Risk-adjusted Screening for Prostate Cancer-Defining the Low-risk Group by Data from the PROBASE Trial.

BACKGROUND: Risk-adjusted screening for prostate cancer (PCa) aims to reduce harms by less frequent retesting, especially in men at a low risk of PCa. Definitions of low risk are based mainly on studies in men starting screening at age 55-60 yr. OBJECTIVE: To identify men at age 45 yr with a low risk of PCa. DESIGN, SETTING, AND PARTICIPANTS: A population-based, risk-adjusted PCa screening trial was conducted in Germany using baseline prostate-specific antigen (PSA) starting in young men (PROBASE). INTERVENTION: PSA measurements starting at the age of 45 yr. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The incidence of PCa within 5 yr was assessed in men with screen-negative baseline PSA <1.5 ng/ml compared with those with PSA 1.5-≤3.0 ng/ml. RESULTS AND LIMITATIONS: Of 23301 men who received a first PSA test at age 45 yr, 0.79% had a screen-positive PSA value of ≥3 ng/ml. Among the 89% of men who had a screen-negative baseline PSA value of <1.5 ng/ml, only 0.45% received a positive PSA test ≥3 ng/ml upon retesting after 5 yr. By contrast, for those with a screen-negative baseline PSA value of 1.5-3 ng/ml, 13% surpassed 3 ng/ml upon biennial testing within the next 4 yr. The incidence of PCa in subsequent screening rounds increased with increasing baseline PSA levels, from 0.13 per 1000 person-years for men with initial PSA level of <1.5 ng/ml to 8.0 per 1000 person-years for those with PSA levels of 1.5-3.0 ng/ml. A limitation is a follow-up time of only 5 yr, so far. CONCLUSIONS: Men with baseline PSA <1.5 ng/ml at age 45 yr are at a very low risk of PCa over the next 5 yr. PATIENT SUMMARY: The PROBASE study showed that men with baseline prostate-specific antigen (PSA) <1.5 ng/ml at age 45 yr have a very low prostate cancer detection rate over 5 yr and do not need PSA retesting during this time.

Neuropilin2 in Mesenchymal Stromal Cells as a Potential Novel Therapeutic Target in Myelofibrosis.

Bone marrow fibrosis in myeloproliferative neoplasm (MPN), myelodysplastic syndromes (MDS), MPN/MDS overlap syndromes and acute myeloid leukemia (AML) is associated with poor prognosis and early treatment failure. Myelofibrosis (MF) is accompanied by reprogramming of multipotent bone marrow mesenchymal stromal cells (MSC) into osteoid and fiber-producing stromal cells. We demonstrate NRP2 and osteolineage marker NCAM1 (neural cell adhesion molecule 1) expression within the endosteal niche in normal bone marrow and aberrantly in MPN, MDS MPN/MDS overlap syndromes and AML (n = 99), as assessed by immunohistochemistry. Increased and diffuse expression in mesenchymal stromal cells and osteoblasts correlates with high MF grade in MPN (p < 0.05 for NRP2 and NCAM1). Single cell RNA sequencing (scRNAseq) re-analysis demonstrated NRP2 expression in endothelial cells and partial co-expression of NRP2 and NCAM1 in normal MSC and osteoblasts. Potential ligands included transforming growth factor ß1 (TGFB1) from osteoblasts and megakaryocytes. Murine ThPO and JAK2V617F myelofibrosis models showed co-expression of Nrp2 and Ncam1 in osteolineage cells, while fibrosis-promoting MSC only express Nrp2. In vitro experiments with MC3T3-E1 pre-osteoblasts and analysis of Nrp2-/- mouse femurs suggest that Nrp2 is functionally involved in osteogenesis. In summary, NRP2 represents a potential novel druggable target in patients with myelofibrosis.

Pathology skills lab: use of macroscopic tumor models in pathology teaching.

BACKGROUND: The shortage of pathologists in Germany, coupled with an aging workforce, requires innovative approaches to attract medical students to the field. Medical education must address different learning styles to ensure that all students are successful. METHODS: The pilot project "Practical Pathology" aims to enhance students' understanding of pathology by providing hands-on experience in macroscopic gross analysis through the use of tumor dummies built from scratch. RESULTS: An evaluation survey, completed by 63 participating students provided positive feedback on the course methodology, its relevance to understanding the pathology workflow, and its improvement over traditional teaching methods. The majority of students recognized the importance of hands-on training in medical education. Students with previous work experience rated the impact of the course on knowledge acquisition even more positively. CONCLUSION: The course improved students' understanding of pathological processes and potential sources of clinical-pathological misunderstanding. An increase in motivation for a potential career in the field of pathology was observed in a minority of students, although this exceeded the percentage of pathologists in the total medical workforce.

NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer.

PURPOSE: The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC. MATERIALS AND METHODS: We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs. RESULTS: NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers. CONCLUSION: NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

Elucidating the need for prostate cancer risk calculators in conjunction with mpMRI in initial risk assessment before prostate biopsy at a tertiary prostate cancer center.

OBJECTIVE: Utilizing personalized risk assessment for clinically significant prostate cancer (csPCa) incorporating multiparametric magnetic resonance imaging (mpMRI) reduces biopsies and overdiagnosis. We validated both multi- and univariate risk models in biopsy-naïve men, with and without the inclusion of mpMRI data for csPCa detection. METHODS: N = 565 men underwent mpMRI-targeted prostate biopsy, and the diagnostic performance of risk calculators (RCs), mpMRI alone, and clinical measures were compared using receiver operating characteristic curve (ROC) analysis and decision curve analysis (DCA). Subgroups were stratified based on mpMRI findings and quality. RESULTS: csPCa was detected in 56.3%. PI-RADS score achieved the highest area under the curve (AUC) when comparing univariate risk models (AUC 0.82, p < 0.001). Multivariate RCs showed only marginal improvement in csPCa detection compared to PI-RADS score alone, with just one of four RCs showing significant superiority. In mpMRI-negative cases, the non-MRI-based RC performed best (AUC 0.80, p = 0.016), with the potential to spare biopsies for 23%. PSA-density and multivariate RCs demonstrated comparable performance for PI-RADS 3 constellation (AUC 0.65 vs. 0.60-0.65, p > 0.5; saved biopsies 16%). In men with suspicious mpMRI, both mpMRI-based RCs and the PI-RADS score predicted csPCa excellently (AUC 0.82-0.79 vs. 0.80, p > 0.05), highlighting superior performance compared to non-MRI-based models (all p < 0.002). Quality-assured imaging consistently improved csPCa risk stratification across all subgroups. CONCLUSION: In tertiary centers serving a high-risk population, high-quality mpMRI provides a simple yet effective way to assess the risk of csPCa. Using multivariate RCs reduces multiple biopsies, especially in mpMRI-negative and PI-RADS 3 constellation.

Key learnings from concordant systematic biopsies in prostate-specific membrane antigen positron emission tomography/computed tomography-guided prostate biopsies: Enhancing targeting accuracy.

BACKGROUND: Prostate cancer (PCa) diagnosis and staging have evolved with the advent of 68Ga-Prostate-specific membrane antigen positron emission tomography/computed tomography (PSMA-PET/CT). This study investigates the role of complementary systematic biopsies (SB) during PSMA-PET/CT-guided targeted prostate biopsies (PET-TB) for PCa detection, grading, and distribution. We address the uncertainty surrounding the necessity of SB in conjunction with PET-TB. METHODS: We analyzed PCa grading and distribution in 30 men who underwent PET-TB and SB because of contraindication to magnetic resonance imaging or high clinical suspicion of PCa. Tumor distribution was assessed in relation to the PET-highlighted lesions. Standardized reporting schemes, encompassing SUVmax, PRIMARY score, and miTNM classification, were evaluated. RESULTS: 80% of patients were diagnosed with PCa, with 70% classified as clinically significant (csPCa). SB detected more csPCa cases than PET-TB, but the differences were not statistically significant. Discordant results were observed in 25% of cases, where SB outperformed PET-TB. Spatial analysis revealed that tumor-bearing cores from SB were often located in close proximity to the PET-highlighted region. Reporting schemes showed potential for csPCa detection with significantly increased SUVmax in csPCA patients. Subsequent follow-up data underscored the importance of SB in precise PCa grading and staging. CONCLUSIONS: While PET-TB can simplify prostate biopsy and reduce invasiveness by core number, SB cannot be omitted yet due to potential PET-TB targeting errors. Factors such as limited spatial resolution and fusion inaccuracies contribute to the need for SB. Standardization in reporting schemes currently cannot compensate for targeting errors highlighting the need for refinement.

Hypermethylated SHOX2 in circulating cell-free DNA post renal cell carcinoma surgery as TNM-independent biomarker for recurrence risk.

INTRODUCTION: Adjuvant immune checkpoint inhibitor trials in renal cell carcinoma (RCC) call for improved recurrence risk stratification. Due to limitations of circulating tumor DNA (ctDNA) use in RCC, the use of hypermethylated SHOX2 gene (mSHOX2) in circulating cell-free DNA is explored as a surrogate marker for identifying high-risk patients after RCC surgery. METHODS: Liquid biopsies were collected post-surgery from 45 RCC patients (mean duration 4.3 days). Real-time polymerase chain reaction was used to analyze SHOX2 methylation in circulating cell-free DNA. Patients were categorized as mSHOX2 positive or negative by cut-off. Metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival (OS) were assessed using Cox regression and Log-rank analyses (median follow-up time: 60 months). RESULTS: 17 patients were mSHOX2 positive, showing unfavorable OS/CSS (Log-rank P = 0.004 and 0.02) and nearly 6-fold higher recurrence risk (hazard ratio 5.89, 95% CI 1.46-23.8). Multivariable Cox analysis confirmed mSHOX2 as an independent recurrence risk factor, disregarding TNM-based stratification. CONCLUSIONS: mSHOX2 effectively identifies high-risk RCC patients post-surgery, indicating minimal residual disease. This easy to implement biomarker has potential for guiding of adjuvant therapy decisions.

Ring Finger Protein 34 (RNF34) as a Prognostic Biomarker for Clear Cell Renal Cell Carcinoma.

INTRODUCTION: Ring finger proteins play pivotal roles in diverse cellular processes and are implicated in contribution to cancer. Ring finger protein 34 (RNF34) has antiapoptotic and oncogenic properties. RNF34 is upregulated during carcinogenesis and tumor progression in the colorectal adenoma-carcinoma sequence and was already described to mediate chemoresistance. In clear cell renal cell carcinoma (ccRCC), however, the role and expression patterns of RNF34 are unknown. METHODS: First, we investigated the association of RNF34 mRNA expression with clinicopathological parameters and survival using data obtained from The Cancer Genome Atlas (TCGA) ccRCC cohort (N = 533). To assess RNA34 protein expression, we performed immunohistochemical (IHC) staining of an established ccRCC cohort (University of Bonn) in a tissue microarray (TMA) format. This validation cohort contains 109 primary ccRCC samples. IHC data were associated with clinicopathological parameters and overall survival (Kaplan-Meier analysis). Adjustment for covariables was done using the Cox regression model. RESULTS: RNF34 expression is correlated with adverse clinicopathological parameters. Survival analysis revealed an association between RNF34 expression and shortened survival. Cox regression analysis confirmed RNF34 expression as an independent prognostic parameter. CONCLUSION: Our study provides evidence for RNF34 as a prognostic biomarker in ccRCC and points toward a major role of this protein in renal cell carcinoma carcinogenesis.

Expression of the microtubule-associated protein 2 (MAP2) as a potential independent prognostic marker in prostate cancer.

PURPOSE: Investigation of Microtubuli-associated Protein 2 (MAP2) expression and its clinical relevance in prostate cancer. MATERIAL AND METHODS: MAP2 expression was immunohistochemically analysed on radical prostatectomy specimens using whole block sections (n = 107) and tissue microarrays (TMA; n = 310). The staining intensity was evaluated for carcinoma, benign tissue and prostatic intraepithelial neoplasia. Expression data were correlated with clinicopathological parameters and biochemical recurrence-free survival. Additionally, MAP2 protein expression was quantitatively analysed in the serum of histologically confirmed prostate carcinoma patients and the control group using a commercial enzyme-linked immunosorbent assay. RESULTS: MAP2 staining was significantly stronger in neoplastic tissue than in non-neoplastic prostatic glands, both in whole block sections (p < 0.01) and in TMA sections (p < 0.05). TMA data revealed significantly stronger MAP2 staining in high-grade tumors. Survival analysis showed a significant correlation between strong MAP2 staining in carcinoma and shortened biochemical recurrence-free survival after prostatectomy (p < 0.001). Multivariate Cox regression analysis confirmed MAP2 as an independent predictor for an unfavourable course. Mean MAP2 serum levels for non-PCA vs. PCA patients differed significantly (non-PCA = 164.7 pg/ml vs. PCA = 242.5 pg/ml, p < 0.001). CONCLUSION: The present data support MAP2 as a novel biomarker in PCA specimens. MAP2 is correlated with tumor grade and MAP2 high-expressing PCA is associated with an increased risk of biochemical recurrence after radical prostatectomy. Future studies are necessary to evaluate MAP2 as a valuable immunohistochemical biomarker in preoperative PCA diagnostic procedures, in particular with regard to treatment modalities.

Clinical implications of AGR2 in primary prostate cancer: Results from a large-scale study.

Human anterior gradient-2 (AGR2) has been implicated in carcinogenesis of various solid tumours, but the expression data in prostate cancer are contradictory regarding its prognostic value. The objective of this study is to evaluate the expression of AGR2 in a large prostate cancer cohort and to correlate it with clinicopathological data. AGR2 protein expression was analysed immunohistochemically in 1023 well-characterized prostate cancer samples with a validated antibody. AGR2 expression levels in carcinomas were compared with matched tissue samples of adjacent normal glands. AGR2 expression levels were dichotomized and tested for statistical significance. Increased AGR2 expression was found in 93.5% of prostate cancer cases. AGR2 levels were significantly higher in prostate cancer compared with normal prostate tissue. A gradual loss of AGR2 expression was associated with increasing tumour grade (ISUP), and AGR2 expression is inversely related to patient survival, however, multivariable significance is not achieved. AGR2 is clearly upregulated in the majority of prostate cancer cases, yet a true diagnostic value appears unlikely. In spite of the negative correlation of AGR2 expression with increasing tumour grade, no independent prognostic significance was found in this large-scale study.

Multiparametric Magnetic Resonance Imaging in Prostate Cancer Screening at the Age of 45 Years: Results from the First Screening Round of the PROBASE Trial.

BACKGROUND: Magnetic resonance imaging (MRI) has been suggested as a tool for guiding biopsy recommendations in prostate cancer (PC) screening. OBJECTIVE: To determine the performance of multiparametric MRI (mpMRI) in young men at age 45 yr who participated in a PC screening trial (PROBASE) on the basis of baseline prostate-specific antigen (PSA). DESIGN, SETTING, AND PARTICIPANTS: Participants with confirmed PSA ≥3 ng/ml were offered mpMRI followed by MRI/transrectal ultrasound fusion biopsy (FBx) with targeted and systematic cores. mpMRI scans from the first screening round for men randomised to an immediate PSA test in PROBASE were evaluated by local readers and then by two reference radiologists (experience >10 000 prostate MRI examinations) blinded to the histopathology. The PROBASE trial is registered as ISRCTN37591328 OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The local and reference Prostate Imaging-Data and Reporting System (PI-RADS) scores were compared, and the sensitivity, negative predictive value (NPV), and accuracy were calculated for both readings for different cutoffs (PI-RADS 3 vs 4). RESULTS AND LIMITATIONS: Of 186 participants, 114 underwent mpMRI and FBx. PC was detected in 47 (41%), of whom 33 (29%) had clinically significant PC (csPC; International Society of Urological Pathology grade group ≥2). Interobserver reliability between local and reference PI-RADS scores was moderate (k = 0.41). At a cutoff of PI-RADS 4, reference reading showed better performance for csPC detection (sensitivity 79%, NPV 91%, accuracy of 85%) than local reading (sensitivity 55%, NPV 80%, accuracy 68%). Reference reading did not miss any PC cases for a cutoff of PI-RADS <3. If PI-RADS ≥4 were to be used as a biopsy cutoff, mpMRI would reduce negative biopsies by 68% and avoid detection of nonsignificant PC in 71% of cases. CONCLUSIONS: Prostate MRI in a young screening population is difficult to read. The MRI accuracy of for csPC detection is highly dependent on reader experience, and double reading might be advisable. More data are needed before MRI is included in PC screening for men at age 45 yr. PATIENT SUMMARY: Measurement of prostate specific antigen (PSA) is an effective screening test for early detection of prostate cancer (PC) and can reduce PC-specific deaths, but it can also lead to unnecessary biopsies and treatment. Magnetic resonance imaging (MRI) after a positive PSA test has been proposed as a way to reduce the number of biopsies, with biopsy only recommended for men with suspicious MRI findings. Our results indicate that MRI accuracy is moderate for men aged 45 years but can be increased by a second reading of the images by expert radiologists. For broad application of MRI in routine screening, double reading may be advisable.

Molecular Alterations in Intraductal Carcinoma of the Prostate.

Intraductal carcinoma of the prostate is most commonly associated with high-grade invasive prostate cancer. However, isolated IDC-P without adjacent cancer or high-grade cancer is also well known. Common genetic alterations present in IDC-P with adjacent high-grade prostate cancer are those described in high-grade tumors, such as PTEN loss (69-84%). In addition, the rate of LOH involving TP53 and RB1 is significantly higher. IDC-P is common in the TCGA molecular subset of SPOP mutant cancers, and the presence of SPOP mutations are more likely in IDC-P bearing tumors. IDC-P without adjacent high-grade cancers are by far less common. They are less likely to have PTEN loss (47%) and rarely harbor an ERG fusion (7%). Molecular alterations that may predispose a person to the development of IDC-P include the loss of BRCA2 and PTEN as well as mutations in SPOP. However, the causative nature of these genetic alterations is yet to be validated.

Optimized complete cytoreduction in ovarian cancer through intraoperative real-time tumor visualization by 5-ALA - a case report.

Introduction: Complete macroscopic cytoreduction represents the most important prognostic parameter for overall survival in ovarian cancer. This dogma remains tenacious despite significant improvements in adjuvant systemic treatment. Hence, optimization of surgical therapy is an overarching goal to improve patients' outcomes. In this context, intraoperative tumor-specific imaging might facilitate optimized cytoreduction. In neurosurgery, intraoperative 5-aminolevulinic acid (5-ALA) guided imaging is applied in clinical routine to assess surgical resection margins. Here, we report the case of a patient with ovarian cancer in whom intraoperative 5-ALA tumor visualization led to optimized complete cytoreduction. Objective: Intraoperative administration of 5-ALA led to improved complete cytoreduction by identification and resection of additional ovarian cancer tumor manifestations. Case: The 39-year-old patient, Jehovah`s witness, presented to our department with a left sided ovarian mass, suspicious of ovarian cancer, based on clinical examination, sonographic suspicious features and a CA12-5 elevation. The patient's medical history and family history was unremarkable. Preoperative CT imaging of the thorax and abdomen showed no pathology besides the adnexal mass. Surgery was performed by a midline laparotomy with hysterectomy, bilateral adnexectomy, pelvic peritonectomy, omentectomy, ureterolysis, diaphragm stripping, adhesiolysis and the collection of peritoneal and rectal samples. Intraoperative 5-ALA imaging using a dedicated excitation and detection loupe system (Reveal, DVI) led to tumor detection at the diaphragm, the omentum and the rectum that was not detectable by palpation and visualization using white light. The pathology results revealed that the 5-ALA positive samples (diaphragm, rectum and omentum) obtained by intraoperative 5-ALA were positive for ovarian cancer. Conclusion: Intraoperative administration of 5-ALA represents a promising approach to improve complete cytoreduction in ovarian cancer surgery thereby improving clinical outcomes. Hence, further research and clinical trials are required to investigate the potential of intraoperative 5-ALA imaging in ovarian cancer debulking surgery and its impact on long-term clinical outcomes.

Digital Rectal Examination Is Not a Useful Screening Test for Prostate Cancer.

BACKGROUND: Annual digital rectal examination (DRE) is recommended as a stand-alone screening test for prostate cancer (PCa) in Germany for 45+ yr olds. DRE diagnostic performance in men as young as 45 yr old has not been proved by a screening trial. OBJECTIVE: To determine DRE diagnostic performance in a screening trial. DESIGN, SETTING, AND PARTICIPANTS: This analysis was conducted within the multicentric, randomized PROBASE trial, which enrolled >46 000 men at age 45 to test risk-adapted prostate-specific antigen (PSA) screening for PCa. INTERVENTION: (1) DRE was analyzed as a one-time, stand-alone screening offer at age 45 in 6537 men in one arm of the trial and (2) PCa detection by DRE was evaluated at the time of PSA-screen-driven biopsies (N = 578). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: (1) True-/false-positive detection rates of DRE as compared with PSA screening and (2) DRE outcome at the time of a prostate biopsy were evaluated. RESULTS AND LIMITATIONS: (1) A prospective analysis of 57 men with suspicious DRE at age 45 revealed three PCa. Detection rate by DRE was 0.05% (three of 6537) as compared with a four-fold higher rate by PSA screening (48 of 23 301, 0.21%). The true-positive detection rate by DRE relative to screening by PSA was 0.22 (95% confidence interval [CI] = [0.07-0.72]) and the false-positive detection rate by DRE was 2.2 (95% CI = [1.50-3.17]). (2) Among PSA-screen-detected PCa cases, 86% had unsuspicious DRE (sensitivity relative to PSA was 14%), with the majority of these tumors (86%) located in the potentially accessible zones of the prostate as seen by magnetic resonance imaging. CONCLUSIONS: The performance of stand-alone DRE to screen for PCa is poor. DRE should not be recommended as a PCa screening test in young men. Furthermore, DRE does not improve the detection of PSA-screen-detected PCa. PATIENT SUMMARY: Our report demonstrated the poor diagnostic performance of digital rectal examination in the screening for prostate cancer in young men.

Web-grading-a tool to test personal grading of renal and prostate cancer.

Only a few pathologists have the opportunity to verify their personal grading through objective assessment. This study introduces a web-based grading platform to facilitate and validate the grading of renal cell carcinoma and prostate cancer. Two representative images of two clinically annotated cohorts of 100 cases each of prostate and renal cell carcinoma were used. Each participant was asked to grade a tumor series utilizing a three tiered grading system. Finally, a Kaplan-Meier curve was drawn, and the log-rank test was used for statistical testing of the p-value. The grading of 22 participants (68%) achieved prognostic significance. Further analysis highlighted that only two pathologists were able to reliably separate low- and high-grade tumors from intermediate grades. The limitations of this study are the low number of participants in each of the cohorts and the potential selection bias of the tumor images. This web-based grading portal facilitates the assessment of the validity of grading by individual pathologists. The observation that most participants can only successfully identify high- or low-grade tumors but cannot discriminate between more subtle intermediate grades does indicate that there is a need for the development of more formal training programs for tumor grading.

CTLA4 DNA methylation is associated with CTLA-4 expression and predicts response to immunotherapy in head and neck squamous cell carcinoma.

BACKGROUND: The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value. METHODS: We analyzed CTLA4 promoter methylation in tumors of HNSCC patients (N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort (N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine. RESULTS: Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3+, CD4+, CD8+, and CD45+ immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines. CONCLUSIONS: Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.

Circulating Cell-Free SEPT9 DNA Methylation in Blood Is a Biomarker for Minimal Residual Disease Detection in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND: Tumorous SEPT9 (septin 9, SEPTIN9) circulating cell-free DNA (ccfDNA) methylation in blood plasma is a powerful biomarker for diagnosis, molecular staging, prognosis, and recurrence monitoring in head and neck squamous cell carcinoma (HNSCC) patients. The present study aimed to evaluate the clinical performance of SEPT9 ccfDNA methylation to detect post-surgical minimal residual disease (MRD) in patients with localized or locally advanced HNSCC treated with curative intent. METHODS: We applied quasi-digital methylation-specific real-time PCR to quantify SEPT9 ccfDNA methylation levels 2 to 30 days post-surgically in plasma from n = 219 prospectively enrolled HNSCC patients. We tested the associations of SEPT9 ccfDNA methylation with clinicopathological parameters and used Kaplan-Meier and Cox proportional hazards analyses for univariate, pairwise bivariate, and multivariate analyses of disease-free survival. RESULTS: Of 219 patients, 26.5% (58/219) were post-surgically SEPT9 ccfDNA methylation positive. SEPT9 ccfDNA methylation positivity was significantly associated with tumor site, American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC; 8th edition) tumor stage, nodal category and extracapsular extension, lymphatic and vascular invasion, and surgical margin. Bivariate Cox proportional hazards analysis proved post-surgical SEPT9 ccfDNA methylation positivity to be an independent prognostic factor tested together with AJCC/UICC tumor stage (SEPT9: hazard ratio [HR] = 2.43, 95% CI, 1.37-4.30, P = 0.002; AJCC/UICC stage: HR = 1.48, 95% CI, 1.11-1.98, P = 0.008). CONCLUSIONS: Post-surgical SEPT9 ccfDNA methylation may aid to identify high-risk HNSCC patients who could benefit from an intensified adjuvant treatment and surveillance.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice.

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53flox mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53flox mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients.

Inflammatory fibroid polyp of the renal pelvis: first report at an extra-gastrointestinal site with molecular confirmation.

Inflammatory fibroid polyps (IFP) are rare and benign mesenchymal tumours of the gastrointestinal tract. They are submucosal spindle cell lesions with an eosinophilic-rich inflammatory infiltrate and mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene. In this report, we present the case of a 74-year-old female with a solid tumour of the kidney, which presented as a bland proliferation of spindle cells with thin-walled blood vessels and an inflammatory infiltrate with eosinophilic granulocytes. Immunohistochemistry revealed a positivity for vimentin and a weak staining for CD99 and CD34 in the spindle cells. Because of the morphological similarity to IFPs of the gastrointestinal tract, a molecular pathology analysis was performed. This identified an oncogenic mutation in exon 18 of the PDGFRA gene, which is characteristic for inflammatory fibroid polyps of the gastrointestinal tract. To the best of our knowledge, this is the first case of an IFP in the urogenital tract.