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Collision tumors with squamous cell carcinoma (SCC) and malignant melanoma components are extremely rare. We report a case of a penile collision tumor with a component of HPV-associated SCC and malignant melanoma in a 61 year-old man. The patient underwent a partial penectomy and bilateral inguinal sentinel lymph node surgery. Microscopy showed a biphasic tumor with a component of SCC with warty-basaloid morphology, positive for HPV16 and 43, and a malignant melanoma associated with a BRAF mutation. This case posed diagnostic and therapeutic challenges due to the absence of a precise TNM classification and documented treatment guidelines for this tumor type.

Background: The tumor immune microenvironment (TIME) plays a critical role in tumor progression, metastatic potential, and therapeutic response. Brightfield multiplex immunohistochemistry (BF-mIHC) is essential for in-depth TIME analysis, however, manual cell enumeration remains impractical for routine pathology. The contribution of Computer Vision is crucial for reliable TIME characterization, but the state of the art still lacks validated tools. In this study, we developed and validated a deep learning (DL) model for automated immune cell quantification in penile squamous cell carcinoma (PSCC) and non–small cell lung carcinoma (NSCLC).

Design: The study involves 13 slides of PSCC and 5 of NSCLC. Slides undergo BF-mIHC staining and digitization at ×20 (0.504 μm/pixel) by a Leica Aperio AT2 scanner. The BF-mIHC protocol stains CD4 in green, CD8 in DAB, CD163 (PSCC) and CD20 (NSCLC) in purple, CD68 in teal, FOXP3 (only PSCC) in yellow. Ten WSIs are randomly chosen for training the model and 8 are used for a blinded test. All WSIs are sampled in hotspot square patches (n.360) of 512×512 pixels. To generate a ground-truth dataset of stained cells, all patches are manually annotated by 2 expert pathologists. The 277 patches of the training WSIs are augmented by geometric and color transforms (1108 final patches). A multiclass U-Net with a ResNet-34 encoder is trained with a NVIDIA RTX3060 and validated using 20% of the patches. Final evaluation of performance is conducted on the 83 patches extracted from the 8 test WSIs.

Results: Training the DL model for 2000 epochs takes 60.19 hours. After training, the validation Dice score reaches 0.93. On the test subset, the DL model achieves both accuracy and specificity for background detection of 82%. Accuracy for chromogens is 87% for green, 97% for DAB, 98% for purple, and 99% for both teal and yellow. Sensitivity for chromogens is 81% for green, 85% for DAB, 77% for purple, 82% for teal, and 85% for yellow. Specificity for all chromogens is 99%. Computational time of automated detection and counting on new image patches (512×512 pixels) takes 2 seconds each.

Conclusions: This study develops a DL model validated for the automated characterization of the TIME in PSCC and NSCLC. This workflow enables a reproducible, objective, and high-throughput analysis of WSIs. The establishment of a robust and standardized tool for TIME profiling expands the clinical utility and translational relevance of BF-mIHC in diagnostic pathology.

BACKGROUND: Penile squamous cell carcinoma is an uncommon neoplasm that arises via two carcinogenic pathways, one linked to HPV infection and the other to chronic inflammation and p53 alterations.

OBJECTIVE/METHODS: We assessed the distribution of HPV genotypes in penile tumors and subsequent inguinal metastases in a cohort of 343 patients, analyzing their concordance with p16 stain and histological subtype, as well as the predictive significance of HPV tumor status and the immunohistochemical expression of p16 and p53 in inguinal lymph node metastasis (ILNM).

RESULTS: The overall prevalence of HPV in primary penile tumors was 42.9%, with high-risk HPV genotypes detected in 95.2% of HPV-positive cases. HPV16 was the most prevalent genotype identified in both primary tumors and metastases. However, other genotypes, including the low-risk HPV82, were also found in metastatic disease. We observed good concordance between HPV tumor status and histological subtype and very good concordance between HPV tumor status and p16 staining, with Cohen's kappa (κ) values of 0.80 (p-value < 0.001) and 0.83 (p < 0.001), respectively.

CONCLUSIONS: In most HPV-positive metastatic cases, the same HPV genotypes were detected in both the metastasis and the penile tumor. Both p16 staining and histological subtype can serve as surrogates for molecular HPV testing in lower resourced settings. In this dataset, no significant association was found between HPV status, p16 expression, or p53 expression and the presence of ILNM, suggesting their limited utility as predictive markers for ILNM in penile cancer.

Introduction & Objectives: The diagnostic workup for suspected prostate cancer is often emotionally distressing and may negatively affect patients’ well-being. Considerable variability exists in waiting times across diagnostic pathways, yet evidence on how such delays influence psychological distress is limited. Objective: To assess whether a fast-track diagnostic workup reduces psychological and physiological stress among men with suspected prostate cancer.

Materials & Methods: In this randomized clinical trial, 304 men referred for suspected prostate cancer at the Urology Department, Örebro University Hospital (Sweden), were randomized to either a fast-track diagnostic workup—aiming for the shortest possible waiting time—or usual care. Primary outcomes were patient-reported symptoms of distress (anxiety, depression, perceived stress, and sleep disruption); secondary outcomes included physiological stress markers, such as diurnal salivary cortisol. Data were collected from randomization to 12 months post-enrollment.

Results: Men in the fast-track group had a significantly shorter waiting time to their first consultation (median 7 days) compared with the usual care group (median 35 days). During the first six months, participants in the fast-track arm reported lower levels of distress, anxiety, and perceived stress, and improved sleep quality. Among those undergoing prostate biopsy, the strongest intervention effect was seen one to four weeks post-procedure. For men subsequently diagnosed with prostate cancer, the effect was most pronounced one week after biopsy. Differences between groups were no longer evident at 12 months. Diurnal cortisol profiles indicated a more favorable stress pattern in th efast-track group, especially during the period surrounding diagnostic disclosure.

Conclusions: A fast-track diagnostic workup for suspected prostate cancer substantially reduces psychological and physiological stress during the diagnostic phase. These findings support the implementation of accelerated diagnostic pathways in urological practice to improve patient experience and potentially reduce stress-related health risks.

BACKGROUND: Penile cancer (PeCa) is a rare but aggressive disease where lymph node metastases (LNM) represent the most significant prognostic factor. Accurate identification of LNM remains a clinical priority, but traditional imaging and clinical parameters often fail to detect occult LNM. Soluble immune checkpoint proteins (sICs) have recently emerged as potential non-invasive biomarkers in various malignancies, although unexplored in PeCa. The primary aim of this study was to explore the value of a panel of 14 sICs for predicting LNM in PeCa. The secondary aim was to compare plasma sIC levels between PeCa patients and cancer-free controls.

METHODS: Using ProcartaPlex immunoassays, BTLA, IDO, LAG-3, HVEM, PD-1, PD-L1, PD-L2, TIM-3, CD80, CTLA-4, GITR, CD27, CD28, and CD137 were measured in plasma from 284 PeCa patients and 45 cancer-free controls. PeCa patients were divided into a training set (n=202) and a test set (n=82). A prediction model for LNM was created using logistic regression.

RESULTS: Overall accuracy of the prediction model reached 77.5% (95% CI: 70.9 - 83.3) for the training set, yielding 8.9% sensitivity and 99.3% specificity in predicting LNM. Upon validation using the test set, the accuracy decreased to 62.2% (95% CI: 50.8-72.7) with 17.9% sensitivity and 85.2% specificity. When comparing PeCa patients and cancer-free controls, four inhibitory sICs (IDO, TIM-3, CD80, and CTLA-4) were found at significantly higher levels in the PeCa group. Due to the rarity of the disease, the main limitation of the study is the small number of patients with LNM.

CONCLUSION: Our study provides no evidence that sICs can predict LNM in PeCa, although four inhibitory sICs were significantly elevated in PeCa patients compared to cancer-free controls, suggesting systemic immunosuppression associated with tumor presence, consistent with findings in other malignancies. Studies with larger cohorts are warranted to clarify the prognostic significance of sICs in PeCa.

BACKGROUND AND OBJECTIVE: To improve treatment for patients with penile cancer, there is a need for prognostic and treatment predictive biomarkers. The objective of this study was to examine the expression of checkpoint proteins (programmed cell death ligand 1 [PD-L1], T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain [TIGIT], and cluster of differentiation 155 [CD155]) and human papillomavirus (HPV) status in primary tumors of penile cancer patients with indication for perioperative oncological treatment. As a secondary aim, we evaluated the associations between these biomarkers and penile cancer-specific survival.

METHODS: Fifty-two patients who underwent surgical treatment during 2009-2018 were included. HPV status was determined by polymerase chain reaction, and tissue microarray sections from primary tumors were subjected to immunohistochemistry to evaluate the expression of PD-L1, TIGIT, and CD155.

KEY FINDINGS AND LIMITATIONS: PD-L1, TIGIT, and CD155 were expressed widely. Specifically, 75% of patients had PD-L1-positive tumors, 80% had TIGIT-positive tumors, and 98% had CD155-positive tumors. Additionally, 47% of patients had HPV-positive tumors. Patients with HPV-positive tumors had better survival than those with HPV-negative tumors. Patients with indication for perioperative oncological therapy who received such treatment and whose tumors exhibited low PD-L1 expression demonstrated better survival than those with higher PD-L1 expression levels. The main limitations of this study include the small number of patients, retrospective design, and use of tissue microarrays rather than whole tissue sections.

CONCLUSIONS AND CLINICAL IMPLICATIONS: We found high expressions of the investigated checkpoint proteins, suggesting an immunosuppressed tumor microenvironment in patients with advanced penile cancer. These findings imply that checkpoint proteins could serve as prognostic and treatment predictive biomarkers. Patients with HPV-positive tumors had better prognosis.

PATIENT SUMMARY: In this report, we investigated tumor tissue from patients with penile cancer and identified a high prevalence of proteins that play an important role in the immune system's defense against cancer. The findings suggest that these proteins can be important in understanding and developing treatments for patients with lymph node metastasized penile cancer.

Inguinal lymph node surgery is a standard treatment for penile cancer patients with intermediate or high risk for lymph node metastasis (LNM) according to European Association of Urology (EAU) risk grading. We are proposing a more objective histological prognostic grading system for inguinal LNM in these patients. We assessed worst pattern of invasion, lymphocytic host response, lymphovascular invasion, and perineural invasion in a population-based cohort of 306 penile cancer patients. Patients were classified into low, intermediate, and high risk for inguinal LNM. There was a significant association both between risk groups and pT stage (p < 0.001) and between risk groups and LNM. Univariate logistic regression showed 25.43 times higher odds of LNM for patients in the intermediate risk group compared with the low risk group (odds ratio (OR) 25.43; 95% confidence interval (CI): 5.94-108.97) and a 177.13 times higher odds in the high risk group compared to the low risk group (OR 177.13; 95% CI: 40.09-782.51). When comparing our histological risk grading with the EAU grading, we found a higher sensitivity, of 51.28% (95% CI: 45.68-56.88) versus 37.09% (95% CI: 31.68-42.50), as well as a higher area under the curve (0.86; 95% CI: 0.81-0.89; versus 0.65; 95% CI: 0.58-0.71) with our grading system. While our grading classified 111 patients as low risk, only 31 were considered low risk for LNM according to the EAU risk classification. The new histological risk grading system shows a higher sensitivity and includes a higher number of patients in the low risk group in whom lymph node surgery could be avoided, reducing morbidity and costs.

The reliability of molecular diagnostic and prognostic tools is contingent on the quality of biospecimens, which are often collected during surgical procedures. This study investigated the impact of surgical manipulation on gene expression in the urinary bladder mucosa during radical cystectomy. Seventeen patients with urinary bladder cancer were enrolled, and paired pre- and post-surgery biopsies were analyzed. Pre-surgical biopsies were obtained in situ under anesthesia, while post-surgical biopsies were collected ex vivo following bladder removal. Total RNA was extracted, and gene expression was assessed using qPCR arrays, measuring the expression of 374 inflammation-related genes. The findings from the exploratory phase were further validated by analyzing key genes in an independent patient cohort using TaqMan® gene-specific assays. Exploratory analysis revealed significant differential expression in 27 genes, with key genes such as IL6, FOS, and PTGS2 being upregulated post-surgery. Validation of five selected genes in an independent cohort confirmed these findings. This study reinforces the necessity of accounting for surgery-induced alterations in gene expression when analyzing tissue samples collected intraoperatively. By elucidating the molecular impact of surgical interventions, this work provides critical insights for refining experimental methodologies and enhancing the interpretability of gene expression studies in clinical and research settings.

Introduction & Objectives: Immune checkpoint proteins are recognized as critical regulators of the immune system and play an important role in the defence against cancer. Unfortunately, cancer cells evade the immune destruction by overexpressing inhibitory checkpoint proteins, hence creating a favourable tumour microenvironment. Recently, it has been proposed that immune checkpoint proteins can be detected in soluble form in body fluids. This is important since a blood sample is less invasive than a tissue sample and hypotheticall yhas the potential to be more representative. The aim of the study was to evaluate the concentrations of soluble immune inhibitory checkpoint proteins (sICs) in men with and without penile cancer.

Materials & Methods: The circulating levels of ten soluble immune inhibitory checkpoint proteins (TIM-3, CD152, HVEM, IDO, LAG-3, BTLA, CD80, PD-1, PD-L1 and PD-L2) were assessed in plasma of 206 men with penile cancer (cases) and 46 men free from penile cancer (controls) using a multiplex Luminex assay. Mean concentrations of sICs were calculated and bootstrap resampling together with Welch's t-tests were used to assess differential expression and distribution of sICs between cases and controls.

Results: HVEM was detectable in 87% of the samples. PD-L1 was only detectable in 43.3% and therefore excluded from further analyses. The remaining markers were detectable in all samples. When comparing the mean concentrations of soluble immune inhibitory checkpoint proteins, nine sICs were found in higher concentrations in cases compared to controls, out of which seven reached statistical significance (Table 1).

Conclusions: Our study indicates that men with penile cancer have higher concentrations of soluble immune inhibitory checkpoint proteins in plasma compared to penile cancer-free men. Further studies are needed to evaluate their prognostic value