Publications by authors named "Markus Eckstein"

89 Publications

Tumor budding correlates with tumor invasiveness and predicts worse survival in pT1 non-muscle-invasive bladder cancer.

Sci Rep 2021 Sep 9;11(1):17981. Epub 2021 Sep 9.

Department of Urology, University of Regensburg, Caritas St. Josef Medical Center, Landshuter Str. 65, 93053, Regensburg, Germany.

Tumor budding is defined as a single cell or a cluster of up to 5 tumor cells at the invasion front. Due to the difficulty of identifying patients at high risk for pT1 non-muscle-invasive bladder cancer (NMIBC) and the difficulties in T1 substaging, tumor budding was evaluated as a potential alternative and prognostic parameter in these patients. Tumor budding as well as growth pattern, invasion pattern and lamina propria infiltration were retrospectively evaluated in transurethral resection of the bladder (TURB) specimens from 92 patients with stage pT1 NMIBC. The presence of tumor budding correlated with multifocal tumors (p = 0.003), discontinuous invasion pattern (p = 0.039), discohesive growth pattern (p < 0.001) and extensive lamina propria invasion (p < 0.001). In Kaplan-Meier analysis, tumor budding was associated with significantly worse RFS (p = 0.005), PFS (p = 0.017) and CSS (p = 0.002). In patients who received BCG instillation therapy (n = 65), the absence of tumor budding was associated with improved RFS (p = 0.012), PFS (p = 0.011) and CSS (p = 0.022), with none of the patients suffering from progression or dying from the disease. Tumor budding is associated with a more aggressive and invasive stage of pT1 NMIBC and a worse outcome. This easy-to-assess parameter could help stratify patients into BCG therapy or early cystectomy treatment groups.
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http://dx.doi.org/10.1038/s41598-021-97500-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8429693PMC
September 2021

The Prognostic Value of FGFR3 Expression in Patients with T1 Non-Muscle Invasive Bladder Cancer.

Cancer Manag Res 2021 20;13:6567-6578. Epub 2021 Aug 20.

Department of Urology and Pediatric Urology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany.

Purpose: Fibroblast growth factor receptor 3 (FGFR3) alterations are frequent in non-muscle-invasive bladder cancer (NMIBC), although current data regarding the prognostic and therapeutic relevance are inconsistent. We analyzed the prognostic role of FGFR3 mRNA expression in stage T1 NMIBC.

Patients And Methods: The mRNA expression of FGFR3 and cyclin-dependent kinase inhibitor 2A (CDKN2A) was measured by RT-qPCR in 80 patients with stage T1 NMIBC treated with transurethral resection of the bladder and correlated with clinical data and KRT5 and KRT20 expression, used as surrogate markers for basal and luminal subtypes, respectively.

Results: FGFR3 and CDKN2A transcript levels were not correlated. FGFR3 expression was associated with the expression of KRT5 (p=0.002) and KRT20 (p < 0.001). CDKN2A expression was negatively correlated with KRT5 (p=0.030). In Kaplan-Meier analysis and univariable Cox regression analysis, high FGFR3 expression was associated with significantly reduced recurrence-free survival (RFS) (HR=3.78; p < 0.001) and improved overall survival (OS) (HR=0.50; p=0.043), while high CDKN2A expression was associated with reduced OS (HR=2.34; p=0.034). Patient age was the only clinicopathological parameter associated with reduced OS (HR=2.29; p=0.022). No parameter was an independent prognostic factor in multivariable analysis. Next, we stratified the patients depending on their lineage differentiation. In univariable analysis, the prognostic effect of FGFR3 and CDKN2A was observed primarily in patients demonstrating high expression of KRT5 or KRT20, whereas high FGFR3 expression was associated with significantly reduced RFS, irrespective of instillation therapy.

Conclusion: Stage T1 NMIBC patients with high FGFR3 expression show shorter RFS but better OS than patients with low FGFR3 expression.
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http://dx.doi.org/10.2147/CMAR.S318893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384147PMC
August 2021

promoter hypomethylation is a negative prognostic biomarker at initial diagnosis but predicts response and favorable outcome to anti-PD-1 based immunotherapy in clear cell renal cell carcinoma.

J Immunother Cancer 2021 Aug;9(8)

Center for Integrated Oncology Aachen/Bonn/Cologne/Düsseldorf (CIO-ABCD), Bonn, Germany

Background: In metastatic clear cell renal cell carcinoma (ccRCC), different combination therapies, each including anti-PD-1 immune checkpoint blockade (ICB), are applied as first-line treatment. Robust predictive biomarkers for rational upfront therapy decisions are lacking, although they are urgently needed. Recently, we showed that promoter methylation predicts response to ICB in melanoma. Here, we aimed to investigate methylation in ccRCC and its utility to serve as a predictive biomarker for anti-PD-1 based ICB in metastatic ccRCC.

Methods: methylation was analyzed with regard to transcriptional gene activity (mRNA expression), intratumoral immune cell composition, and clinical course in two ccRCC cohorts obtained from The Cancer Genome Atlas (TCGA cohort, n=533) and the University Hospital Bonn (UHB Non-ICB Cohort, n=116). In addition, methylation as well as CD8 T cell infiltrates and PD-L1 expression were evaluated in pre-treatment samples from a multicenter cohort (RCC-ICB Cohort, n=71). Patients included in the RCC-ICB Cohort were treated with either first line anti-PD-1 based combination therapy (n=25) or monotherapy post-tyrosine kinase inhibition in second line or later. Analyses were performed with regard to treatment response according to RECIST, progression-free survival (PFS), event-free survival (EFS), and overall survival (OS) following treatment initiation.

Results: promoter hypomethylation was significantly correlated with mRNA expression, lymphocyte infiltration, and poor OS in both primary ccRCC cohorts (TCGA: HR 0.30 (95% CI 0.18 to 0.49), p<0.001; UHB Non-ICB: HR 0.35 (95% CI 0.16 to 0.75), p=0.007). In contrast, promoter hypomethylation predicted response and, accordingly, favorable outcomes (PFS and OS) in patients with ICB-treated ccRCC, overcompensating the negative prognostic value of hypomethylation at initial diagnosis. Moreover, in multivariable Cox regression, promoter hypomethylation remained an independent predictor of improved outcome in ICB-treated ccRCC after co-adjustment of the International Metastatic Renal Cell Carcinoma Database Consortium score (HR 3.00 (95% CI 1.47 to 6.28), p=0.003).

Conclusions: Our study suggests methylation as a powerful predictive biomarker for immunotherapy response in metastatic RCC.
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http://dx.doi.org/10.1136/jitc-2021-002949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395367PMC
August 2021

Utility of stromal tumor infiltrating lymphocyte scoring (sTILs) for risk stratification of patients with muscle-invasive urothelial bladder cancer after radical cystectomy.

Urol Oncol 2021 Aug 20. Epub 2021 Aug 20.

Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, Germany; BRIDGE-Consortium Germany e.V., Mannheim, Germany; Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany. Electronic address:

Background: Multi-omics analyses of muscle-invasive bladder cancer (MIBC) demonstrated that specific patterns of tumor infiltrating lymphocytes (TILs) associates with improved outcomes in patients treated with radical cystectomy. However, methodologies for simple and robust quantification of TILs, especially for daily practice purposes, are lacking. Thus, we investigated the feasibility of stromal TIL scoring on hematoxylin/eosin stained (HE) slides in MIBC.

Materials And Methods: sTILs were scored on HE whole slides of 241 MIBC patients treated with radical cystectomy and adjuvant chemotherapy. Median infiltration of 10% was used as objective cut-off. Additionally, immunohistochemistry was performed on spatially organized tissue microarrays to quantify key immune cell populations objectively for correlational analyses with sTIL scoring results (CD3/Pan-T-cells, CD8/cytotoxic T-Cells, CD56/NK-cells, CD68/macrophages). sTILs amounts were correlated with clinicopathological features, recurrence-free (RFS), disease-specific (DSS), and overall survival (OS).

Results: sTIL amounts correlated moderately to strongly with quantitatively estimated amounts of pan-T-cells (r = 0.73, P <0.0001), cytotoxic T-cells (r = 0.73, P <0.0001), NK-cells (r = 0.68, P <0.0001), macrophages (r = 0.55, P <0.0001) and with pan-cytotoxic immune infiltration (r = 0.78, P <0.0001), thus reflecting overall infiltration with key immune cell populations. sTIL infiltration ≥10% was associated with significantly higher 5-year OS (45.5% vs. 19.8%), DSS (56.6% vs. 25.6%) and RFS (56.2% vs. 18.9%; P <0.0001 for all three comparisons) rates, and lower pT-stage (P = 0.015), lower pN-stage (P = 0.028), lower rates of lymphovascular invasion (P = 0.0003) and blood vessel invasion (P = 0.01) when compared to sTIL infiltration of <10%. Multivariable regressions models confirmed sTILs as strongest independent predictor for improved outcomes following radical cystectomy.

Conclusions: HE based sTIL scoring is a reliable tool to assess MIBC inflammation status and to stratify the survival of MIBC patients undergoing radical cystectomy. sTIL amount is an independent predictor for improved survival, and might be an useful, routinely applicable tool to identify patients benefiting from perioperative platinum-based chemotherapy and checkpoint inhibitor therapy. However, external validation of our data is required.
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http://dx.doi.org/10.1016/j.urolonc.2021.07.025DOI Listing
August 2021

Neoadjuvant Immunotherapy of Oral Squamous Cell Carcinoma: Case Report and Assessment of Histological Response.

Front Oncol 2021 21;11:720951. Epub 2021 Jul 21.

Department of Oral and Maxillofacial Surgery, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Background: The treatment of oral cancer remains challenging due to its infiltrative nature and a high tendency for tumour relapse leading to an overall poor prognosis. In the case of early recurrence, the patient's prognosis deteriorates dramatically, with survival rate dropping to below 30%. Minimal improvements in survival trends in recurrent and advanced stage tumours have been reported in recent decades. Neoadjuvant immunotherapy may represent a new therapeutic approach changing the standard of care in advanced oral cancer therapy.

Case Presentation: We describe the case of a woman in her late 30's who presented in mid-2019 with oral squamous cell carcinoma (OSCC) localized to the floor of the mouth. After initial R0 resection, selective neck dissection, and adjuvant brachytherapy, an early recurrence of OSCC located between the hyoid bone and the mandible was diagnosed at the end of 2019. An off-label treatment regimen was performed with neoadjuvant use of Pembrolizumab 19 days prior to salvage surgery. Radiological and histological assessment of T-cell and programmed cell death protein 1 ligand 1 (PD-L1) expression was performed before and after checkpoint inhibitor application. Neoadjuvant immunotherapy resulted in increased T-cell infiltration and PD-L1 expression, as well as a significant tumour necrosis rate. One cycle of Pembrolizumab led to significant regressive tumour changes with increases in immune infiltration, sclerosis, and necrosis of 75% of the tumour mass with only 25% vital tumour cells remaining. By June 2020, the patient remained without recurrence.

Conclusions: The case presented outlines the potential effects of neoadjuvant immunotherapy in recurrent or advanced OSCC prior to definitive surgical tumour treatment. The benefit of additional adjuvant treatment after histologic response will be discussed. The case is also analysed considering ongoing clinical trials of neoadjuvant immunotherapy for head and neck malignancies.
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http://dx.doi.org/10.3389/fonc.2021.720951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8334861PMC
July 2021

Combination of GP88 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Is an Independent Prognostic Factor for Bladder Cancer Patients.

Cells 2021 Jul 15;10(7). Epub 2021 Jul 15.

Bridge Consortium, 68135 Mannheim, Germany.

Urothelial bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide and accounts for approximately 3% of global cancer diagnoses. We are interested in prognostic markers that may characterize tumor cells (TCs) and immune cells (ICs) and their relationship in BCa. A potential candidate marker that meets these criteria is progranulin (GP88), which is expressed separately in TCs and ICs. We analyzed GP88 expression by immunohistochemistry (IHC) in 196 muscle-invasive BCa samples using a tissue microarray. The immunoreactive score for GP88 staining in TCs and the percentage of GP88-positive ICs was determined. An easy cutoff for the staining status of TCs (positive vs. negative) and ICs (0% vs. >0%) and, more generally, negative vs. positive GP88 staining could be applied. We detected 93 patients (47.4%) and 92 patients (46.9%) with GP88-positive TCs or ICs, respectively. The IHC results were correlated with clinicopathological and survival data. Positive GP88 staining in TCs appeared to be an independent poor prognostic factor for disease-specific survival (DSS) (RR (relative risk) = 1.74; = 0.009) and recurrence-free survival (RFS) (RR = 1.92; = 0.002). In contrast, negative GP88 staining in ICs was an independent negative predictor for overall survival (OS) (RR = 2.18; < 0.001), DSS (RR = 2.84; < 0.001) and RFS (RR = 2.91; < 0.001) in multivariate Cox's regression analysis. When combining GP88 staining in TCs and ICs, a specific combination of GP88-positive TCs and GP88-negative ICs was associated with a 2.54-fold increased risk of death, a 4.21-fold increased risk of disease-specific death and a 4.81-fold increased risk of recurrence compared to GP88-negative TCs and GP88-positive ICs. In summary, GP88 positivity in TCs is a negative prognostic factor for DSS and RFS. In addition, GP88 positivity can mark ICs that are associated with a good prognosis (OS, DSS and RFS). The combination of GP88 staining in TCs and ICs appears to be a significant independent prognostic biomarker in muscle-invasive BCa.
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http://dx.doi.org/10.3390/cells10071796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306318PMC
July 2021

Validation of a classification and scoring system for the diagnosis of laryngeal and pharyngeal squamous cell carcinomas by confocal laser endomicroscopy.

Braz J Otorhinolaryngol 2021 Jul 20. Epub 2021 Jul 20.

University Hospital Aachen, RWTH Aachen University, Department of Otorhinolaryngology, Head and Neck Surgery, Germany. Electronic address:

Introduction: Confocal laser endomicroscopy is an optical imaging technique that allows in vivo, real-time, microscope-like images of the upper aerodigestive tract's mucosa. The assessment of morphological tissue characteristics for the correct differentiation between healthy and malignant suspected mucosa requires strict evaluation criteria.

Objective: This study aims to validate an eight-point score for the correct assessment of malignancy.

Methods: We performed confocal laser endomicroscopy between March and October 2020 in 13 patients. 197 sequences (11.820 images) originated from the marginal area of pharyngeal and laryngeal carcinomas. Specimens were taken at corresponding locations and analyzed in H&E staining as a standard of reference. A total of six examiners evaluated the sequences based on a scoring system; they were blinded to the histopathological examination. The primary endpoints are sensitivity, specificity, and accuracy. Secondary endpoints are interrater reliability and receiver operator characteristics.

Results: Healthy mucosa showed epithelium with uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture. Confocal laser endomicroscopy of malignant cells demonstrated a disorganized arrangement of variable cellular morphology. We calculated an accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 83.2%, 81.3%, 85.5%, 86.7%, and 79.7%, respectively, with a κ-value of 0.64, and an area under the curve of 0.86.

Conclusion: The results confirm that this scoring system is applicable in the laryngeal and pharyngeal mucosa to classify benign and malignant tissue. A scoring system based on defined and reproducible characteristics can help translate this experimental method to broad clinical practice in head and neck diagnosis.
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http://dx.doi.org/10.1016/j.bjorl.2021.06.002DOI Listing
July 2021

High expression of ERBB2 is an independent risk factor for reduced recurrence-free survival in patients with stage T1 non-muscle-invasive bladder cancer.

Urol Oncol 2021 Jul 27. Epub 2021 Jul 27.

Department of Urology and Pediatric Urology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany; Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, Germany.

Introduction: Molecular markers associated with breast cancer are assumed to be associated with outcome in non-muscle-invasive bladder cancer (NMIBC).

Materials And Methods: We retrospectively investigated the association of the mRNA expression of estrogen receptor 1 (ESR1) and 2 (ESR2), progesterone receptor (PGR), MKI67, and HER2 (ERBB2) with recurrence-free (RFS), cancer-specific (CSS), and overall survival (OS) in 80 patients with stage T1 NMIBC.

Results: High expression of ESR2 (P = 0.003), ERBB2 (P < 0.001), and MKI67 (P = 0.029) was associated with shorter RFS. Only high ERBB2 was an independent prognostic factor for reduced RFS (HR = 2.98; P = 0.009). When sub stratifying the cohort, high ESR2 was associated with reduced RFS (P < 0.001), CSS (P = 0.037) and OS (P = 0.006) in patients without instillation therapy. High ESR2 was associated with reduced CSS (P = 0.018) and OS (P = 0.029) in females and with shorter RFS in both sexes (males: P = 0.035; females: P = 0.010). Patients with high ERBB2 showed reduced CSS (P = 0.011) and OS (P = 0.042) in females and reduced CSS (P = 0.012) in those without instillation, while RFS was significantly reduced irrespective of sex or instillation.

Conclusion: High mRNA expression of ERBB2 is an independent predictor of reduced RFS in patients with stage T1 NMIBC. High ERBB2 and ESR2 are associated with reduced outcomes, especially in females and patients without instillation therapy.
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http://dx.doi.org/10.1016/j.urolonc.2021.06.021DOI Listing
July 2021

Prognostic impact of molecular muscle-invasive bladder cancer subtyping approaches and correlations with variant histology in a population-based mono-institutional cystectomy cohort.

World J Urol 2021 Jul 14. Epub 2021 Jul 14.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Krankenhausstr. 8-10, 91054, Erlangen, Germany.

Purpose: Recently discovered molecular classifications for urothelial bladder cancer appeared to be promising prognostic and predictive biomarkers. The present study was conducted to evaluate the prognostic impact of molecular subtypes assessed by two different methodologies (gene and protein expression), to compare these two approaches and to correlate molecular with histological subtypes in a consecutively collected, mono-institutional muscle-invasive bladder cancer (MIBC) cohort.

Methods: 193 MIBC were pathologically re-evaluated and molecular subtypes were assessed on mRNA (NanoString technology, modified 21-gene-containing MDACC approach) and protein levels (immuno-histochemical [IHC] analysis of CK5, CK14, CD44, CK20, GATA3 and FOXA1). Descriptive statistical methods and uni-/multi-variable survival models were employed to analyze derived data.

Results: Neither gene expression nor protein-based subtyping showed significant associations with disease-specific (DSS) or recurrence-free survival (RFS). Agreement between mRNA (reference) and protein-based subtyping amounted 68.6% for basal, 76.1% for luminal and 50.0% for double-negative tumors. Histological subtypes associated with RFS in uni-variable (P = 0.03), but not in multivariable survival analyses. Tumors with variant histology predominantly showed luminal subtypes (gene expression subtyping: 36/55 cases, 65.5%; protein subtyping: 44/55 cases, 80.0%). Squamous differentiation significantly associated with basal subtypes (gene expression subtyping: 44/45 squamous cases, 97.8%; protein subtyping: 36/45 cases, 80.0%).

Conclusion: In our consecutive cystectomy cohort, neither gene, protein expression-based subtyping, nor histological subtypes associated with DSS or RFS in multi-variably adjusted survival analyses. Application of a limited IHC subtyping marker panel showed high concordance of 83.9% with gene expression-based subtyping, thus underlining the utility for subtyping in pathological routine diagnostics. In addition, histological MIBC subtypes are strong indicators for intrinsic subtypes.
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http://dx.doi.org/10.1007/s00345-021-03788-1DOI Listing
July 2021

High Androgen Receptor mRNA Expression Is Associated with Improved Outcome in Patients with High-Risk Non-Muscle-Invasive Bladder Cancer.

Life (Basel) 2021 Jun 30;11(7). Epub 2021 Jun 30.

Department of Urology and Pediatric Urology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

The role of the androgen receptor (AR) in non-muscle-invasive bladder cancer (NMIBC) remains controversial. We retrospectively analyzed the mRNA expression of AR using RT-qPCR in 95 patients with high-risk NMIBC treated with a bladder-sparing approach and correlated AR with clinical data and recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS). The mRNA expression of AR and KRT5, i.e., the basal-like subtype, was strongly correlated (rs = 0.456; < 0.001). AR ( = 0.053) and KRT5 ( = 0.029) mRNA expression was negatively correlated with tumor grade. Kaplan-Meier analyses indicated significantly prolonged CSS ( = 0.020) and OS ( = 0.015) and a trend towards longer RFS ( = 0.051) in patients with high AR expression. High KRT5 expression was associated with significantly longer RFS ( = 0.033), CSS ( = 0.029) and OS ( = 0.030), while high KRT20 expression was associated with reduced RFS ( = 0.042). In multivariable analysis, none of the molecular markers was an independent prognostic factor. When performing a substratification with regard to molecular markers and clinicopathological parameters, high AR expression showed improved OS in patients with high KRT20 mRNA expression ( = 0.041). Women showed significantly longer OS in cases with high AR expression ( = 0.011). High AR was associated with significantly improved CSS in males ( = 0.044) and patients with instillation therapy ( = 0.040), while OS was improved regardless of instillation therapy. Younger patients with high AR expression had significantly improved RFS ( = 0.021), CSS ( = 0.014) and OS ( = 0.007). RFS was also improved in patients with high AR and low expression of either KRT5 ( = 0.003) or KRT20 ( = 0.014), but not in patients with high expression of KRT5 or KRT20. In conclusion, high AR mRNA expression is correlated with KRT5 mRNA expression and is associated with an improved outcome in high-risk NMIBC.
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http://dx.doi.org/10.3390/life11070642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306811PMC
June 2021

Risk factors associated with positive surgical margins' location at radical cystectomy and their impact on bladder cancer survival.

World J Urol 2021 Jul 1. Epub 2021 Jul 1.

Department of Surgical Oncology (Urology), Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Purpose: To evaluate the risk factors associated with positive surgical margins' (PSMs) location and their impact on disease-specific survival (DSS) in patients with bladder cancer (BCa) undergoing radical cystectomy (RC).

Methods: We analyzed a large multi-institutional cohort of patients treated with upfront RC for non-metastatic (cT1-4aN0M0) BCa. Multivariable binomial logistic regression analyses were used to assess the risk of PSMs at RC for each location after adjusting for clinicopathological covariates. The Kaplan-Meier method was used to estimate DSS stratified by margins' status and location. Log-rank statistics and Cox' regression models were used to determine significance.

Results: A total of 1058 patients were included and 108 (10.2%) patients had PSMs. PSMs were located at soft-tissue, ureter(s), and urethra in 57 (5.4%), 30 (2.8%) and 21 (2.0%) patients, respectively. At multivariable analysis, soft-tissue PSMs were independently associated with pathological stage T4 (pT4) (Odds ratio (OR) 6.20, p  <  0.001) and lymph-node metastases (OR 1.86, p  =  0.04). Concomitant carcinoma-in-situ (CIS) was an independent risk factor for ureteric PSMs (OR 6.31, p  =  0.003). Finally, urethral PSMs were independently correlated with pT4-stage (OR 5.10, p  =  0.01). The estimated 3-years DSS rates were 58.2%, 32.4%, 50.1%, and 40.3% for negative SMs, soft-tissue-, ureteric- and urethral PSMs, respectively (log-rank; p  <  0.001).

Conclusions: PSMs' location represents distinct risk factors' patterns. Concomitant CIS was associated with ureteric PSMs. Urethral and soft-tissue PSM showed worse DSS rates. Our results suggest that clinical decision-making paradigms on adjuvant treatment and surveillance might be adapted based on PSM and their location.
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http://dx.doi.org/10.1007/s00345-021-03776-5DOI Listing
July 2021

Impact of intraepithelial capillary loops and atypical vessels in confocal laser endomicroscopy for the diagnosis of laryngeal and hypopharyngeal squamous cell carcinoma.

Eur Arch Otorhinolaryngol 2021 Jun 29. Epub 2021 Jun 29.

Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich-Alexander-Universität Erlangen-Nürnberg, University Hospital, Waldstrasse 1, 91054, Erlangen, Germany.

Purpose: Confocal laser endomicroscopy (CLE) allows surface imaging of the laryngeal and pharyngeal mucosa in vivo at a thousand-fold magnification. This study aims to compare irregular blood vessels and intraepithelial capillary loops in healthy mucosa and squamous cell carcinoma (SCC) via CLE.

Materials And Methods: We included ten patients with confirmed SCC and planned total laryngectomy in this study between March 2020 and February 2021. CLE images of these patients were collected and compared with the corresponding histology in hematoxylin and eosin staining. We analyzed the characteristic endomicroscopic patterns of blood vessels and intraepithelial capillary loops for the diagnosis of SCC.

Results: In a total of 54 sequences, we identified 243 blood vessels which were analyzed regarding structure, diameter, and Fluorescein leakage, confirming that irregular, corkscrew-like vessels (24.4% vs. 1.3%; P < .001), dilated intraepithelial capillary loops (90.8% vs. 28.7%; P < .001), and increased capillary leakage (40.7% vs. 2.5%; P < .001), are significantly more frequently detected in SCC compared to the healthy epithelium. We defined a vessel diameter of 30 μm in capillary loops as a cut-off value, obtaining a sensitivity, specificity, PPV, and NPV and accuracy of 90.6%, 71.3%, 57.4%, 94.7%, and 77.1%, respectively, for the detection of malignancy based solely on capillary architecture.

Conclusion: Capillaries within malignant lesions are fundamentally different from those in healthy mucosa regions. The capillary architecture is a significant feature aiding the identification of malignant mucosa areas during in-vivo, real-time CLE examination.
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http://dx.doi.org/10.1007/s00405-021-06954-8DOI Listing
June 2021

Cytokeratin 5 and cytokeratin 20 inversely correlate with tumour grading in Ta non-muscle-invasive bladder cancer.

J Cell Mol Med 2021 Aug 29;25(16):7890-7900. Epub 2021 Jun 29.

Organ Systems, KU Leuven, Leuven, Belgium.

Cytokeratin 5 is a marker of basal molecular subtypes of muscle-invasive bladder cancer (MIBC), which correlates with worse overall survival compared to luminal subtypes. Our observations have not confirmed CK5 as a marker of high-grade (HG) disease in Ta non-muscle-invasive bladder cancer (NMIBC). Therefore, to understand the basal-luminal immunohistochemistry profile in Ta NMIBC, we performed immunohistochemistry for CK5, P40, P63 (basal), GATA3 and CK20 (luminal) and studied the correlation with HG and clinical outcome in 109 patients with Ta NMIBC. HG and low-grade (LG) diseases were scored in each patient. Four different CK5 patterns were evaluated: absent (median 41.3%), normal (72.5%), rising (84.4%) and full thickness (23.9%). The median percentage of GATA3 was 100%. HG disease and CK5 expression and rising CK5 pattern had a significant inverse correlation, whereas HG disease and CK20 expression had a significant positive correlation. We also found a significant inverse correlation between CK5 expression and CK20 expression. Quantitative PCR confirmed that the presence of CK5 correlated with up-regulation of CK5 RNA. None of the markers could differentiate patients with regard to clinical outcome. Our results suggest a role for CK5 and CK20 in differentiating between LG and HG disease in Ta NMIBC.
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http://dx.doi.org/10.1111/jcmm.16712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358875PMC
August 2021

IQGAP3, a YAP Target, Is Required for Proper Cell-Cycle Progression and Genome Stability.

Mol Cancer Res 2021 Jun 28. Epub 2021 Jun 28.

Experimental Renal and Cardiovascular Research, Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.

Controlling cell proliferation is critical for organism development, tissue homeostasis, disease, and regeneration. IQGAP3 has been shown to be required for proper cell proliferation and migration, and is associated to a number of cancers. Moreover, its expression is inversely correlated with the overall survival rate in the majority of cancers. Here, we show that expression is elevated in cervical cancer and that in these cancers high expression is correlated with an increased lethality. Furthermore, we demonstrate that is a target of YAP, a regulator of cell cycle gene expression. IQGAP3 knockdown resulted in an increased percentage of HeLa cells in S phase, delayed progression through mitosis, and caused multipolar spindle formation and consequentially aneuploidy. Protein-protein interaction studies revealed that IQGAP3 interacts with MMS19, which is known in Drosophila to permit, by competitive binding to Xpd, Cdk7 to be fully active as a Cdk-activating kinase (CAK). Notably, IQGAP3 knockdown caused decreased MMS19 protein levels and XPD knockdown partially rescued the reduced proliferation rate upon IQGAP3 knockdown. This suggests that IQGAP3 modulates the cell cycle via the MMS19/XPD/CAK axis. Thus, in addition to governing proliferation and migration, IQGAP3 is a critical regulator of mitotic progression and genome stability. IMPLICATIONS: Our data indicate that, while IQGAP3 inhibition might be initially effective in decreasing cancer cell proliferation, this approach harbors the risk to promote aneuploidy and, therefore, the formation of more aggressive cancers.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0639DOI Listing
June 2021

Prognostic Role of FGFR Alterations and FGFR mRNA Expression in Metastatic Urothelial Cancer Undergoing Checkpoint Inhibitor Therapy.

Urology 2021 Jun 19. Epub 2021 Jun 19.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen.

Objective: To examine the disease-specific survival(DSS) after checkpoint inhibitor(CPI) therapy based on FGFR alterations and FGFR mRNA expression levels in patients with metastatic urothelial cancer(mUCa) within a multi-center cohort.

Methods: Within a cohort of 72 patients with mUCa from five academic centers in Germany FGFR alterations, as well as FGFR1-4 mRNA expression levels in tumor samples from the primary tumor or metastatic sites. Spearman rank correlations, logistic regression, as well as Kaplan-Meier survival analyses and univariate Cox proportional hazards regression models were employed to examine the impact of different FGFR patterns on the DSS after CPI treatment.

Results: FGFR3 mutations or gene fusions (gene alterations) were detected in 16.9% of all samples. Patients with or without FGFR3 gene alterations did not show different oncological outcomes undergoing CPI treatment. Low expression of FGFR2 mRNA alone, as well as the combination of either low FGFR2mRNA expression and FGFR3 gene alteration or high FGFR3mRNA expression (P = 0.027), identified a subgroup of patients with unfavorable outcomes, comprising 40% of the total cohort. This trend was also observed in univariate Cox proportional hazards regression analysis(FGFR3 gene alteration: Hazard ratio(HR) 5.33, 95%Confidence interval(CI)1.76-15.0, P = 0.004; FGFR3mRNA expression:HR 3.04, 95%CI 1.40-7.13, P = 0.005).

Conclusion: Assessment of FGFR mRNA expression identified a high-risk subgroup of patients with mUCa. These patients showing overexpression of FGFR3 mRNA were found to have unfavorable DSS after CPI treatment. Using this approach may be suitable for identifying a patient population with poor response to CPI treatment, which may benefit from early FGFR inhibition.
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http://dx.doi.org/10.1016/j.urology.2021.05.055DOI Listing
June 2021

KRT20, KRT5, ESR1 and ERBB2 Expression Can Predict Pathologic Outcome in Patients Undergoing Neoadjuvant Chemotherapy and Radical Cystectomy for Muscle-Invasive Bladder Cancer.

J Pers Med 2021 May 26;11(6). Epub 2021 May 26.

Department of Urology, Marien Hospital, Ruhr-University Bochum, 44625 Herne, Germany.

Patients with muscle-invasive bladder cancer (MIBC) that underwent neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) show improved overall survival, especially those with pathological complete response (pCR). The response to NAC according to molecular subtypes has been discussed. Molecular targets such as estrogen receptor (ESR1) and human epidermal growth factor receptor 2 (ERBB2) play an important role in breast cancer management and have also been associated with urothelial bladder cancer. Hence, the association of Keratin 20 (KRT20) Keratin 5 (KRT5), ESR1, and ERBB2 mRNA expression in MIBC at transurethral resection (TUR-BT) with pCR after NAC was analyzed retrospectively. Formalin-fixed paraffin-embedded tumour tissue samples from TUR-BT of 54 patients (42 males, 12 females, median age of 64) with MIBC were analyzed for KRT20, KRT5, ESR1, and ERBB2 mRNA expression. After NAC, RC was performed, and the specimens were evaluated for pCR. Statistical analyses comprised nonparametric and chi testing, partition models, and Spearman correlation analyses. After NAC, 22 out of 54 patients (40.7%) had pCR. Tumours with an elevated expression of markers associated with luminal differentiation (KRT20, ERBB2, ESR1) were associated with a higher chance of pCR (55% vs. 15.8%, = 0.009). Elevated ERBB2 expression was positively correlated with luminal expression features such as KRT20, and negatively with basal characteristics such as KRT5. Patients with MIBC showing a high expression of ERBB2, ESR1, or KRT20 have a significantly higher chance of pCR following NAC. These findings might improve patient selection for NAC in MIBC.
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http://dx.doi.org/10.3390/jpm11060473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229873PMC
May 2021

Integration of Spatial PD-L1 Expression with the Tumor Immune Microenvironment Outperforms Standard PD-L1 Scoring in Outcome Prediction of Urothelial Cancer Patients.

Cancers (Basel) 2021 May 12;13(10). Epub 2021 May 12.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Krankenhausstr. 8-10, 91054 Erlangen, Germany.

Background: Immune therapy has gained significant importance in managing urothelial cancer. The value of PD-L1 remains a matter of controversy, thus requiring an in-depth analysis of its biological and clinical relevance.

Methods: A total of 193 tumors of muscle-invasive bladder cancer patients (MIBC) were assessed with four PD-L1 assays. PD-L1 scoring results were correlated with data from a comprehensive digital-spatial immune-profiling panel using descriptive statistics, hierarchical clustering and uni-/multivariable survival analyses.

Results: PD-L1 scoring algorithms are heterogeneous (agreements from 63.1% to 87.7%), and stems from different constellations of immune and tumor cells (IC/TC). While Ventana IC5% algorithm identifies tumors with high inflammation and favorable baseline prognosis, CPS10 and the TCarea25%/ICarea25% algorithm identify tumors with TC and IC expression. Spatially organized immune phenotypes, which correlate either with high PD-L1 IC expression and favorable prognosis or constitutive PD-L1 TC expression and poor baseline prognosis, cannot be resolved properly by PD-L1 algorithms. PD-L1 negative tumors with relevant immune infiltration can be detected by sTILs scoring on HE slides and digital CD8 scoring.

Conclusions: Contemporary PD-L1 scoring algorithms are not sufficient to resolve spatially distributed MIBC immune phenotypes and their clinical implications. A more comprehensive view of immune phenotypes along with the integration of spatial PD-L1 expression on IC and TC is necessary in order to stratify patients for ICI.
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http://dx.doi.org/10.3390/cancers13102327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150350PMC
May 2021

Imaging, histopathological degree of degeneration and clinical findings - Do these correlate in patients with temporomandibular joint disorders.

J Stomatol Oral Maxillofac Surg 2021 May 12. Epub 2021 May 12.

Department of Oral and Maxillofacial Surgery, University Hospital Erlangen, Friedrich-Alexander-University Erlangen Nuremberg, Glückstraße 11, 91054 Erlangen, Germany; Section of Oral and Maxillofacial Surgery, Department of Otorhinolaryngology, University Hospital Augsburg, Stenglinstraße 2, 86156 Augsburg, Germany.

The gold standard for temporomandibular joint imaging is magnetic resonance imaging, although there are still pathological findings that cannot be seen in MRI but in surgery and the subsequent histological analysis only. The main goal of this investigation was to validate the MRI score used by histopathological findings as well as clinical findings. In this retrospective study 39 patients were included; 38 of which underwent unilateral and 1 underwent bilateral discectomy. MRI findings were graded according to the score by Wurm. Histopathological analysis was performed in hematoxylin-eosin staining and graded in accordance with the scores by Krenn and by Leonardi. For valuation of preoperative pain values of the temporomandibular joint operated on the numeric rating scale was utilized. Correlations were verified by Spearman-Rho. The MRI scores on average showed significantly lower scores for the discs of the operated temporomandibular joint than for the discs of the non-operated side(p<.01). No significant correlations between MRI findings, histopathological findings and pain intensities could be observed. Thus unsuspicious morphology of the TMJ and the articular disc in MRI is no guarantee for the absence of cartilage-degeneration. Further investigations utilizing T2 cartilage mapping could possibly show better correlations between the temporomandibular joint's degree of degeneration and imaging results.
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http://dx.doi.org/10.1016/j.jormas.2021.05.002DOI Listing
May 2021

Endogenous Retroviral-K Envelope Is a Novel Tumor Antigen and Prognostic Indicator of Renal Cell Carcinoma.

Front Oncol 2021 22;11:657187. Epub 2021 Apr 22.

Department of Gynecology and Obstetrics, University Hospital Erlangen, Comprehensive Cancer Center, European Metropolitan Area Erlangen-Nuremberg (CCC ER-EMN), Friedrich-Alexander-University Erlangen-Nuernberg, Erlangen, Germany.

Renal cell carcinoma (RCC) is one of the ten most common cancers for men and women with an approximate 75% overall 5-year survival. Sixteen histological tumor subtypes exist and the most common are papillary, chromophobe and clear cell renal cell carcinoma (ccRCC) representing 85% of all RCC. Although epigenetically silenced, endogenous retroviral (ERV) genes become activated in tumors and function to ignite immune responses. Research has intensified to understand ERV protein function and their role as tumor antigens and targets for cancer (immune) therapy. ERV-K env is overexpressed and implicated as a therapeutic target for breast cancer, however studies in RCC are limited. In this investigation a human RCC tissue microarray (TMA) (n=374) predominantly consisting of the most common histological tumor subtypes was hybridized with an ERV-K env antibody and correlated with patient clinical data. TMA results showed the highest amount of ERV-K env protein expression and the strongest significant membrane expression in ccRCC versus other RCC subtypes. High ERV-K env total protein expression of all tumor subtypes significantly correlated with low tumor grading and a longer disease specific survival using multivariable analyses. Cell proliferation and invasion were assayed using the kidney cell lines HEK293 with wild-type p53 and a ccRCC cell line MZ1257RC mutated for p53. Transfecting these cell lines with a codon optimized overexpressing CMV vector was performed with or without 5'-Aza-2'-deoxycytidine (Aza) treatment to sustain promoter de-methylation. MZ1257RC showed induction of expression and significantly increased both proliferation and invasion in the presence or absence of Aza. HEK293 cells demonstrated a restriction of expression and invasion with no changes in proliferation in the absence of Aza. However, in the presence of Aza despite increased expression, an inhibition of HEK293 proliferation and a further restriction of invasion was found. This study supports ERV-K env as a single prognostic indicator for better survival of RCC, which we propose represents a new tumor antigen. In addition, ERV-K env significantly regulates proliferation and invasion depending on p53 status and Aza treatment.
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http://dx.doi.org/10.3389/fonc.2021.657187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100683PMC
April 2021

Implementation of Double Immune Checkpoint Blockade Increases Response Rate to Induction Chemotherapy in Head and Neck Cancer.

Cancers (Basel) 2021 Apr 19;13(8). Epub 2021 Apr 19.

Department of Radiation Oncology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054 Bayern, Germany.

To determine whether a single dose of double immune checkpoint blockade (induction chemoimmunotherapy (ICIT)) adds benefit to induction single-cycle platinum doublet (induction chemotherapy (IC)) in locally advanced head and neck squamous cell carcinoma (HNSCC), patients treated with cisplatin 30 mg/m d1-3 and docetaxel 75 mg/m d1 combined with durvalumab 1500 mg fixed dose d5 and tremelimumab 75 mg fixed dose d5 (ICIT) within the CheckRad-CD8 trial were compared with a retrospective cohort receiving the same chemotherapy (IC) without immunotherapy. The endpoint of this analysis was the complete response rate (CR). A total of 53 patients were treated with ICIT and 104 patients with IC only. CR rates were 60.3% for ICIT and 40.3% for IC ( = 0.018). In the total population ( = 157), the most important predictor to achieve a CR was treatment type (OR: 2.21 for ICIT vs. IC; = 0.038, multivariate analysis). The most diverse effects in CR rates between ICIT and IC were observed in younger (age ≤ 60) patients with HPV-positive OPSCCs (82% vs. 33%, = 0.176), while there was no difference in older patients without HPV-positive OPSCCs (53% vs. 48%). The analysis provides initial evidence that ICIT could result in higher CR rates than IC. Young patients with HPV-positive OPSCCs may have the greatest benefit from additional immune checkpoint inhibitors.
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http://dx.doi.org/10.3390/cancers13081959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073509PMC
April 2021

Immune Checkpoint Inhibitors in Urothelial Carcinoma: Recommendations for Practical Approaches to PD-L1 and Other Potential Predictive Biomarker Testing.

Cancers (Basel) 2021 Mar 20;13(6). Epub 2021 Mar 20.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

Immuno-oncology (IO) agents (anti-programmed cell death 1 (PD-1) and anti-programmed cell death-ligand 1 (PD-L1)) are approved as first- and second-line treatments for metastatic UC. PD-L1 expression levels in UC tumors help clinicians determine which patients are more likely to respond to IO therapies. Assays for approved IO agents use different antibodies, immunohistochemical protocols, cutoffs (defining "high" vs. "low" PD-L1 expression), and scoring algorithms. The robust control of pre-analytical and analytical standards is needed to obtain high-quality PD-L1 results. To better understand the status and perspectives of biomarker-guided patient selection for anti-PD-1 and anti-PD-L1 agents in UC, three workshops were held from December 2018 to December 2019 in Italy, Malaysia, and Spain. The primary goal was to develop recommendations for best practice approaches to PD-L1 testing in UC. Recommendations pertaining to the interpretation and reporting of the results of PD-L1 assays from experienced pathologists and oncologists from around the globe are included. A test request form for pathology laboratories was developed as a critical first step for oncologists/urologists to encourage communication between clinicians and pathologists, ensuring fast and high-quality test results. In this era of personalized medicine, we briefly discuss novel biomarkers being evaluated for IO agents in UC.
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http://dx.doi.org/10.3390/cancers13061424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003923PMC
March 2021

Questionnaire-based detection of immune-related adverse events in cancer patients treated with PD-1/PD-L1 immune checkpoint inhibitors.

BMC Cancer 2021 Mar 24;21(1):314. Epub 2021 Mar 24.

Department of Radiation Oncology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsstraße 27, 91054, Erlangen, Germany.

Background: Immune checkpoint inhibitors (ICI) have become standard treatment in different tumor entities. However, safe treatment with ICI targeting the PD-1/PD-L1 axis requires early detection of immune-related adverse events (irAE). There exist different questionnaires of drug manufacturers for the detection of irAE that have not been validated so far.

Methods: The prospective non-interventional ST-ICI trial studied treatment with PD-1/PD-L1 ICI alone or combined with radiotherapy. In the current analysis, the detection rate of self-reported irAE with a patient questionnaire containing 41 different questions was compared to clinician-reported irAE.

Results: Between April 2017 and August 2019, a total of 104 patients were prospectively enrolled. NSCLC (44%) and HNSCC (42%) were the most frequent tumor entities. A total of 784 questionnaires were collected. A total of 29 irAE were reported by clinicians. The most frequent irAE was hypothyroidism (9%), followed by skin reactions (5%), hepatitis (4%), diarrhea (3%), and pneumonitis (3%). Questions that became significantly more often positive at time points of clinician-reported irAE were "weight change", "difficulty to grip things", "bloody or mucous stool" and "insomnia". Self-reported organ-specific questions detected at least 50% of clinician-reported irAE of gastrointestinal, lung, endocrine, and skin irAE. It was not possible to detect hepatic irAE with the questionnaire.

Conclusion: Questionnaires can help to detect gastrointestinal, lung, endocrine, or skin irAE, but not hepatic irAE. Questions on "weight change" and "insomnia" may help to increase the detection rate of irAE, besides organ-specific questions. These results are a valuable contribution to the future development of a specific and practicable questionnaire for early self-reported detection of irAE during ICI therapy in cancer patients.

Trial Registration: ClinicalTrials.gov, NCT03453892 . Registered on 05 March 2018.
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http://dx.doi.org/10.1186/s12885-021-08006-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992796PMC
March 2021

Tumour-Infiltrating Lymphocytes (TILs) and PD-L1 Expression Correlate with Lymph Node Metastasis, High-Grade Transformation and Shorter Metastasis-Free Survival in Patients with Acinic Cell Carcinoma (AciCC) of the Salivary Glands.

Cancers (Basel) 2021 Feb 25;13(5). Epub 2021 Feb 25.

Department of Otorhinolaryngology, Head & Neck Surgery, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Waldstrasse 1, 91054 Erlangen, Germany.

Objectives: The aim of this study was to assess the number of tumour-infiltrating lymphocytes (TILs) and the expression of (PD-L1) in Acinic Cell Carcinoma (AciCC) of the salivary glands, to enable a correlation with clinico-pathological features and to analyse their prognostic impact.

Methods: This single centre retrospective study represents a cohort of 36 primary AciCCs with long-term clinical follow-up. Immunohistochemically defined immune cell subtypes, i.e., those expressing T-cell markers (CD3, CD4 and CD8) or a B-cell marker (CD20) were characterized on tumour tissue sections. The number of TILs was quantitatively evaluated using software for digital bioimage analysis (QuPath). PD-L1 expression on the tumour cells and on immune cells was assessed immunohistochemically employing established scoring criteria: tumour proportion score (TPS), Ventana immune cell score (IC-Score) and combined positive score (CPS).

Results: Higher numbers of tumour-infiltrating T- and B-lymphocytes were significantly associated with high-grade transformation. Furthermore, higher counts of T-lymphocytes correlated with node-positive disease. There was a significant correlation between higher levels of PD-L1 expression and lymph node metastases as well as the occurrence of high-grade transformation. Moreover, PD-L1 CPS was associated with poor prognosis regarding metastasis-free survival ( = 0.049).

Conclusions: The current study is the first to demonstrate an association between PD-L1 expression and lymph node metastases as well as grading in AciCCs. In conclusion, increased immune cell infiltration of T and B cells as well as higher levels of PD-L1 expression in AciCC in association with high-grade transformation, lymph node metastasis and unfavourable prognosis suggests a relevant interaction between tumour cells and immune cell infiltrates in a subset of AciCCs, and might represent a rationale for immune checkpoint inhibition.
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http://dx.doi.org/10.3390/cancers13050965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956490PMC
February 2021

Prospective development and validation of a liquid immune profile-based signature (LIPS) to predict response of patients with recurrent/metastatic cancer to immune checkpoint inhibitors.

J Immunother Cancer 2021 02;9(2)

Department of Radiation Oncology, Universitätsklinikum Erlangen, Erlangen, Germany.

Background: The predictive power of novel biological markers for treatment response to immune checkpoint inhibitors (ICI) is still not satisfactory for the majority of patients with cancer. One should identify valid predictive markers in the peripheral blood, as this is easily available before and during treatment. The current interim analysis of patients of the ST-ICI cohort therefore focuses on the development and validation of a liquid immune profile-based signature (LIPS) to predict response of patients with metastatic cancer to ICI targeting the programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) axis.

Methods: A total of 104 patients were prospectively enrolled. 54 immune cell subsets were prospectively analyzed in patients' peripheral blood by multicolor flow cytometry before treatment with ICI (pre-ICI; n=89), and after the first application of ICI (n=65). Pre-ICI, patients were randomly allocated to a training (n=56) and a validation cohort (n=33). Univariate Cox proportional hazards regression analysis and least absolute shrinkage and selection operator Cox model were used to create a predictive immune signature, which was also checked after the first ICI, to consider the dynamics of changes in the immune status.

Results: Whole blood samples were provided by 89 patients pre-ICI and by 65 patients after the first ICI. We identified a LIPS which is based on five immune cell subtypes: CD14 monocytes, CD8+/PD-1 T cells, plasmacytoid dendritic cells, neutrophils, and CD3/CD56/CD16 natural killer (NK)T cells. The signature achieved a high accuracy (C-index 0.74 vs 0.71) for predicting overall survival (OS) benefit in both the training and the validation cohort. In both cohorts, the low-risk group had significantly longer OS than the high-risk group (HR 0.26, 95% CI 0.12 to 0.56, p=0.00025; HR 0.30, 95% CI 0.10 to 0.91, p=0.024, respectively). Regarding the whole cohort, LIPS also predicted progression-free survival (PFS). The identified LIPS was not affected by clinicopathological features with the exception of brain metastases. NKT cells and neutrophils of the LIPS can be used as dynamic predictive biomarkers for OS and PFS after first administration of the ICI.

Conclusion: Our study identified a predictive LIPS for survival of patients with cancer treated with PD-1/PD-L1 ICI, which is based on immune cell subsets in the peripheral whole blood.

Trial Registration Number: NCT03453892.
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http://dx.doi.org/10.1136/jitc-2020-001845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888377PMC
February 2021

Intraoperative free margins assessment of oropharyngeal squamous cell carcinoma with confocal laser endomicroscopy: a pilot study.

Eur Arch Otorhinolaryngol 2021 Feb 13. Epub 2021 Feb 13.

Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital, University of Erlangen-Nuremberg, Germany, Waldstrasse 1, 91054, Erlangen, Germany.

Purpose: This pilot study aimed to assess the feasibility of intraoperative assessment of safe margins with confocal laser endomicroscopy (CLE) during oropharyngeal squamous cell carcinoma (OPSCC) surgery.

Methods: We included five consecutive patients confirmed OPSCC and planned tumor resection in September and October 2020. Healthy appearing mucosa in the marginal zone, and the tumor margin, were examined with CLE and biopsy during tumor resection. A total of 12,809 CLE frames were correlated with the gold standard of hematoxylin and eosin staining. Three head and neck surgeons and one pathologist were asked to identify carcinoma in a sample of 169 representative images, blinded to the histological results.

Results: Healthy mucosa showed epithelium with uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture. CLE optical biopsy of OPSCC demonstrated a disorganized arrangement of variable cellular morphology. We calculated an accuracy, sensitivity, specificity, PPV, and NPV of 86%, 90%, 79%, 88%, and 82%, respectively, with inter-rater reliability and κ-value of 0.60.

Conclusion: CLE can be easily integrated into the intraoperative setting, generate real-time, in-vivo microscopic images of the oropharynx for evaluation and demarcation of cancer. It can eventually contribute to a less radical approach by enabling a more precise evaluation of the cancer margin.
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http://dx.doi.org/10.1007/s00405-021-06659-yDOI Listing
February 2021

Promoter Mutation Analysis of Whole-Organ Mapping Bladder Cancers.

Genes (Basel) 2021 02 5;12(2). Epub 2021 Feb 5.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

Background: Multifocal occurrence is a main characteristic of urothelial bladder cancer (UBC). Whether urothelial transformation is caused by monoclonal events within the urothelium, or by polyclonal unrelated events resulting in several tumor clones is still under debate. promoter mutations are the most common somatic alteration identified in UBC. In this study, we analyzed different histological tissues from whole-organ mapping bladder cancer specimens to reveal mutational status, as well as to discern how tumors develop.

Methods: Up to 23 tissues from nine whole-organ mapping bladder tumor specimens, were tested for promoter mutations including tumor associated normal urothelium, non-invasive urothelial lesions (hyperplasia, dysplasia, metaplasia), carcinoma in situ (CIS) and different areas of muscle invasive bladder cancers (MIBC). The mutational DNA hotspot region within the promoter was analyzed by SNaPshot analysis including three hot spot regions (-57, -124 or -146). Telomere length was measured by the Relative Human Telomere Length Quantification qPCR Assay Kit.

Results: promoter mutations were identified in tumor associated normal urothelium as well as non-invasive urothelial lesions, CIS and MIBC. Analysis of separate regions of the MIBC showed 100% concordance of promoter mutations within a respective whole-organ bladder specimen. Polyclonal events were observed in five out of nine whole-organ mapping bladder cancers housing tumor associated normal urothelium, non-invasive urothelial lesions and CIS where different promoter mutations were found compared to MIBC. The remaining four whole-organ mapping bladders were monoclonal for mutations. No significant differences of telomere length were observed.

Conclusions: Examining multiple whole-organ mapping bladders we conclude that promoter mutations may be an early step in bladder cancer carcinogenesis as supported by mutations detected in tumor associated normal urothelium as well as non-invasive urothelial lesions. Since mutated promoter regions within non-invasive urothelial lesions are not sufficient alone for the establishment of cancerous growth, this points to the contribution of other gene mutations as a requirement for tumor development.
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http://dx.doi.org/10.3390/genes12020230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915609PMC
February 2021

Bladder Tumor Subtype Commitment Occurs in Carcinoma Driven by Key Signaling Pathways Including ECM Remodeling.

Cancer Res 2021 03 20;81(6):1552-1566. Epub 2021 Jan 20.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.

Basal and luminal subtypes of invasive bladder tumors have significant prognostic and predictive impacts for patients. However, it remains unclear whether tumor subtype commitment occurs in noninvasive urothelial lesions or in carcinoma (CIS) and which gene pathways are important for bladder tumor progression. To understand the timing of this commitment, we used gene expression and protein analysis to create a global overview of 36 separate tissues excised from a whole bladder encompassing urothelium, noninvasive urothelial lesions, CIS, and invasive carcinomas. Additionally investigated were matched CIS, noninvasive urothelial lesions, and muscle-invasive bladder cancers (MIBC) from 22 patients. The final stage of subtype commitment to either a luminal or basal MIBC occurred at the CIS transition. For all tissues combined, hierarchical clustering of subtype gene expression revealed three subtypes: "luminal," "basal," and a "luminal p53-/extracellular matrix (ECM)-like" phenotype of ECM-related genes enriched in tumor-associated urothelium, noninvasive urothelial lesions, and CIS, but rarely invasive, carcinomas. A separate cohort of normal urothelium from noncancer patients showed significantly lower expression of ECM-related genes compared with tumor-associated urothelium, noninvasive urothelial lesions, and CIS. A PanCancer Progression Panel of 681 genes unveiled pathways specific for the luminal p53-/ECM-like cluster, for example, ECM remodeling, angiogenesis, epithelial-to-mesenchymal transition, cellular discohesion, cell motility involved in tumor progression, and cell proliferation and oncogenic ERBB2/ERBB3 signaling for invasive carcinomas. In conclusion, this study provides insights into bladder cancer subtype commitment and associated signaling pathways, which could help predict therapy response and enhance our understanding of therapy resistance. SIGNIFICANCE: This study demonstrates that CIS is the stage of commitment for determining MIBC tumor subtype, which is relevant for patient prognosis and therapy response.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2336DOI Listing
March 2021

Feasibility of intraoperative assessment of safe surgical margins during laryngectomy with confocal laser endomicroscopy: A pilot study.

Auris Nasus Larynx 2021 Aug 16;48(4):764-769. Epub 2021 Jan 16.

Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich-Alexander-Universität Erlangen-Nürnberg, University Hospital, Waldstraße 1, 91054 Erlangen, Germany. Electronic address:

Objective: This pilot study aimed to assess the feasibility of intraoperative assessment of safe margins with Confocal Laser Endomicroscopy (CLE) during planned partial or total laryngectomy.

Methods: Eight patients with confirmed larynx squamous cell carcinoma (SCC) and planned partial or total laryngectomy were included in this study in March 2020. Two head and neck surgeons and one pathologist were asked to classify carcinoma or healthy epithelium in a sample of 94 representative sequences (5.640 images), blinded to the histological results (H&E staining).

Results: Healthy mucosa areas showed epithelium with cells of uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture. CLE optical biopsy of SCC demonstrated a disorganized arrangement of variable cellular morphology. We calculated an accuracy, sensitivity, specificity, PPV, and NPV of 80.1%, 72.3%, 87.9%, 85.7%, and 76.1%, respectively. A distinct transition between healthy appearing tissue and suspicious lesions could also be detected.

Conclusion: CLE can be easily integrated into the intraoperative setting, generate real-time, in-vivo microscopic images of the larynx for evaluation and demarcation of cancer. If validated in further studies, CLE could eventually contribute to a less radical approach by enabling a more precise evaluation of the cancer margin.
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http://dx.doi.org/10.1016/j.anl.2021.01.005DOI Listing
August 2021

Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy.

Cells 2021 01 15;10(1). Epub 2021 Jan 15.

Department of Urology and Pediatric Urology, University Hospital Erlangen, FAU Erlangen-Nürnberg, 91054 Erlangen, Germany.

Bladder cancer (BCa) is the tenth most commonly diagnosed malignant cancer worldwide. Although adjuvant chemotherapy following radical cystectomy is a common therapy for muscle invasive bladder cancer patients, no applicable biomarkers exist to predict which patients will benefit from chemotherapy. In this study, we examined three immune cell markers, the chemokine CC motif ligand 2 (CCL2), the pan macrophage marker cluster of differentiation 68 (CD68) and the M2 macrophage marker cluster of differentiation 163 (CD163), using immunohistochemistry to determine their predictive value for the chemotherapy response in different nodal stage (pN0 vs. pN1 + 2) and tumor stage subgroups (pT2 vs. pT3 + 4). The prognosis was studied in terms of the overall survival (OS), disease-specific survival (DSS), and recurrence-free-survival (RFS) in 168 muscle invasive BCa patients. Chemotherapy was associated with a poorer prognosis in patients with a higher expression of the immune markers CCL2 (RFS), CD68 (DSS and RFS), and CD163 (DSS and RFS) in the N0 group and with poorer survival in patients with a higher expression of the immune markers CCL2 (OS, DSS, and RFS), CD68 (OS, DSS, and RFS), and CD163 (OS, DSS, and RFS) in the pT2 group when compared with treatments without chemotherapy. In contrast, chemotherapy was associated with a better prognosis in patients with a low expression of the immune markers CCL2 (DSS and RFS), CD68 (OS, DSS, and RFS), and CD163 (OS) in the N1 + 2 group. In addition, chemotherapy was associated with improved survival in patients with a low expression of the immune marker CD68 (OS and DSS) and there was a trend for a better prognosis in patients with a low expression of CD163 (OS) in the pT3 + 4 group compared to patients not treated with chemotherapy. Interestingly, CD68 appeared to be the most applicable immune marker to stratify patients by the outcome of chemotherapy in the nodal stage and tumor stage groups. Overall, we suggest that, in addition to the clinical factors of tumor stage and nodal stage, it is also meaningful to consider the abundance of immune cells, such as macrophages, to better predict the response to chemotherapy for BCa patients after radical treatment.
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http://dx.doi.org/10.3390/cells10010159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829767PMC
January 2021
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