be classified based on morphology alone. Nevertheless, some diagnoses are difficult, and further investigations are needed for correct tumor subtyping. Besides histochemical investigations, high-mass-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect new diagnostic biomarkers and hence improve the diagnostic. PATIENTS AND METHODS: Formalin-fixed paraffin embedded tissue specimens from clear cell renal cell carcinoma (ccRCC, n = 552), papillary renal cell carcinoma (pRCC, n = 122), chromophobe renal cell carcinoma (chRCC, n = 108), and renal oncocytoma (rO, n = 71) were analyzed by high-mass-resolution MALDI fourier-transform ion cyclotron resonance (FT-ICR) MSI. The SPACiAL pipeline was executed for automated co-registration of histological and molecular features. Pathway enrichment and pathway topology analysis were performed to determine significant differences between RCC subtypes. RESULTS: We discriminated the four histological subtypes (ccRCC, pRCC, chRCC, and rO) and established the subtype-specific pathways and metabolic profiles. rO showed an enrichment of pentose phosphate, taurine and hypotaurine, glycerophospholipid, amino sugar and nucleotide sugar, fructose and mannose, glycine, serine, and threonine pathways. ChRCC is defined by enriched pathways including the amino sugar and nucleotide sugar, fructose and mannose, glycerophospholipid, taurine and hypotaurine, glycine, serine, and threonine pathways. Pyrimidine, amino sugar and nucleotide sugar, glycerophospholipids, and glutathione pathways are enriched in ccRCC. Furthermore, we detected enriched phosphatidylinositol and glycerophospholipid pathways in pRCC. CONCLUSION: In summary, we performed a classification system with a mean accuracy in tumor discrimination of 85.13%. Furthermore, we detected tumor-specific biomarkers for the four most common primary renal tumors by MALDI-MSI. This method is a useful tool in differential diagnosis and biomarker detection.

Profile of the multicenter cohort of the German Cancer Consortium's Clinical Communication Platform.

Treatment concepts in oncology are becoming increasingly personalized and diverse. Successively, changes in standards of care mandate continuous monitoring of patient pathways and clinical outcomes based on large, representative real-world data. The German Cancer Consortium's (DKTK) Clinical Communication Platform (CCP) provides such opportunity. Connecting fourteen university hospital-based cancer centers, the CCP relies on a federated IT-infrastructure sourcing data from facility-based cancer registry units and biobanks. Federated analyses resulted in a cohort of 600,915 patients, out of which 232,991 were incident since 2013 and for which a comprehensive documentation is available. Next to demographic data (i.e., age at diagnosis: 2.0% 0-20 years, 8.3% 21-40 years, 30.9% 41-60 years, 50.1% 61-80 years, 8.8% 81+ years; and gender: 45.2% female, 54.7% male, 0.1% other) and diagnoses (five most frequent tumor origins: 22,523 prostate, 18,409 breast, 15,575 lung, 13,964 skin/malignant melanoma, 9005 brain), the cohort dataset contains information about therapeutic interventions and response assessments and is connected to 287,883 liquid and tissue biosamples. Focusing on diagnoses and therapy-sequences, showcase analyses of diagnosis-specific sub-cohorts (pancreas, larynx, kidney, thyroid gland) demonstrate the analytical opportunities offered by the cohort's data. Due to its data granularity and size, the cohort is a potential catalyst for translational cancer research. It provides rapid access to comprehensive patient groups and may improve the understanding of the clinical course of various (even rare) malignancies. Therefore, the cohort may serve as a decisions-making tool for clinical trial design and contributes to the evaluation of scientific findings under real-world conditions.

The other colon cancer: a population-based cohort study of appendix tumour trends and prognosis.

AIM: Appendiceal neoplasms are rare subtypes of colorectal tumours that mainly affect younger patients some 20 years earlier than other colon tumours. The aim of this study was to gain more insight into the histological subtypes of this rare disease and include cases previously excluded, such as mucinous neoplasia. METHOD: The cohort study included 1097 patients from the Munich Cancer Registry (MCR) diagnosed between 1998 and 2020. Joinpoint analysis was used to determine trend in incidence. Baseline demographic comparisons and survival analyses using competing risk and univariate/multivariate methods were conducted according to tumour histology: adenocarcinoma (ADENO), neuroendocrine neoplasia (NEN), mixed adeno-neuroendocrine carcinoma (MANEC), and low- (LAMN) and high-grade mucinous neoplasia (HAMN). RESULTS: Up to 2016 the number of cases increased significantly [annual per cent change (APC) = 6.86, p < 0.001] followed by a decline in the following years (APC = -14.82, p = 0.014; average APC = 2.5, p = 0.046). Comparison of all patients showed that NEN (48.4%) and mucinous neoplasms (11.6%) had a considerably better prognosis than ADENO (36.0%) and MANEC (3.0%, p < 0.0001). A multivariate analysis within the NEN and ADENO subgroups revealed that further histological classification was not prognostically relevant, while older age and regional tumour spread at diagnosis were associated with a poor prognosis. ADENO histology with high tumour grade and appendectomy only was also associated with poorer survival. CONCLUSION: Appendiceal neoplasms are histologically heterogeneous; however, this diversity becomes less relevant compared with the marked difference from cancers of the remaining colon. The previously observed increase in cases appears to be abating; fewer cases of appendicitis and/or appendectomies or changes in histopathological assessment may be behind this trend.

Immunohistochemical expression of the hepatocyte growth factor in chromophobe renal cell carcinoma.

BACKGROUND: The prognostic value of Hepatocyte growth factor (HGF) in non-clear cell renal cell carcinoma (RCC) is still unclear. The aim of this study is to evaluate the prognostic impact of HGF expression in a large cohort of chromophobe RCC (chRCC). METHODS: Patients who underwent renal surgery due to chRCC were recruited. Clinical data was retrospectively evaluated. Tumor specimen were analyzed for HGF expression by immunohistochemistry. RESULTS: 81 chRCC patients were eligible for analysis, thereof 37 (45.7%) patients were positive for HGF. No significant associations were found for HGF expression and clinical attributes in patients with chRCC. Kaplan-Meier analysis revealed no differences in 5-year overall survival (OS) for patients with HGF- compared to HGF+ tumors (95.0% versus 90.9%; p = 0.410). CONCLUSIONS: In chRCC HGF expression is not associated with parameters of aggressiveness or survival.

Characterization of Hormone Receptor and HER2 Status in Breast Cancer Using Mass Spectrometry Imaging.

Immunohistochemical evaluation of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 status stratify the different subtypes of breast cancer and define the treatment course. Triple-negative breast cancer (TNBC), which does not register receptor overexpression, is often associated with worse patient prognosis. Mass spectrometry imaging transcribes the molecular content of tissue specimens without requiring additional tags or preliminary analysis of the samples, being therefore an excellent methodology for an unbiased determination of tissue constituents, in particular tumor markers. In this study, the proteomic content of 1191 human breast cancer samples was characterized by mass spectrometry imaging and the epithelial regions were employed to train and test machine-learning models to characterize the individual receptor status and to classify TNBC. The classification models presented yielded high accuracies for estrogen and progesterone receptors and over 95% accuracy for classification of TNBC. Analysis of the molecular features revealed that vimentin overexpression is associated with TNBC, supported by immunohistochemistry validation, revealing a new potential target for diagnosis and treatment.

Aberrant DNA methylation patterns in microsatellite stable human colorectal cancers define a new marker panel for the CpG island methylator phenotype.

A distinct group of colorectal carcinomas (CRCs) referred to as the "CpG island methylator phenotype" (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status.

Tumor DNA-methylome derived epigenetic fingerprint identifies HPV-negative head and neck patients at risk for locoregional recurrence after postoperative radiochemotherapy.

Biomarkers with relevance for loco-regional therapy are needed in human papillomavirus negative aka HPV(-) head and neck squamous cell carcinoma (HNSCC). Based on the premise that DNA methylation pattern is highly conserved, we sought to develop a reliable and robust methylome-based classifier identifying HPV(-) HNSCC patients at risk for loco-regional recurrence (LR) and all-event progression after postoperative radiochemotherapy (PORT-C). The training cohort consisted of HPV-DNA negative HNSCC patients (n = 128) homogeneously treated with PORT-C in frame of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) multicenter biomarker trial. DNA Methylation analysis was performed using Illumina 450 K and 850 K-EPIC microarray technology. The performance of the classifier was integrated with a series of biomarkers studied in the training set namely hypoxia-, 5-microRNA (5-miR), stem-cell gene-expression signatures and immunohistochemistry (IHC)-based immunological characterization of tumors (CD3/CD8/PD-L1/PD1). Validation occurred in an independent cohort of HPV(-) HNSCC patients, pooled from two German centers (n = 125). We identified a 38-methylation probe-based HPV(-) Independent Classifier of disease Recurrence (HICR) with high prognostic value for LR, distant metastasis and overall survival (P < 10-9 ). HICR remained significant after multivariate analysis adjusting for anatomical site, lymph node extracapsular extension (ECE) and size (T-stage). HICR high-risk tumors were enriched for younger patients with hypoxic tumors (15-gene signature) and elevated 5-miR score. After adjustment for hypoxia and 5-miR covariates, HICR maintained predicting all endpoints. HICR provides a novel mean for assessing the risk of LR in HPV(-) HNSCC patients treated with PORT-C and opens a new opportunity for biomarker-assisted stratification and therapy adaptation in these patients.

CXCL9 inhibits tumour growth and drives anti-PD-L1 therapy in ovarian cancer.

BACKGROUND: Response to immune checkpoint blockade (ICB) in ovarian cancer remains disappointing. Several studies have identified the chemokine CXCL9 as a robust prognosticator of improved survival in ovarian cancer and a characteristic of the immunoreactive subtype, which predicts ICB response. However, the function of CXCL9 in ovarian cancer has been poorly studied. METHODS: Impact of Cxcl9 overexpression in the murine ID8-Trp53-/- and ID8-Trp53-/-Brca2-/- ovarian cancer models on survival, cellular immune composition, PD-L1 expression and anti-PD-L1 therapy. CXCL9 expression analysis in ovarian cancer subtypes and correlation to reported ICB response. RESULTS: CXCL9 overexpression resulted in T-cell accumulation, delayed ascites formation and improved survival, which was dependent on adaptive immune function. In the ICB-resistant mouse model, the chemokine was sufficient to enable a successful anti-PD-L1 therapy. In contrast, these effects were abrogated in Brca2-deficient tumours, most likely due to an already high intrinsic chemokine expression. Finally, in ovarian cancer patients, the clear-cell subtype, known to respond best to ICB, displayed a significantly higher proportion of CXCL9high tumours than the other subtypes. CONCLUSIONS: CXCL9 is a driver of successful ICB in preclinical ovarian cancer. Besides being a feasible predictive biomarker, CXCL9-inducing agents thus represent attractive combination partners to improve ICB in this cancer entity.

pT3 colorectal cancer revisited: a multicentric study on the histological depth of invasion in more than 1000 pT3 carcinomas-proposal for a new pT3a/pT3b subclassification.

BACKGROUND: Pathological TNM staging (pTNM) is the strongest prognosticator in colorectal carcinoma (CRC) and the foundation of its post-operative clinical management. Tumours that invade pericolic/perirectal adipose tissue generally fall into the pT3 category without further subdivision. METHODS: The histological depth of invasion into the pericolic/perirectal fat was digitally and conventionally measured in a training cohort of 950 CRCs (Munich). We biostatistically calculated the optimal cut-off to stratify pT3 CRCs into novel pT3a (≤3 mm)/pT3b (>3 mm) subgroups, which were then validated in two independent cohorts (447 CRCs, Bayreuth/542 CRCs, Mainz). RESULTS: Compared to pT3a tumours, pT3b CRCs showed significantly worse disease-specific survival, including in pN0 vs pN+ and colonic vs. rectal cancers (DSS: P < 0.001, respectively, pooled analysis of all cohorts). Furthermore, the pT3a/pT3b subclassification remained an independent predictor of survival in multivariate analyses (e.g. DSS: P < 0.001, hazard ratio: 4.41 for pT3b, pooled analysis of all cohorts). While pT2/pT3a CRCs showed similar survival characteristics, pT3b cancers remained a distinct subgroup with dismal survival. DISCUSSION: The delineation of pT3a/pT3b subcategories of CRC based on the histological depth of adipose tissue invasion adds valuable prognostic information to the current pT3 classification and implementation into current staging practices of CRC should be considered.