Ex Vivo Fluorescence Confocal Microscopy of MRI-Guided Targeted Prostate Biopsies for Rapid Detection of Clinically Significant Carcinomas-A Feasibility Study.

BACKGROUND: MRI-guided prostate biopsies from visible tumor-specific lesions (TBx) can be used to diagnose clinically significant carcinomas (csPCa) requiring treatment more selectively than conventional systematic biopsies (SBx). Ex vivo fluorescence confocal microscopy (FCM) is a novel technique that can be used to examine TBx prior to conventional histologic workup. METHODS: TBx from 150 patients were examined with FCM on the day of collection. Preliminary findings were reported within 2 h of collection. The results were statistically compared with the final histology. RESULTS: 27/40 (68%) of the csPCa were already recognized in the intraday FCM in accordance with the results of conventional histology. Even non-significant carcinomas (cisPCa) of the intermediate and high-risk groups (serum prostate-specific antigen (PSA) > 10 or 20 ng/mL) according to conventional risk stratifications were reliably detectable. In contrast, small foci of cisPCa were often not detected or were difficult to distinguish from reactive changes. CONCLUSION: The rapid reporting of preliminary FCM findings helps to reduce the psychological stress on patients, and can improve the clinical management of csPCa. Additional SBx can be avoided in individual cases, leading to lower rates of complications and scarring in the future surgical area. Additional staging examinations can be arranged without losing time. FCM represents a promising basis for future AI-based diagnostic algorithms.

MRI-Guided Targeted and Systematic Prostate Biopsies as Prognostic Indicators for Prostate Cancer Treatment Decisions.

The standard procedure for the diagnosis of prostate carcinoma involves the collection of 10-12 systematic biopsies (SBx) from both lobes. MRI-guided targeted biopsies (TBx) from suspicious foci increase the detection rates of clinically significant (cs) PCa. We investigated the extent to which the results of the TBx predicted the tumor board treatment decisions. SBx and TBx were acquired from 150 patients. Risk stratifications and recommendations for interventional therapy (prostatectomy and radiotherapy) or active surveillance were established by interdisciplinary tumor boards. We analyzed how often TBx alone were enough to correctly classify the tumors as well as to indicate interventional therapy and how often the findings of SBx were crucial for therapy decisions. A total of 28/39 (72%) favorable risk tumors were detected in TBx, of which 11/26 (42%) very-low-risk tumors were not detected and 8/13 (62%) low-risk tumors were undergraded. A total of 36/44 (82%) intermediate-risk PCa were present in TBx, of which 4 (9%) were underdiagnosed as a favorable risk tumor. A total of 12/13 (92%) high-risk carcinomas were detected and correctly grouped in TBx. The majority of csPCa were identified by the sampling of TBx alone. The tumor size was underestimated in a proportion of ISUP grade 1 tumors. Systematic biopsy sampling is therefore indicated for the next AS follow-up in these cases.

Ex Vivo Fluorescence Confocal Microscopy (FCM) Ensures Representative Tissue in Prostate Cancer Biobanking: A Feasibility Study.

BACKGROUND: Biobanking of prostate carcinoma is particularly challenging due to the actual cancer within the organ often without clear margins. Frozen sections are to date the only way to examine the biobank material for its tumor content. We used ex vivo fluorescence confocal microscopy (FCM) to analyze biobank samples prior to cryoasservation. METHODS: 127 punch biopsies were acquired from prostatectomy-specimens from 40 patients. These biopsies were analyzed with a Vivascope 2500-G4 prior to their transfer to the biobank. In difficult cases, larger samples of the prostatectomy specimens were FCM scanned in order to locate tumor foci. After patient acquisition, all samples were taken from the biobank and analyzed. We compared the results of the FCM examinations with the results of conventional histology and measured the DNA content. RESULTS: With upstream FCM, the tumor content of biobank samples could be determined with high confidence. The detection rate of representative biobank samples was increased due to the rapid feedback. The biobank samples were suitable for further molecular analysis. CONCLUSION: FCM allows for the first time lossless microscopic analysis of biobank samples prior to their cryoasservation and guarantees representative tumor and normal tissue for further molecular analysis.

Ex Vivo Fluorescence Confocal Microscopy (FCM) of Prostate Biopsies Rethought: Opportunities of Intraoperative Examinations of MRI-Guided Targeted Biopsies in Routine Diagnostics.

BACKGROUND: The diagnosis of prostate carcinoma (PCa) requires time- and material-consuming histopathological examinations. Ex vivo fluorescence confocal microscopy (FCM) can detect carcinoma foci in diagnostic biopsies intraoperatively. METHODS: MRI-guided and systematic biopsies were identified in a dataset of our previously published study cohort. Detection rates of clinically relevant tumors were determined in both groups. A retrospective blinded trial was performed to determine how many tumors requiring intervention were detectable via FCM analysis of MRI-guided targeted biopsies alone. RESULTS: MRI-guided targeted biopsies revealed tumors more frequently than systematic biopsies. Carcinomas in need of intervention were reliably represented in the MRI-guided biopsies and were identified in intraoperative FCM microscopy. Combined with serum PSA levels and clinical presentation, 91% of the carcinomas in need of intervention were identified. CONCLUSIONS: Intraoperative FCM analysis of MRI-guided biopsies is a promising approach for the efficient diagnosis of PCa. The method allows a timely assessment of whether a tumor disease requiring intervention is present and can reduce the psychological stress of the patient in the waiting period of the histological finding. Furthermore, this technique can lead to reduction of the total number of biopsies needed for the diagnosis of PCa.

Organ manifestations of COVID-19: what have we learned so far (not only) from autopsies?

The use of autopsies in medicine has been declining. The COVID-19 pandemic has documented and rejuvenated the importance of autopsies as a tool of modern medicine. In this review, we discuss the various autopsy techniques, the applicability of modern analytical methods to understand the pathophysiology of COVID-19, the major pathological organ findings, limitations or current studies, and open questions. This article summarizes published literature and the consented experience of the nationwide network of clinical, neuro-, and forensic pathologists from 27 German autopsy centers with more than 1200 COVID-19 autopsies. The autopsy tissues revealed that SARS-CoV-2 can be found in virtually all human organs and tissues, and the majority of cells. Autopsies have revealed the organ and tissue tropism of SARS-CoV-2, and the morphological features of COVID-19. This is characterized by diffuse alveolar damage, combined with angiocentric disease, which in turn is characterized by endothelial dysfunction, vascular inflammation, (micro-) thrombosis, vasoconstriction, and intussusceptive angiogenesis. These findings explained the increased pulmonary resistance in COVID-19 and supported the recommendations for antithrombotic treatment in COVID-19. In contrast, in extra-respiratory organs, pathological changes are often nonspecific and unclear to which extent these changes are due to direct infection vs. indirect/secondary mechanisms of organ injury, or a combination thereof. Ongoing research using autopsies aims at answering questions on disease mechanisms, e.g., focusing on variants of concern, and future challenges, such as post-COVID conditions. Autopsies are an invaluable tool in medicine and national and international interdisciplinary collaborative autopsy-based research initiatives are essential.

Ex Vivo Fluorescence Confocal Microscopy in Specimens of the Liver: A Proof-of-Concept Study.

Ex vivo Fluorescence Confocal Microscopy (FCM) is a technique providing high-resolution images of native tissues. The method is increasingly used in surgical settings in areas of dermatology and urology. Only a few publications exist about examinations of tumors and non-neoplastic lesions of the liver. We report on the application of FCM in biopsies, surgical specimens and autopsy material (33 patients, 39 specimens) of the liver and compare the results to conventional histology. Our preliminary examinations indicated a perfect suitability for tumor diagnosis (ĸ = 1.00) and moderate/good suitability for the assessment of inflammation (ĸ = 0.4-0.6) with regard to their severity and localization. Macro-vesicular steatosis was reliably detected, micro-vesicular steatosis tended to be underestimated. Cholestasis and eosinophilic granules in granulocytes were not represented in the scans. The tissue was preserved as native material and maintained its quality for downstream histological, immunohistological and molecular examinations. In summary, FCM is a material sparing method that provides rapid feedback to the clinician about the presence of tumor, the degree of inflammation and structural changes. This can lead to faster therapeutic decisions in the management of liver tumors, treatment of hepatitis or in liver transplant medicine.

Diagnostic Performance of Ex Vivo Fluorescence Confocal Microscopy in the Assessment of Diagnostic Biopsies of the Prostate.

BACKGROUND: Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumors in dermatological specimens. METHODS: We compare intraoperative diagnoses of the real-time application of FCM in pre-therapeutic prostate biopsies (35 patients, total number of biopsy specimens: n = 438) with the findings of conventional histology. RESULTS: Prostate carcinoma was reliably diagnosed in all patients. Depending on scan quality and experience of the examiner, smaller lesions of well differentiated carcinoma (ISUP1) could not be consistently differentiated from reactive changes. Furthermore, in some cases there was difficulty to distinguish ISUP grade 2 from ISUP grade 1 tumors. ISUP grades 3-5 were reliably detected in FCM. CONCLUSIONS: Despite some limitations, FCM seems to be an effective tool for the timely assessment of prostate biopsies enabling reliable diagnosis of prostate cancer in patients requiring therapy.

Feasibility study for ex vivo fluorescence confocal microscopy (FCM) on diagnostic prostate biopsies.

BACKGROUND: Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumours in dermatological specimens. Preliminary results found good feasibility when this technique was used to examine prostate cancer (PCa) specimens. METHODS: We report on the application of FCM in magnet resonance imaging (MRI)-fused prostate biopsies (10 patients, total number of biopsy specimens: n=121) and compare the results to conventional histology. RESULTS: Specific structures of the prostatic tissue were very well represented in the FCM images comparable to conventional histology. Prostate carcinoma was diagnosed with good sensitivity (79/68%) and high specificity (100%) by two pathologists with substantial/almost perfect levels of agreement with the results of conventional histology (kappa 0.79/0.86). Depending on the quality of the scans, malignant lesions of 1.8 mm and more in diameter were reliably diagnosed. Smaller lesions were rated as suspect for malignancy, but could not be consistently differentiated from reactive changes. Optimal image qualities were achieved in focus depths of up to 50 µm, whereas deeper scans led to insufficient representation of cytological features. Pre-treatment with acridine orange (AO) did not alter immunoreactivity of the tissue or its feasibility for fluorescence in situ hybridization (FISH) analyses and adequate amounts of DNA could be extracted for further polymerase chain reaction (PCR)-based examinations. CONCLUSIONS: FCM seems to be a promising tool for the timely diagnosis in cases of PCa in patients requiring therapy. In particular, this technique is a material-sparing method that conserves the biopsies as unfixed material for further analysis such as molecular tumour companion diagnosis.

First case of postmortem study in a patient vaccinated against SARS-CoV-2.

A previously symptomless 86-year-old man received the first dose of the BNT162b2 mRNA COVID-19 vaccine. He died 4 weeks later from acute renal and respiratory failure. Although he did not present with any COVID-19-specific symptoms, he tested positive for SARS-CoV-2 before he died. Spike protein (S1) antigen-binding showed significant levels for immunoglobulin (Ig) G, while nucleocapsid IgG/IgM was not elicited. Acute bronchopneumonia and tubular failure were assigned as the cause of death at autopsy; however, we did not observe any characteristic morphological features of COVID-19. Postmortem molecular mapping by real-time polymerase chain reaction revealed relevant SARS-CoV-2 cycle threshold values in all organs examined (oropharynx, olfactory mucosa, trachea, lungs, heart, kidney and cerebrum) except for the liver and olfactory bulb. These results might suggest that the first vaccination induces immunogenicity but not sterile immunity.

Hepatic Vasculopathy and Regenerative Responses of the Liver in Fatal Cases of COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 (COVID-19). It may impact the heart, brain, kidney, and liver.1 Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19.2,3 Histologic evaluation has been limited to small numbers of COVID-19 cases with no control subjects2,4 and demonstrated largely heterogeneous patterns of pathology.2,3.

Uncommon finding of a "black node" in a patient with malignant melanoma and arthroplasty: A case report.

Postarthroplasty lymphadenopathy should be kept in mind particularly in patients with tumor (eg, melanoma) with a long-term history of total joint replacement therapy. Microscopy is mandatory in establishing diagnosis and is thus helpful for therapy.

Biofunctionalization of synthetic bone substitutes with angiogenic stem cells: Influence on regeneration of critical-size bone defects in an in vivo murine model.

PURPOSE: The aim of this study was to investigate the influence of human bone marrow-derived endothelial progenitor cells (EPC) on vascularization and bone regeneration in combination with a bone-substitute material (BSM) in a critical-size bone defect in a murine model. Critical-size bone defects were performed and the defects were filled according to the group membership. MATERIALS AND METHODS: Eighteen rats were randomized in two experimental groups: BSM (BoneCeramic) with/without EPC biofunctionalization, and a control group without BSM and EPC. Calvaria bone defects were performed and the defects were filled according to the group membership. After 8 weeks, qualitative tissue response of newly formed bone mass was analyzed by histology, cone beam CT (CBCT) and micro-CT (µCT) scans. Occurrence of tumor masses due to EPC vascularization in peripheral organs was investigated microscopically in histological slides of liver and kidney. RESULTS: The combination of EPC and BSM showed smaller bone defects in the CT scans and the histological evaluation as the single use of BSM without EPC by trend (p = 0.067). Further, a higher amount of blood vessels could be found in histological slices of BSM in combination with EPC. No inflammatory response or tumor formation could be found. CONCLUSION: These findings confirm the biocompatibility of the used BSM and provide evidence that the combination of BSM with EPC might be effective for bone vascularization and regeneration. Using EPC in augmentation sites might overall lead to faster and better bone regeneration and should be further investigated in future studies.

Portal vein leiomyosarcoma: a case report and review of the literature.

BACKGROUND: Leiomyosarcoma of vascular smooth muscle is a very rare entity. A fair number of cases of vascular leiomyosarcomas have been reported, and the vast majority of these tumors arose from the inferior vena cava. CASE PRESENTATION: We report the case of a 71-year-old female patient who presented with recurrent upper abdominal pain. A CT-scan demonstrated a heterogenous mass in the liver hilum. Liver function tests and hematology parameters as well as the tumor markers were normal. Due to the unclear diagnosis a percutaneous biopsy of this mass was performed and revealed leiomyosarcoma. The patient was treated by a right sided hemihepatectomy with portal vein reconstruction and an end-to-side hepatico-jejunostomy. Final histology confirmed complete (R0) resection of a moderately differentiated leiomyosarcoma of the portal vein. After complete (R0) resection of the lesion, the patient remained without any signs of tumor recurrence for a total of 36 months until detection of an unresectable local recurrence. After surgical re-exploration the patient was finally referred to palliative radiotherapy. CONCLUSION: Vascular leiomyosarcoma of the portal vein is an extremely rare tumor entity. We have described a case with no evidence-based neo/adjuvant treatment options, where aggressive surgery achieved a tumor-free margin (R0), performed in a specialized center for sarcoma and hepatobiliary surgery.

High definition plus colonoscopy combined with i-scan tone enhancement vs. high definition colonoscopy for colorectal neoplasia: A randomized trial.

BACKGROUND: High definition endoscopy is the accepted standard in colonoscopy. However, an important problem is missed polyps. AIMS: Our objective was to assess the additional adenoma detection rate between high definition colonoscopy with tone enhancement (digital chromoendoscopy) vs. white light high definition colonoscopy. METHODS: In this prospective randomized trial patients were included to undergo a tandem colonoscopy. The first exam was a white light colonoscopy with removal of all visualized polyps. The second examination was randomly assigned in a 1:1 ratio as either again white light colonoscopy (Group A) or colonoscopy with tone enhancement (Group B). Primary endpoint was the adenoma detection rate during the second withdrawal (sample size calculation - 40 per group). RESULTS: 67 lesions (Group A: n=34 vs. Group B: n=33) in 80 patients (mean age 61 years, male 64%) were identified on the first colonoscopy. The second colonoscopy detected 78 additional lesions: n=60 with tone enhancement vs. n=18 with white light endoscopy (p<0.001). Tone enhancement found more additional adenomas (A n=20 vs. B n=6, p=0.006) and identified significantly more missed adenomas per subject (0.5 vs. 0.15, p=0.006). CONCLUSIONS: High definition plus colonoscopy with tone enhancement detected more adenomas missed by white light colonoscopy.

Frequency and characterization of benign lesions in patients undergoing surgery for the suspicion of solid pancreatic neoplasm.

OBJECTIVES: A diagnosis of benign lesions (BLs) is reported in 5% to 21% of pancreatoduodenectomies performed for neoplasms; no data for body-tail resections are available. The aims were to investigate the frequency and characterize the BLs mimicking cancer in the head and the body-tail of the pancreas. METHODS: This study is a retrospective review of pancreatic specimenscollected from 2005 to 2011 in the pathology database of Mainz (Germany). Patients with final diagnosis excluding malignancy were analyzed by histology, imaging, and clinical aspects. RESULTS: Among 373 patients, 33 patients (8.8%) were diagnosed with a benign disease: 25 (8.4%) of 298 in the pancreatic head and 8 (10.7%) of 75 in the body-tail resections. Paraduodenal pancreatitis was diagnosed in 13 (3.5%) of 373 patients; autoimmune pancreatitis (AIP), in 11 (2.9%); "ordinary" chronic pancreatitis, in 6 (1.6%); and accessory spleen, in 3 (0.8%). In pancreatic head resections, the most frequent diagnoses were paraduodenal pancreatitis (13/298, 4.4%) and AIP (9/298, 3%), whereas in the body-tail, the most frequent diagnoses were accessory spleen (3/75, 4%), chronic pancreatitis (3/75, 4%), and AIP (2/75, 2.7%). CONCLUSIONS: Benign lesions are observed with the same frequency inspecimens of the head or the body-tail of the pancreas.

Efficacy of SpyGlass(TM)-directed biopsy compared to brush cytology in obtaining adequate tissue for diagnosis in patients with biliary strictures.

AIM: To evaluate the diagnostic yield (inflammatory activity) and efficiency (size of the biopsy specimen) of SpyGlass(TM)-guided biopsy vs standard brush cytology in patients with and without primary sclerosing cholangitis (PSC). METHODS: At the University Medical Center Mainz, Germany, 35 consecutive patients with unclear biliary lesions (16 patients) or long-standing PSC (19 patients) were screened for the study. All patients underwent a physical examination, lab analyses, and abdominal ultrasound. Thirty-one patients with non-PSC strictures or with PSC were scheduled to undergo endoscopic retrograde cholangiography (ERC) and subsequent peroral cholangioscopy (POC). Standard ERC was initially performed, and any lesions or strictures were localized. POC was performed later during the same session. The Boston Scientific SpyGlass System(TM) (Natick, MA, United States) was used for choledochoscopy. The biliary tree was visualized, and suspected lesions or strictures were biopsied, followed by brush cytology of the same area. The study endpoints (for both techniques) were the degree of inflammation, tissue specimen size, and the patient populations (PSC vs non-PSC). Inflammatory changes were divided into three categories: none, low activity, and high activity. The specimen quantity was rated as low, moderate, or sufficient. RESULTS: SpyGlass(TM) imaging and brush cytology with material retrieval were performed in 29 of 31 (93.5%) patients (23 of the 29 patients were male). The median patient age was 45 years (min, 20 years; max, 76 years). Nineteen patients had known PSC, and 10 showed non-PSC strictures. No procedure-related complications were encountered. However, for both methods, tissues could only be retrieved from 29 patients. In cases of inflammation of the biliary tract, the diagnostic yield of the SpyGlass(TM)-directed biopsies was greater than that using brush cytology. More tissue material was obtained for the biopsy method than for the brush cytology method (P = 0.021). The biopsies showed significantly more inflammatory characteristics and greater inflammatory activity compared to the cytological investigation (P = 0.014). The greater quantity of tissue samples proved useful for both PSC and non-PSC patients. CONCLUSION: SpyGlass(TM) imaging can be recommended for proper inflammatory diagnosis in PSC patients. However, its value in diagnosing dysplasia was not addressed in this study and requires further investigation.

Confocal endomicroscopy identifies loss of local barrier function in the duodenum of patients with Crohn's disease and ulcerative colitis.

BACKGROUND: Increased cell shedding with gap formation and local barrier dysfunction can be identified endomicroscopically in the terminal ileum of patients with inflammatory bowel disease. We aim to evaluate whether these changes are also present in the duodenum of patients with inflammatory bowel disease. METHODS: Fifteen patients with Crohn's disease (CD), 10 patients with ulcerative colitis (UC), and 10 controls underwent fluorescein-aided confocal laser endomicroscopy (CLE). CLE was performed on macroscopically normal antral and duodenal (D1, D2, D3, D4) mucosa. Representative CLE images were prospectively analyzed. Images were scored for the number of epithelial gaps, cell shedding, and the degree of fluorescein leakage into the intestinal lumen. RESULTS: Both CD and UC patients had significantly more epithelial gaps, epithelial cell shedding, and leakage of fluorescein into the duodenal lumen than controls. The degree of cell shedding and epithelial gap formation was similar in CD and UC patients. In all cases, macroscopic endoscopic appearances of the duodenum were normal, and conventional histological analysis showed a mild nonspecific duodenitis in 7 of 15 patients with CD. Patients with UC had a histologically normal duodenum. Gap formation, cell shedding, and fluorescein leakage was similar in CD with active compared with inactive disease, except for D2 shedding. CONCLUSIONS: CLE can detect epithelial damage and barrier loss in the duodenum of CD and UC patients that is not apparent on conventional endoscopy or histology.

Non-pheochromocytoma (PCC)/paraganglioma (PGL) tumors in patients with succinate dehydrogenase-related PCC-PGL syndromes: a clinicopathological and molecular analysis.

OBJECTIVE: Although the succinate dehydrogenase (SDH)-related tumor spectrum has been recently expanded, there are only rare reports of non-pheochromocytoma/paraganglioma tumors in SDHx-mutated patients. Therefore, questions still remain unresolved concerning the aforementioned tumors with regard to their pathogenesis, clinicopathological phenotype, and even causal relatedness to SDHx mutations. Absence of SDHB expression in tumors derived from tissues susceptible to SDH deficiency is not fully elucidated. DESIGN AND METHODS: Three unrelated SDHD patients, two with pituitary adenoma (PA) and one with papillary thyroid carcinoma (PTC), and three SDHB patients affected by renal cell carcinomas (RCCs) were identified from four European centers. SDHA/SDHB immunohistochemistry (IHC), SDHx mutation analysis, and loss of heterozygosity analysis of the involved SDHx gene were performed on all tumors. A cohort of 348 tumors of unknown SDHx mutational status, including renal tumors, PTCs, PAs, neuroblastic tumors, seminomas, and adenomatoid tumors, was investigated by SDHB IHC. RESULTS: Of the six index patients, all RCCs and one PA displayed SDHB immunonegativity in contrast to the other PA and PTC. All immunonegative tumors demonstrated loss of the WT allele, indicating bi-allelic inactivation of the germline mutated gene. Of 348 tumors, one clear cell RCC exhibited partial loss of SDHB expression. CONCLUSIONS: These findings strengthen the etiological association of SDHx genes with pituitary neoplasia and provide evidence against a link between PTC and SDHx mutations. Somatic deletions seem to constitute the second hit in SDHB-related renal neoplasia, while SDHx alterations do not appear to be primary drivers in sporadic tumorigenesis from tissues affected by SDH deficiency.