Does Dual-Energy Computed Tomography Material Decomposition Improve Radiomics Capability to Predict Survival in Head and Neck Squamous Cell Carcinoma Patients? A Preliminary Investigation.

OBJECTIVE: Our study objective was to explore the additional value of dual-energy CT (DECT) material decomposition for squamous cell carcinoma of the head and neck (SCCHN) survival prognostication. METHODS: A group of 50 SCCHN patients (male, 37; female, 13; mean age, 63.6 ± 10.82 years) with baseline head and neck DECT between September 2014 and August 2020 were retrospectively included. Primary tumors were segmented, radiomics features were extracted, and DECT material decomposition was performed. We used independent train and validation datasets with cross-validation and 100 independent iterations to identify prognostic signatures applying elastic net (EN) and random survival forest (RSF). Features were ranked and intercorrelated according to their prognostic importance. We benchmarked the models against clinical parameters. Intraclass correlation coefficients were used to analyze the interreader variation. RESULTS: The exclusively radiomics-trained models achieved similar ( P = 0.947) prognostic performance of area under the curve (AUC) = 0.784 (95% confidence interval [CI], 0.775-0.812) (EN) and AUC = 0.785 (95% CI, 0.759-0.812) (RSF). The additional application of DECT material decomposition did not improve the model's performance (EN, P = 0.594; RSF, P = 0.198). In the clinical benchmark, the top averaged AUC value of 0.643 (95% CI, 0.611-0.675) was inferior to the quantitative imaging-biomarker models ( P < 0.001). A combined imaging and clinical model did not improve the imaging-based models ( P > 0.101). Shape features revealed high prognostic importance. CONCLUSIONS: Radiomics AI applications may be used for SCCHN survival prognostication, but the spectral information of DECT material decomposition did not improve the model's performance in our preliminary investigation.

Radiomics for therapy-specific head and neck squamous cell carcinoma survival prognostication (part I).

BACKGROUND: Treatment plans for squamous cell carcinoma of the head and neck (SCCHN) are individually decided in tumor board meetings but some treatment decision-steps lack objective prognostic estimates. Our purpose was to explore the potential of radiomics for SCCHN therapy-specific survival prognostication and to increase the models' interpretability by ranking the features based on their predictive importance. METHODS: We included 157 SCCHN patients (male, 119; female, 38; mean age, 64.39 ± 10.71 years) with baseline head and neck CT between 09/2014 and 08/2020 in this retrospective study. Patients were stratified according to their treatment. Using independent training and test datasets with cross-validation and 100 iterations, we identified, ranked and inter-correlated prognostic signatures using elastic net (EN) and random survival forest (RSF). We benchmarked the models against clinical parameters. Inter-reader variation was analyzed using intraclass-correlation coefficients (ICC). RESULTS: EN and RSF achieved top prognostication performances of AUC = 0.795 (95% CI 0.767-0.822) and AUC = 0.811 (95% CI 0.782-0.839). RSF prognostication slightly outperformed the EN for the complete (ΔAUC 0.035, p = 0.002) and radiochemotherapy (ΔAUC 0.092, p < 0.001) cohort. RSF was superior to most clinical benchmarking (p ≤ 0.006). The inter-reader correlation was moderate or high for all features classes (ICC ≥ 0.77 (± 0.19)). Shape features had the highest prognostic importance, followed by texture features. CONCLUSIONS: EN and RSF built on radiomics features may be used for survival prognostication. The prognostically leading features may vary between treatment subgroups. This warrants further validation to potentially aid clinical treatment decision making in the future.

Increased HRD score in cisplatin resistant penile cancer cells.

BACKGROUND/INTRODUCTION: Penile cancer is a rare disease in demand for new therapeutic options. Frequently used combination chemotherapy with 5 fluorouracil (5-FU) and cisplatin (CDDP) in patients with metastatic penile cancer mostly results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance against standard therapy could help to understand molecular mechanisms underlying chemotherapy resistance and to identify candidate treatments for an efficient second line therapy. METHODS: We generated a cell line from a humanpapilloma virus (HPV) negative penile squamous cell carcinoma (UKF-PEC-1). This cell line was subject to chronic exposure to chemotherapy with CDDP and / or 5-FU to induce acquired resistance in the newly established chemo-resistant sublines (PEC-1rCDDP2500, adapted to 2500 ng/ml CDDP; UKF-PEC-1r5-FU500, adapted to 500 ng/ml 5- FU; UKF-PEC1rCDDP2500/r5-FU500, adapted to 2500 ng/ml CDDP and 500 ng/ml 5 -FU). Afterwards cell line pellets were formalin-fixed, paraffin embedded and subject to sequencing as well as testing for homologous recombination deficiency (HRD). Additionally, exemplary immunohistochemical stainings for p53 and gammaH2AX were applied for verification purposes. Finally, UKF-PEC-1rCDDP2500, UKF-PEC-1r5-FU500, UKF-PEC1rCDDP2500/r5-FU500, and UKF-PEC-3 (an alternative penis cancer cell line) were tested for sensitivity to paclitaxel, docetaxel, olaparib, and rucaparib. RESULTS AND CONCLUSIONS: The chemo-resistant sublines differed in their mutational landscapes. UKF-PEC-1rCDDP2500 was characterized by an increased HRD score, which is supposed to be associated with increased PARP inhibitor and immune checkpoint inhibitor sensitivity in cancer. However, UKF-PEC-1rCDDP2500 did not display sensitivity to PARP inhibitors.

The routine use of LCD-Array hybridisation technique for HPV subtyping in the diagnosis of penile carcinoma compared to other methods.

BACKGROUND: Routine human papillomavirus (HPV) testing is performed in cervival cancer and is required for classification of some head and neck cancers. In penile cancer a statement on HPV association of the carcinoma is required. In most cases p16 immunohistochemistry as a surrogate marker is applied in this setting. Since differing clinical outcomes for HPV positive and HPV negative tumors are described we await HPV testing to be requested more frequently by clinicians, also in the context of HPV vaccination, where other HPV subtypes are expected to emerge. METHOD: Therefore, a cohort of archived, formalin-fixed paraffin embedded (FFPE) penile neoplasias was stained for p16 and thereafter tested for HPV infection status via PCR based methods. Additionally to Sanger sequencing, we chose LCD-Array technique (HPV 3.5 LCD-Array Kit, Chipron; LCD-Array) for the detection of HPV in our probes expecting a less time consuming and sensitive HPV test for our probes. RESULTS: We found that LCD-Array is a sensitive and feasible method for HPV testing in routine diagnostics applicable to FFPE material in our cohort. Our cohort of penile carcinomas and carcinomas in situ was associated with HPV infection in 61% of cases. We detected no significant association between HPV infection status and histomorphological tumor characteristics as well as overall survival. CONCLUSIONS: We showed usability of molecular HPV testing on a cohort of archived penile carcinomas. To the best of our knowledge, this is the first study investigating LCD-Array technique on a cohort of penile neoplasias.

Patterns of care, toxicity and outcome in the treatment of salivary gland carcinomas: long-term experience from a tertiary cancer center.

BACKGROUND: Salivary gland carcinomas (SGC) cover a heterogeneous group of malignancies with a lack of data of high-level evidence. METHODS: Clinical data of 127 patients treated for SGC at a university cancer center between 2002 and 2017 were analyzed retrospectively. The association of clinicopathological characteristics, treatment modalities, adverse events, and outcome was assessed. RESULTS: Patients received surgery (n = 65), surgery followed by (chemo-)radiotherapy (n = 56), or primary (chemo-)radiotherapy (n = 6). Injury to the cranial nerves or their branches was the most frequent surgical complication affecting 40 patients (33.1%). Ten year overall and progression-free survival rates were 73.2% and 65.4%, respectively. Parotid tumor site, advanced tumor, and positive nodal stage remained independent negative prognostic factors for overall survival, loco-regional and distant tumor control in multivariate analysis. CONCLUSIONS: Optimizing treatment strategies for SGC, depending on distinct clinicopathological factors, remains challenging due to the low incidence rates of the disease.

Anti-EGF Receptor-Based Conversion Chemotherapy in RAS Wild-Type Colorectal Cancer Patients: Impact on Survival and Resection Rates.

BACKGROUND: Initially unresectable colorectal liver metastases can become resectable after chemotherapy. Combination chemotherapy with epidermal growth factor receptor (EGFR) antibodies has shown consistent high response rates in patients with all rat sarcoma (RAS) wild-type tumors. METHODS: Out of a cohort of 424 patients with metastatic colorectal cancer, we identified 30 patients with initially unresectable Kirsten RAS (KRAS) exon 2 wild-type colorectal liver metastases who received neoadjuvant chemotherapy with anti-EGFR agents between January 2008 and February 2014. In all patients, extended RAS analysis (KRAS and NRAS exon 3 codon 59/61 and exon 4 codon 117/146) was carried out retrospectively. RESULTS: RAS mutation analysis identified further KRAS mutations in 4/30 patients (13.3%). In none of these 4 patients a R0 resection was achieved. In contrast, 15/26 (57.7%) RAS wild-type patients were R0 resected. Median overall survival was > 63.3 months in R0-resected patients versus 30.0 months in those with a R1 or R2 resection (HR 0.23; [95% CI 0.10-0.75; p = 0.008). CONCLUSION: Our data suggest that a RAS wild-type and a R0 resection are the strongest predictors for overall survival.

Impact of re-excision of residual adjacent vulvar intraepithelial neoplasia (VIN III) and histological tumour-free margin (hTFM) on survival in primary squamous cell carcinoma of vulva.

BACKGROUND: hTFM in primary vulvar cancer is an important prognostic factor. Ideally, a diameter of > 8 mm should be achieved after primary surgery. The role of VIN III persistence after primary surgery in vulvar cancer is still unclear. The main objective of the current study was to study the role of residual VIN III re-excision and compare differences in disease-free survival among patients with different hTFM and in primary vulvar cancer. METHODS: Forty-two patients with residual adjacent VIN III after primary surgery for vulvar cancer which were operated between 2000 and 2016 in our clinic were enrolled in this retrospective study. Re-excision rates for residual adjacent VIN III were calculated. According to the histological margin patients were divided into three group: < 3, 3-8 and > 8 mm. Univariate and multivariate analyses were conducted using the Kaplan-Meier method and Cox proportional hazards models, respectively. RESULTS: The vast majority of patients had pT1b stage (57.1%), grading G2 (71.4%) and lymph node-negative (45.3%) disease at first diagnosis. The re-excision rate was 57.1%. The 5-year disease-free survival (DFS) rates in patients with < 3, 3-8 and > 8 mm hTFM were 50.0, 50.0 and 81.0%, respectively (p = 0.032). The 5-year DFS rates in patients with re-excision and without re-excision for VIN III were 77.3 and 52.9%, respectively (p = 0.060). In univariate analysis was solely hTFM > 8 mm a prognostic factor for DFS (p = 0.017). CONCLUSIONS: hTFM may be a potential prognostic indicator for DFS in vulvar cancer patients. Re-excision for residual adjacent VIN III could not be established as a prognostic factor for DFS after primary surgery in squamous cell cancer of vulva.

Microsatellite Instability Occurs Rarely in Patients with Cholangiocarcinoma: A Retrospective Study from a German Tertiary Care Hospital.

Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.

Combined p16 and p53 expression in cervical cancer of unknown primary and other prognostic parameters : A single-center analysis.

BACKGROUND AND PURPOSE: Cervical cancer of unknown primary (CUP) represents an uncommon and heterogeneous subentity of head and neck cancer. However, both optimal diagnostics and therapy remain unclear. An improved understanding of the underlying pathology is essential to enable future tailored therapies and optimized outcomes. MATERIALS AND METHODS: We retrospectively analyzed 53 patients with head and neck CUP and 48 available cervical lymph node specimens. All patients have received radiotherapy between 2007 and 2015. Preradiotherapy involved lymph node specimens were analyzed for p16 and p53 immunoreactivity. The prognostic relevance of the combined p16 and p53 status and other clinical parameters were examined by univariate and multivariate analyses. RESULTS: Median patient age was 61.5 years and median irradiation dose to the involved nodal levels was 66 Gy. Of the 48 evaluated specimens, 13 (27%) were p16-positive and 31 (64.6%) p53-positive. After a median follow up of 32.9 months, patients with p16-negative and simultaneously p53-positive tumors showed a significantly inferior tumor-specific survival (TSS) compared to those with either p16+/p53-, p16+/p53+, or p16-/p53- (univariate: p = 0.055, multivariate: p = 0.038). Other factors with an adverse impact on TSS in the univariate analysis were smoking history (p = 0.032) and nodal stage (p = 0.038). CONCLUSIONS: The combined p16- and p53-expression status in cervical metastases of CUP may represent a simple method for risk stratification. Further validation of these biomarkers in large prospective trials is essential to design rational trials for CUP treatment optimization.

Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma are highly dependent on oxidative phosphorylation.

The metabolic properties of lymphomas derived from germinal center (GC) B cells have important implications for therapeutic strategies. In this study, we have compared metabolic features of Hodgkin-Reed-Sternberg (HRS) cells, the tumor cells of classical Hodgkin's lymphoma (cHL), one of the most frequent (post-)GC-derived B-cell lymphomas, with their normal GC B cell counterparts. We found that the ratio of oxidative to nonoxidative energy conversion was clearly shifted toward oxidative phosphorylation (OXPHOS)-linked ATP synthesis in HRS cells as compared to GC B cells. Mitochondrial mass, the expression of numerous key proteins of oxidative metabolism and markers of mitochondrial biogenesis were markedly upregulated in cHL cell lines and in primary cHL cases. NFkappaB promoted this shift to OXPHOS. Functional analysis indicated that both cell growth and viability of HRS cells depended on OXPHOS. The high rates of OXPHOS correlated with an almost complete lack of lactate production in HRS cells not observed in other GC B-cell lymphoma cell lines. Overall, we conclude that OXPHOS dominates energy conversion in HRS cells, while nonoxidative ATP production plays a subordinate role. Our results suggest that OXPHOS could be a new therapeutic target and may provide an avenue toward new treatment strategies in cHL.

Role of CD15 expression in dysplastic and neoplastic tissue of the bile duct - a potential novel tool for differential diagnosis of indeterminate biliary stricture.

BACKGROUND AND AIMS: CD15 is expressed by various cancer types; among these are intrahepatic and perihilar cholangiocarcinoma (CCA). The aim of this study was to elucidate CD15 expression in distal CCA as well as in dysplastic biliary tissue and to determine its prognostic significance. METHODS AND RESULTS: Tissue samples from patients with intrahepatic (iCCA, n = 22), perihilar (pCCA, n = 7) and distal CCA (dCCA, n = 15), who underwent surgical resection in the period from 2010 to 2015 were evaluated for CD15 expression. Tissue of synchronous lymph node metastasis (n = 13), CCA-associated dysplasia (n = 20), dysplasia in intraductal biopsies (n = 10) and benign proliferations (n = 12), as well as inflammatory biliary lesions (n = 28) and non-inflammatory bile ducts (n = 23), were evaluated equally for CD15 expression. CD15 was found to be expressed highly in iCCA (81.8%), pCCA (85.7%), dCCA (73.3%), CCA-associated dysplasia (70.0%), dysplasia in intraductal biopsies (100%) and metastatic tissue (84.6%). CD15 expression was negative in 58 of 64 benign bile duct alterations resulting in an overall sensitivity and specificity of CD15 in CCA of 80.7 and 90.6% patients, respectively. CD15 expression was correlated significantly with a decreased overall survival in patients with CD15-positive CCA associated dysplasia (P = 0.003). However, CD15 expression in the invasive tumour component was not correlated with clinical outcome. CONCLUSION: CD15 is a sensitive and specific marker for intraepithelial and invasive neoplasias of the bile duct. Therefore, it can be helpful in the delineation of dysplastic and neoplastic biliary cells from non-neoplastic tissue, which frequently causes a diagnostic problem in indeterminate biliary stricture.

The G protein-coupled receptor 55 ligand l-α-lysophosphatidylinositol exerts microglia-dependent neuroprotection after excitotoxic lesion.

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein-coupled cannabinoid receptor 1 (CB1 ) and microglial G protein-coupled cannabinoid receptor 2 (CB2 ). However, pharmacological experiments with CB1 /CB2 -deficient mice unraveled the existence of further, so-called non-CB1 /non-CB2 G protein-coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l-α-Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI-mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca(2+) and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI-mediated modulation of microglia function.

A Comparison of Two Different FFPE Tissue Dissection Methods for Routine Diagnostics in Molecular Pathology: Manual Macrodissection versus Automated Microdissection Using the Roche "AVENIO Millisect" System.

Currently, in routine diagnostics, most molecular testing is performed on formalin-fixed, paraffin-embedded tissue after a histomorphological assessment. In order to find the best possible and targeted individual therapy, knowing the mutational status of the tumour is crucial. The "AVENIO Millisect" system Roche introduced an automation solution for the dissection of tissue on slides. This technology allows the precise and fully automated dissection of the tumour area without wasting limited and valuable patient material. In this study, the digitally guided microdissection was directly compared to the manual macrodissection regarding the precision and duration of the procedure, their DNA concentrations as well as DNA qualities, and the overall costs in 24 FFPE samples. In 21 of 24 cases (87.5%), the DNA yields of the manually dissected samples were higher in comparison to the automatically dissected samples. Shorter execution times and lower costs were also benefits of the manual scraping process. Nevertheless, the DNA quality achieved with both methods was comparable, which is essential for further molecular testing. Therefore, it could be used as an additional tool for precise tumour enrichment.

Uncommon Coexistence of Pleomorphic Adenoma and Warthin's Tumor in a Painfully Swollen Left Parotid Gland: A Surgical Case Report.

BACKGROUND Benign pleomorphic adenoma is the most common primary tumor of the salivary glands and mainly arises in the parotid gland. Warthin's tumor, or papillary cystadenoma lymphomatosum, represents <30% of benign parotid tumors. The simultaneous occurrence of multiple parotid tumors is rarely described - depending on the corresponding histology (different/identical), the time of their occurrence (synchronous/metachronous), as well as their location (unilateral/bilateral), multiple parotid tumors can be further sub-classified. CASE REPORT We describe the case of a 54-year-old female patient with progressive and painful swelling of the left parotid gland for the last 6 months. During extra-oral examination, a bulging, displaceable mass of approximately 3 cm was determined. A subsequent MRI (magnetic resonance imaging) examination revealed a multifocal lesion but failed to provide a decisive clue as to the tumor entity of the lesion, and a lateral (superficial) parotidectomy was performed. Postoperative histomorphological interpretation allowed the final pathological diagnosis of synchronous, unilateral occurrence of a pleomorphic adenoma as well as a Warthin's tumor. CONCLUSIONS This report presents a rare case of synchronous unilateral parotid tumors and supports that benign pleomorphic adenoma and Warthin's tumor are the most common associations. Since clinical examination, MRI imaging, and even cytological assessment could be misleading in the detection of synchronous ipsilateral multiple parotid gland tumors, our report also highlights the importance of timely and accurate diagnosis with histopathology to plan surgery and to exclude malignant transformation, which is a rare but important association with both types of primary salivary gland tumor.

Sequencing of BRCA1/2-alterations using NGS-based technology: annotation as a challenge.

In this study, the molecular profile of different BRCA-associated tumor types was assessed with regard to the classification and annotation of detected BRCA1/2 variants. The aim was to establish guidelines in order to facilitate the interpretation of BRCA1/2 alterations in routine diagnostics. Annotation of detected variants was evaluated compared to background mutations found in normal tissue samples and manually reviewed according to distinct online databases. This retrospective study included 48 samples (45 tumors, three non-tumors), which were sequenced with the GeneReader (QIAGEN). Thereof ten samples were additionally analyzed with the Ion S5™ (Thermo Fisher) and 20 samples with the MiSeq™ (Illumina®) to compare the different NGS devices, as well as the sequencing results and their quality. The analysis showed that the individual NGS platforms detected different numbers of BRCA1/2 alterations in the respective tumor sample. In addition, the GeneReader revealed variability in the detection and classification of pathogenic alterations within the platform itself as well as in comparison with the other platforms or online databases. The study concluded that the Ion S5™ in combination with the Oncomine™ Comprehensive Assay v3 is most recommendable for current and prospective requirements of molecular analysis in routine diagnostics. In addition to the two BRCA1/2 genes, a broad number of other genes (BRCAness genes and genes involved in the repair pathway) is covered by the panel, which may open up new treatment options for patients depending on the respective eligibility criteria.

Tumor-associated immune cell infiltrate density in penile squamous cell carcinomas.

Penile squamous cell carcinomas are rare tumor entities throughout Europe. Early lymphonodal spread urges for aggressive therapeutic approaches in advanced tumor stages. Therefore, understanding tumor biology and its microenvironment and correlation with known survival data is of substantial interest in order to establish treatment strategies adapted to the individual patient. Fifty-five therapy naïve squamous cell carcinomas, age range between 41 and 85 years with known clinicopathological data, were investigated with the use of tissue microarrays (TMA) regarding the tumor-associated immune cell infiltrate density (ICID). Slides were stained with antibodies against CD3, CD8 and CD20. An image analysis software was applied for evaluation. Data were correlated with clinicopathological characteristics and overall survival. There was a significant increase of ICID in squamous cell carcinomas of the penis in relation to tumor adjacent physiological tissue. Higher CD3-positive ICID was significantly associated with lower tumor stage in our cohort. The ICID was not associated with overall survival. Our data sharpens the view on tumor-associated immune cell infiltrate in penile squamous cell carcinomas with an unbiased digital and automated cell count. Further investigations on the immune cell infiltrate and its prognostic and possible therapeutic impact are needed.