Identification of patients at risk for biochemical recurrence after radical prostatectomy with intra-operative frozen section.

OBJECTIVE: To identify patients at risk for biochemical recurrence (BCR) of prostate cancer (PCa) after radical prostatectomy (RP) with intra-operative whole-mount frozen section (FS) of the prostate. MATERIAL AND METHODS: We examined differences in BCR between patients with initial negative surgical margins at FS, patients with final negative surgical margins with initial positive margins at FS without residual PCa after secondary tumour resection, and patients with final negative surgical margins with initially positive margins at FS with residual PCa in the secondary tumour resection specimen. Institutional data of 883 consecutive patients undergoing RP were collected. Intra-operative whole-mount FS was routinely used to check for margin status and, if necessary, to resect more periprostatic tissue in order to achieve negative margins. Patients with lymph node-positive disease or final positive surgical margins were excluded from the analysis. Kaplan-Meier curves and multivariable Cox proportional hazards regression analyses adjusting for clinical covariates were employed to examine differences in biochemical recurrence-free survival (BRFS) according to the resection status mentioned above. RESULTS: The median follow-up was 22.4 months. The 1- and 2-year BRFS rates in patients with (81.0% and 72.9%, respectively; P = 0.001) and without residual PCa (90.3% and 82.3%, respectively; P = 0.033) after secondary tumour resection were significantly lower compared to patients with initial R0 status (93.4% and 90.9%, respectively). On multivariable Cox regression only residual PCa in the secondary tumour resection was associated with a higher risk of BCR compared to initial R0 status (hazard ratio 1.99, 95% confidence interval 1.01-3.92; P = 0.046). CONCLUSION: Despite being classified as having a negative surgical margin, patients with residual PCa in the secondary tumour resection specimen face a high risk of BCR. These findings warrant closer post-RP surveillance of this particular subgroup. Further research of this high-risk subset of patients should focus on examining whether these patients benefit from early salvage therapy and how resection status impacts oncological outcomes in the changing landscape of PCa treatment.

Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatography-tandem mass spectrometry-based proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive high-grade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS.

Noninvasive Diagnosis of High-Grade Urothelial Carcinoma in Urine by Raman Spectral Imaging.

The current gold standard for the diagnosis of bladder cancer is cystoscopy, which is invasive and painful for patients. Therefore, noninvasive urine cytology is usually used in the clinic as an adjunct to cystoscopy; however, it suffers from low sensitivity. Here, a novel noninvasive, label-free approach with high sensitivity for use with urine is presented. Coherent anti-Stokes Raman scattering imaging of urine sediments was used in the first step for fast preselection of urothelial cells, where high-grade urothelial cancer cells are characterized by a large nucleus-to-cytoplasm ratio. In the second step, Raman spectral imaging of urothelial cells was performed. A supervised classifier was implemented to automatically differentiate normal and cancerous urothelial cells with 100% accuracy. In addition, the Raman spectra not only indicated the morphological changes that are identified by cytology with hematoxylin and eosin staining but also provided molecular resolution through the use of specific marker bands. The respective Raman marker bands directly show a decrease in the level of glycogen and an increase in the levels of fatty acids in cancer cells as compared to controls. These results pave the way for "spectral" cytology of urine using Raman microspectroscopy.

Dynamic changes of tumor gene expression during repeated pressurized intraperitoneal aerosol chemotherapy (PIPAC) in women with peritoneal cancer.

BACKGROUND: Intraperitoneal chemotherapy is used to treat peritoneal cancer. The pattern of gene expression changes of peritoneal cancer during intraperitoneal chemotherapy has not been studied before. Pressurized intraperitoneal aerosol chemotherapy is a new form of intraperitoneal chemotherapy using repeated applications and allowing repeated tumor sampling during chemotherapy. Here, we present the analysis of gene expression changes during pressurized intraperitoneal aerosol chemotherapy with doxorubicin and cisplatin using a 22-gene panel. METHODS: Total RNA was extracted from 152 PC samples obtained from 63 patients in up to six cycles of intraperitoneal chemotherapy. Quantitative real-time PCR was used to determine the gene expression levels. For select genes, immunohistochemistry was used to verify gene expression changes observed on the transcript level on the protein level. Observed (changes in) expression levels were correlated with clinical outcomes. RESULTS: Gene expression profiles differed significantly between peritoneal cancer and non- peritoneal cancer samples and between ascites-producing and non ascites-producing peritoneal cancers. Changes of gene expression patterns during repeated intraperitoneal chemotherapy cycles were prognostic of overall survival, suggesting a molecular tumor response of peritoneal cancer. Specifically, downregulation of the whole gene panel during intraperitoneal chemotherapy was associated with better treatment response and survival. CONCLUSIONS: In summary, molecular changes of peritoneal cancer during pressurized intraperitoneal aerosol chemotherapy can be documented and may be used to refine individual treatment and prognostic estimations.

Histological changes in endocrine and exocrine pancreatic tissue from patients exposed to incretin-based therapies.

AIMS: Incretin-based therapies have been associated with an increased risk of pancreatitis. Recently, various histological abnormalities have been reported in human pancreatic tissue from brain-dead organ donors who had been exposed to incretin-based drugs. In the present study we examined pancreatic tissue collected at surgery. METHODS: Human pancreatic tissue from 7 type 2-diabetic patients treated with incretin-based drugs (type 2-I), 6 diabetic patients without incretin treatment (type 2-NI), 11 patients without diabetes (no diabetes group) and 9 brain-dead organ donors (BDOD group) was examined. RESULTS: Fractional beta-cell area was reduced in the type 2-NI group compared to the group without diabetes (P < .05), but there was no difference compared to the type 2-I patients. Alpha-cell area (P = .30), beta-cell replication (P = .17) and alpha-cell replication (P = .91) were not different. There were also no differences in acinar cell (P = .13) and duct cell replication (P = .099). Insulin-positive duct cells were more frequent in the type 2-I and the BDOD groups (P = .034). No co-expression of insulin and glucagon was detected. Pancreatic intraepithelial neoplasia (PanIN) lesions were very rare, all low-grade (PanIN 1a and 1b) and tended to occur more frequently in the type 2-I group (P = .084). CONCLUSIONS: The present results did not reveal marked histological abnormalities in the pancreas of incretin-treated patients with type 2 diabetes. Low numbers of specimens available and a large inter-individual variability of the findings warrant caution regarding the interpretation of histological data concerning drug effects on the human pancreas.

A framework for falsifiable explanations of machine learning models with an application in computational pathology.

In recent years, deep learning has been the key driver of breakthrough developments in computational pathology and other image based approaches that support medical diagnosis and treatment. The underlying neural networks as inherent black boxes lack transparency and are often accompanied by approaches to explain their output. However, formally defining explainability has been a notorious unsolved riddle. Here, we introduce a hypothesis-based framework for falsifiable explanations of machine learning models. A falsifiable explanation is a hypothesis that connects an intermediate space induced by the model with the sample from which the data originate. We instantiate this framework in a computational pathology setting using hyperspectral infrared microscopy. The intermediate space is an activation map, which is trained with an inductive bias to localize tumor. An explanation is constituted by hypothesizing that activation corresponds to tumor and associated structures, which we validate by histological staining as an independent secondary experiment.

Advances in Digital Pathology: From Artificial Intelligence to Label-Free Imaging.

BACKGROUND: Digital pathology, in its primary meaning, describes the utilization of computer screens to view scanned histology slides. Digitized tissue sections can be easily shared for a second opinion. In addition, it allows tissue image analysis using specialized software to identify and measure events previously observed by a human observer. These tissue-based readouts were highly reproducible and precise. Digital pathology has developed over the years through new technologies. Currently, the most discussed development is the application of artificial intelligence to automatically analyze tissue images. However, even new label-free imaging technologies are being developed to allow imaging of tissues by means of their molecular composition. SUMMARY: This review provides a summary of the current state-of-the-art and future digital pathologies. Developments in the last few years have been presented and discussed. In particular, the review provides an outlook on interesting new technologies (e.g., infrared imaging), which would allow for deeper understanding and analysis of tissue thin sections beyond conventional histopathology. KEY MESSAGES: In digital pathology, mathematical methods are used to analyze images and draw conclusions about diseases and their progression. New innovative methods and techniques (e.g., label-free infrared imaging) will bring significant changes in the field in the coming years.

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

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 chi2 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%, p = 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.

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

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.

Label-free, automated classification of microsatellite status in colorectal cancer by infrared imaging.

Challenging histopathological diagnostics in cancer include microsatellite instability-high (MSI-H) colorectal cancer (CRC), which occurs in 15% of early-stage CRC and is caused by a deficiency in the mismatch repair system. The diagnosis of MSI-H cannot be reliably achieved by visual inspection of a hematoxylin and eosin stained thin section alone, but additionally requires subsequent molecular analysis. Time- and sample-intensive immunohistochemistry with subsequent fragment length analysis is used. The aim of the presented feasibility study is to test the ability of quantum cascade laser (QCL)-based infrared (IR) imaging as an alternative diagnostic tool for MSI-H in CRC. We analyzed samples from 100 patients with sporadic CRC UICC stage II and III. Forty samples were used to develop the random forest classifier and 60 samples to verify the results on an independent blinded dataset. Specifically, 100% sensitivity and 93% specificity were achieved based on the independent 30 MSI-H- and 30 microsatellite stable (MSS)-patient validation cohort. This showed that QCL-based IR imaging is able to distinguish between MSI-H and MSS for sporadic CRC - a question that goes beyond morphological features - based on the use of spatially resolved infrared spectra used as biomolecular fingerprints.