Artificial Intelligence to Predict the BRAF V595E Mutation in Canine Urinary Bladder Urothelial Carcinomas

In dogs, the BRAF mutation (V595E) is common in bladder and prostate cancer and represents a specific diagnostic marker. Recent advantages in artificial intelligence (AI) offer new opportunities in the field of tumour marker detection. While AI histology studies have been conducted in humans to detect BRAF mutation in cancer, comparable studies in animals are lacking. In this study, we used commercially available AI histology software to predict BRAF mutation in whole slide images (WSI) of bladder urothelial carcinomas (UC) stained with haematoxylin and eosin (HE), based on a training (n = 81) and a validation set (n = 96). Among 96 WSI, 57 showed identical PCR and AI-based BRAF predictions, resulting in a sensitivity of 58% and a specificity of 63%. The sensitivity increased substantially to 89% when excluding small or poor-quality tissue sections. Test reliability depended on tumour differentiation (p < 0.01), presence of inflammation (p < 0.01), slide quality (p < 0.02) and sample size (p < 0.02). Based on a small subset of cases with available adjacent non-neoplastic urothelium, AI was able to distinguish malignant from benign epithelium. This is the first study to demonstrate the use of AI histology to predict BRAF mutation status in canine UC. Despite certain limitations, the results highlight the potential of AI in predicting molecular alterations in routine tissue sections

Artificial Intelligence to Predict the BRAF V595E Mutation in Canine Urinary Bladder Urothelial Carcinomas.

In dogs, the BRAF mutation (V595E) is common in bladder and prostate cancer and represents a specific diagnostic marker. Recent advantages in artificial intelligence (AI) offer new opportunities in the field of tumour marker detection. While AI histology studies have been conducted in humans to detect BRAF mutation in cancer, comparable studies in animals are lacking. In this study, we used commercially available AI histology software to predict BRAF mutation in whole slide images (WSI) of bladder urothelial carcinomas (UC) stained with haematoxylin and eosin (HE), based on a training (n = 81) and a validation set (n = 96). Among 96 WSI, 57 showed identical PCR and AI-based BRAF predictions, resulting in a sensitivity of 58% and a specificity of 63%. The sensitivity increased substantially to 89% when excluding small or poor-quality tissue sections. Test reliability depended on tumour differentiation (p < 0.01), presence of inflammation (p < 0.01), slide quality (p < 0.02) and sample size (p < 0.02). Based on a small subset of cases with available adjacent non-neoplastic urothelium, AI was able to distinguish malignant from benign epithelium. This is the first study to demonstrate the use of AI histology to predict BRAF mutation status in canine UC. Despite certain limitations, the results highlight the potential of AI in predicting molecular alterations in routine tissue sections

Better prognostic markers for nonmuscle invasive papillary urothelial carcinomas

Bladder cancer is a common type of cancer, especially among men in developed countries. Most cancers in the urinary bladder are papillary urothelial carcinomas. They are characterized by a high recurrence frequency (up to 70 %) after local resection. It is crucial for prognosis to discover these recurrent tumours at an early stage, especially before they become muscle-invasive. Reliable prognostic biomarkers for tumour recurrence and stage progression are lacking. This is why patients diagnosed with a non-muscle invasive bladder cancer follow extensive follow-up regimens with possible serious side effects and with high costs for the healthcare systems. WHO grade and tumour stage are two central biomarkers currently having great impact on both treatment decisions and follow-up regimens. However, there are concerns regarding the reproducibility of WHO grading, and stage classification is challenging in small and fragmented tumour material. In Paper I, we examined the reproducibility and the prognostic value of all the individual microscopic features making up the WHO grading system. Among thirteen extracted features there was considerable variation in both reproducibility and prognostic value. The only feature being both reasonably reproducible and statistically significant prognostic was cell polarity. We concluded that further validation studies are needed on these features, and that future grading systems should be based on well-defined features with true prognostic value. With the implementation of immunotherapy, there is increasing interest in tumour immune response and the tumour microenvironment. In a search for better prognostic biomarkers for tumour recurrence and stage progression, in Paper II, we investigated the prognostic value of tumour infiltrating immune cells (CD4, CD8, CD25 and CD138) and previously investigated cell proliferation markers (Ki-67, PPH3 and MAI). Low Ki 67 and tumour multifocality were associated with increased recurrence risk. Recurrence risk was not affected by the composition of immune cells. For stage progression, the only prognostic immune cell marker was CD25. High values for MAI was also strongly associated with stage progression. However, in a multivariate analysis, the most prognostic feature was a combination of MAI and CD25. BCG-instillations in the bladder are indicated in intermediate and high-risk non-muscle invasive bladder cancer patients. This old-fashion immunotherapy has proved to reduce both recurrence- and progression-risk, although it is frequently followed by unpleasant side-effects. As many as 30-50% of high-risk patients receiving BCG instillations, fail by develop high-grade recurrences. They do not only suffer from unnecessary side-effects, but will also have a delay in further treatment. Together with colleagues at three different Dutch hospitals, in Paper III, we looked at the prognostic and predictive value of T1-substaging. A T1-tumour invades the lamina propria, and we wanted to separate those with micro- from those with extensive invasion. We found that BCG-failure was more common among patients with extensive invasion. Furthermore, T1-substaging was associated with both high-grade recurrence-free and progression-free survival. Finally, in Paper IV, we wanted to investigate the prognostic value of two classical immunohistochemical markers, p53 and CK20, and compare them with previously investigated proliferation markers. p53 is a surrogate marker for mutations in the gene TP53, considered to be a main characteristic for muscle-invasive tumours. CK20 is a surrogate marker for luminal tumours in the molecular classification of bladder cancer, and is frequently used to distinguish reactive urothelial changes from urothelial carcinoma in situ. We found both positivity for p53 and CK20 to be significantly associated with stage progression, although not performing better than WHO grade and stage. The proliferation marker MAI, had the highest prognostic value in our study. Any combination of variables did not perform better in a multivariate analysis than MAI alone

Transitional cell carcinoma of the bladder : histopathological and biological factors and prognosis

The main purpose of the studies reported in this thesis has been to determine the extent to which the behaviour of TCC can be predicted by histopathological and biological characteristics. The potential additional prognostic value of these factors was evaluated by combining them with other prognostic factors in multivariate analysis. In chapter 2 a two grade system of histological grading .. using simple histological criteria, is proposed. The interobserver variability of the WHO grading system and the two grade system is tested. The extent to which patient survival and progression free survival correlated with the two grade system, is evaluated. The additional value of grading is tested by combining it with other prognostic factors such as stage, age and mitotic index in multivariate analysis. In chapter 3 BM expression in TCC is described in an attempt to evaluate its use for the histopathological identification of microinvasion. Furthermore the usefulness of BM staining for the prediction of the clinical behaviour of TCC is assessed in comparison with grading, staging and ploidy. In chapter 4 the use of a two grade morphometrical grading system for prediction of the clinical behaviour of TCC is described. Also the heterogeneity in the WHO grade II tumours is evaluated using morphometry. In chapter 5 a study is reported in which it is determined whether image cytometry can provide useful parameters which can be used in the prediction of TCC behaviour. Special attention is given to the potential value of rare incidents, e.g. occasional cells with a very high DNA content. In chapter 6 a study is described concerning numerical chromosome aberrations in TCC as assessed by counting chromosomes in metaphase spreads. The modal number of chromosomes and the chromosomal range are used as potential prognostic factors in comparison with histological parameters. In chapter 7 "classical" metaphase chromosome counting is compared with interphase cytogenetics, especially in tumours having diploid and hyperdiploid DNA content. In chapter 8 The findings of these studies are discussed and general conclusions are draw

DIAGNOSIS AND TREATMENT OF UROTHELIAL CARCINOMA OF THE BLADDER

Chapter 1 is a general introduction. Chapter 2 describes currently known methylation markers in urine for diagnosis of bladder cancer and their diagnostic accuracy. In Chapter 3 several methylation markers in urine are evaluated to use for diagnosis of bladder cancer and an optimal two gene panel is proposed. In chapter 4 several additives are tested to preserve urinary DNA prior to the analysis of methylation markers in urine. A protocol for preservation is proposed. In chapter 5 the two systems used for grading bladder cancer are tested to determine their reproducibility, both are poorly reproducible. In chapter 6, several measurents are used to determine whether a more objective method to determine bladder cancer grade is possible. In chapter 7 the value of an immediate instillation of mitomycin C after transurethral resection of a bladder tumor is evaluated in a randomised controlled trial. The study shows that an immediate instillation reduces the risk of recurrent bladder cancer. In chapter 8, we evaluate whether the value of an immediate instillation is different in several subgroups of patients. We found that an immediate instillation is effective in all risk groups. In Chapter 9, the timing of an immediate instillation of mitomycin C is investigated. We found that, altough it is recommended to administer an immediate instillation as soon a possible after transurethral resection of a bladder tumour, patients may still benefit from an instillation 1 day after transurethral resection. In chapter 10, interleukin-2 instillations are tested in patients with bladder cancer with and without a marker lesion. The study was closed prematurely so no soli conclusions can be made. However no clear benefit of a marker lesion was detected. In part 3 bladder preservation using brachytherapiy in high risk non muscle invasive bladder cancer, and muscle invasive bladder cancer is evaluatied. We found that brachytherapy is a good alternative to cystectomy in selected patients, without compromising survival. Additionally, the implantaion of the brachytherapey cathethers can safely be conducted using a robot-assisted laparoscopic approach

Superficial bladder cancer diagnosis — the deliberate choice between fluorescent diagnosis and optical biopsy

Bladder carcinoma in situ (CIS) is a potentially invasive tumor whose early detection is a key step to ensuring the preservation of the bladder, reducing mortality, and improving the quality of patients’ life. The early diagnosis of bladder cancer requires a sensitive technique that can detect the lesion to determine its stage and grade. ALA induced-PpIX makes it possible to detect tumors with 90% sensitivity. ALA hexyl ester (hALA) increases the sensitivity to 95%. Macroscopic techniques require a histological biopsy to define the tumor invasiveness. Imaging with Fibered Optic Confocal Fluorescence Microscopy allows the optical sectioning of examined tissues providing images with subcellular resolution after labeling with adequate fluorescent dye chosen based on the sensitivity of the used device. Available fluorescent agents are compatible with used devices; however, their toxicity and mutagenesis studies are unsatisfactory. During imaging, an optical fiber is introduced into the bladder via the urethra and placed in contact with the bladder wall. The distinction between the different types of epithelial cells is based on the cell size, morphology, and signal intensity. Although not fully adopted for clinical application, the FOCM represents a real asset that reduces invasiveness and complements the fluorescence-based endoscopy

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated