Bone marrow haematopoiesis in patients with COVID-19.

AIMS: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection broadly affects organ homeostasis, including the haematopoietic system. Autopsy studies are a crucial tool for investigation of organ-specific pathologies. Here we perform an in-depth analysis of the impact of severe coronavirus disease 2019 (COVID-19) on bone marrow haematopoiesis in correlation with clinical and laboratory parameters. METHODS AND RESULTS: Twenty-eight autopsy cases and five controls from two academic centres were included in the study. We performed a comprehensive analysis of bone marrow pathology and microenvironment features with clinical and laboratory parameters and assessed SARS-CoV-2 infection of the bone marrow by quantitative polymerase chain reaction (qPCR) analysis. In COVID-19 patients, bone marrow specimens showed a left-shifted myelopoiesis (19 of 28, 64%), increased myeloid-erythroid ratio (eight of 28, 28%), increased megakaryopoiesis (six of 28, 21%) and lymphocytosis (four of 28, 14%). Strikingly, a high proportion of COVID-19 specimens showed erythrophagocytosis (15 of 28, 54%) and the presence of siderophages (11 of 15, 73%) compared to control cases (none of five, 0%). Clinically, erythrophagocytosis correlated with lower haemoglobin levels and was more frequently observed in patients from the second wave. Analysis of the immune environment showed a strong increase in CD68+ macrophages (16 of 28, 57%) and a borderline lymphocytosis (five of 28, 18%). The stromal microenvironment showed oedema (two of 28, 7%) and severe capillary congestion (one of 28, 4%) in isolated cases. No stromal fibrosis or microvascular thrombosis was found. While all cases had confirmed positive testing of SARS-CoV-2 in the respiratory system, SARS-CoV-2 was not detected in the bone marrow by high-sensitivity PCR, suggesting that SARS-CoV-2 does not commonly replicate in the haematopoietic microenvironment. CONCLUSIONS: SARS-CoV-2 infection indirectly impacts the haematological compartment and the bone marrow immune environment. Erythrophagocytosis is frequent and associated with lower haemoglobin levels in patients with severe COVID-19.

COVID-19 Infections in Gonads: Consequences on Fertility?

COVID-19 may influence human fertility and sexuality in several ways. Different cell types in gonads show a constitutive expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), which provide potential entry pathways for SARS-CoV-2. In addition to the biological effects of a COVID-19 infection on the gonads, the impact of the ongoing COVID-19 pandemic on mental health issues and sexual behavior may affect reproduction. This review summarizes the current knowledge on the influence of COVID-19 on the gonads and discusses possible consequences on human fertility. In this context, the close interaction between the hypothalamic-pituitary-adrenal axis and the hypothalamic-pituitary-gonadal axis in response to COVID-19-related stress is discussed. Some women noticed changes in their menstrual cycle during the COVID-19 pandemic, which could be due to psychological stress, for example. In addition, occasional cases of reduced oocyte quality and ovarian function are described after COVID-19 infection. In men, COVID-19 may cause a short-term decrease in fertility by damaging testicular tissue and/or impairing spermatogenesis. Moreover, decreased ratio testosterone/LH and FSH/LH in COVID-19 compared to aged-matched healthy men has been reported. Available data do not suggest any effect of the available SARS-CoV-2 vaccines on fertility. The effects of long COVID on human fertility have been reported and include cases with premature ovarian failure and oligomenorrhoea in women and erectile dysfunction in men. Despite the increasing knowledge about the effects of COVID-19 infections on human gonads and fertility, the long-term consequences of the COVID-19 pandemic cannot yet be assessed in this context.

Hypoxia and Ezrin Expression in Primary Melanoma Have High Prognostic Relevance.

Hypoxia affects tumor aggressiveness and activates pathways associated with epithelial mesenchymal transition (EMT) which are crucial for tumor progress. In this study, the correlation of hypoxia and EMT with sentinel lymph node status and tumor-specific survival was investigated in primary melanomas. CD34 for capillary count and Hypoxia inducible factor-1α (HIF-1α) as hypoxia indicators as well as Ezrin and L1-Cell Adhesion Molecule (L1CAM), both critical proteins contributing to EMT, were analyzed using immunohistochemistry in 49 melanoma patients with long follow-up (F/U, mean 110 months; range 12−263 months). We found a significant correlation between Breslow tumor thickness and Ezrin expression (p = 0.018). L1CAM expression in primary melanoma was significantly associated with HIF-1α expression (p < 0.0001) and sentinel lymph node metastasis (p = 0.011). Furthermore, low capillary count, reflecting hypoxic condition, was significantly associated with Ezrin expression (p = 0.047) and decreased tumor-specific survival (p = 0.035). In addition, patients with high Ezrin expression in their primary melanoma had a dramatic loss of life early in their F/U period (mean survival time 29 months; range 15−44 month). Our results highlight the relevance of Ezrin, L1CAM and HIF-1α as prognostic markers in melanoma patients. Additionally, we demonstrate that hypoxia in primary melanoma affects EMT and is at least partly responsible for early metastatic dissemination.

Molecular pathology in breast disease: diagnostic, prognostic, and therapeutic tools.

Molecular testing in breast cancer gained increasing attention and importance as specific molecular results can tailor not only oncological decisions on systemic adjuvant or neoadjuvant or in metastatic setting, but increasingly serve in diagnostic routine histopathological services to differentiate between morphologically overlapping or ambiguous histological pictures. Diagnostic tools involve in most cases a broad spectrum of immunohistochemical panels, followed by entity-specific in situ hybridization probes and in given cases NGS-based sequencing. Workflow of which methodology is applied and in which order depends on the specific entity resp. on the given differential diagnosis in question. Regarding prognostic/predictive molecular testing, the choice of assay and the workflow are based on clinical algorithms and on the evidence of targeted therapies following the molecular alterations. In this review paper, we aim to address the use of molecular technics in [1] the histological diagnostic setting (such as subtyping of invasive carcinomas/malignant spindle cell tumors and sarcomas and some B3 lesions) and [2] in the context of adjuvant or neoadjuvant or other clinical settings with special focus of targeted therapies.

Clinically Undiagnosed Diseases in Autopsies: Frequency and Risk Factors.

CONTEXT.­: Autopsies can reveal clinically undiagnosed diseases. However, the frequency of first diagnoses at autopsy and their association with clinically known risk factors are not well understood because of lack of systematic analyses addressing this topic. OBJECTIVE.­: To perform a large retrospective cohort analysis on the frequency of clinically undiagnosed postmortem findings and correlate these with patients' risk factors. DESIGN.­: Six hundred forty-eight consecutive and complete autopsies of adults (age >18 years), performed in the University Hospital Zurich, Switzerland, during a 3-year time period were retrospectively analyzed. Clinical diagnoses and postmortem findings were compared in order to identify clinically undiagnosed lesions and clarify their correlation with common risk factors. RESULTS.­: In 633 of 648 patients (98%), at least one clinically undiagnosed finding was identified at autopsy. The most common nonneoplastic entities were bronchopneumonia (198; 31%), coronary artery disease (155; 24%) and acute or subacute myocardial infarction (94; 15%), and the most common malignancies were prostate cancer in men (14; 2.2%), followed by kidney cancer (10; 1.5%), gastrointestinal stromal tumor (10; 1.5%), and lung carcinoma (9; 1.4%) in both genders. Clinically undiagnosed cardiac amyloidosis was demonstrated in 8% (52 of 648) of patients and was significantly associated with age, hypertension, chronic kidney disease, coronary artery disease, and hypertensive cardiomyopathy. CONCLUSIONS.­: Autopsy is a useful investigation for the detection of clinically undiagnosed entities. In our cohort, cardiac amyloidosis showed the highest number of underlying risk factors, but was clinically underdiagnosed. Our findings underline the necessity of improved clinical detection of cardiac amyloidosis, especially in light of emerging therapeutic options. Moreover, we characterize the most common entities prone to clinical underdiagnosis.

Pre-clinical phaeochromocytoma and paraganglioma models: Cell lines, animal models, and a human primary culture model.

While the establishment of human phaeochromocytoma and paraganglioma (PPGL) cell lines has proven to be particularly difficult over several decades of research, there are other reliable pre-clinical PPGL models currently available. This review provides a summary of these models, together with our recently established personalised drug screening platform using patient-derived PPGL primary cultures. Such currently available PPGL models include murine and rat PPGL cell lines, of which only one cell line (PC12) is publicly accessible through a cell repository, and PPGL animal models, of which the patient-derived xenograft models are promising but complex to establish. We have developed next-generation implementation of human PPGL primary cultures, enabling reliable and personalised drug screening and an individualised analysis of tumour drug responsivity based on the tumour's unique genetic, biochemical, immunohistochemical and clinical profile. Overall, reliable PPGL models, including patient-derived primary culture models, are essential to advance pre-clinical research in the field of PPGLs.

Postoperative Myocardial Infarction Due to Coronary Embolization of Valve Tissue After Surgical Mitral Valve Replacement.

We present the case of a 61-year-old man with known Morbus Barlow disease, who presented with postoperative myocardial infarction and cardiac arrest within 1 hour after minimally invasive mitral valve surgery owing to coronary artery occlusion by native mitral valve tissue.

Opposing effects of cannabidiol in patient-derived neuroendocrine tumor, pheochromocytoma/paraganglioma primary cultures.

CONTEXT: Treatment options for advanced neuroendocrine tumors (NETs), pheochromocytomas and paragangliomas (together PPGLs) are still limited. In recent years, anti-tumor effects of cannabinoids have been reported; however, there are only very limited data available in NETs or PPGLs. OBJECTIVE: Investigation of the effects of cannabidiol (CBD) on patient-derived human NET/PPGL primary cultures and on NET/PPGL cell lines. METHODS: We established primary cultures derived from 46 different patients with PPGLs (n = 35) or NETs (n = 11) who underwent tumor resection at two centers. Treatment of patient primary cultures with clinically relevant doses (5 µM) and slightly higher doses (10 µM) of CBD was performed. RESULTS: We found opposing effects of 5 µM CBD: significant anti-tumor effects in 5/35 (14%) and significant tumor-promoting effects in 6/35 (17%) of PPGL primary cultures. In terms of anti-tumor effects, cluster 2-related PPGLs showed significantly stronger responsivity to CBD compared to cluster 1-related PPGLs (p = 0.042). Of the cluster 2-related tumors, NF1 PPGLs showed strongest responsivity (4/5 PPGL primary cultures with a significant decrease in cell viability were NF1-mutated). We also found opposing effects of 10 µM CBD in PPGLs and NETs: significant anti-tumor effects in 9/33 of PPGL (27%) and 3/11 of NET (27%) primary cultures, significant tumor-promoting effects in 6/33 of PPGL (18%) and 2/11 of NET (18%) primary cultures. CONCLUSIONS: We suggest a potential novel treatment option for some NETs/PPGLs, but also provide evidence for caution when applying cannabinoids as supportive therapy for pain or appetite management to cancer patients, and possibly as health supplements.

Granulomas in bone marrow biopsies: clinicopathological significance and new perspectives.

Bone marrow granulomas in trephine biopsies are a rare and usually incidental finding. Possible causes include infectious (especially tuberculous and rarer non-tuberculous mycobacteria, but also many other bacterial, viral, fungal and parasitic agents) and non-infectious causes (especially medications, autoimmune disease, sarcoidosis, haematological and non-haematological malignancy). Necrotising granulomas are generally suggestive of an infectious aetiology (tuberculosis being the most common), whereas fibrin ring granulomas are associated with Q-fever and Epstein Barr Virus, although exceptions are possible. Every case suspicious for infectious aetiology should undergo further analysis like special staining (Ziehl-Neelsen for acid-fast rods) or molecular studies. The histomorphology should always be clinically correlated. In cases in which no infectious cause can be identified, untargeted metagenomics may represent a valid diagnostic tool that may become standard in the near future for bone marrow diagnostics. In this review, we have analysed the published data from 1956 up to today, and we report aspects of epidemiology, aetiology, diagnostic algorithms, differential diagnosis and the role of metagenomics in bone marrow biopsies with granulomas.

PD-L1 and HIF-2α Upregulation in Head and Neck Paragangliomas after Embolization.

Hypoxia activates pathways associated with tumor progression, metastatic spread, and alterations in the immune microenvironment leading to an immunosuppressive phenotype. In particular, the upregulation of PD-L1, a target for therapy with checkpoint inhibitors, is well-studied in several tumors. However, the relationship between hypoxia and PD-L1 regulation in pheochromocytomas and paragangliomas (PPGL), and especially in paragangliomas treated with embolization, is still largely unexplored. We investigated the expression of the hypoxia-marker HIF-2α and of PD-L1 in a PPGL-cohort with and without embolization as potential biomarkers that may predict the response to treatment with HIF-2α and checkpoint inhibitors. A total of 29 tumor samples from 25 patients who were operated at a single center were included and analyzed utilizing immunohistochemistry (IHC) for PD-L1 and HIF-2α. Embolization prior to surgery was performed in seven (24%) tumors. PD-L1 expression in tumor cells of head and neck paragangliomas (HNPGLs) receiving prior embolization (median PD-L1 positivity: 15%) was significantly higher as compared to PD-L1 expression in HNPGLs without prior embolization (median PD-L1 positivity: 0%) (p = 0.008). Consistently, significantly more HNPGLs with prior embolization were positive for HIF-2α (median nuclear HIF-2α positivity: 40%) as compared to HNPGLs without prior embolization (median nuclear HIF-2α positivity: 0%) (p = 0.016). Our results support the hypothesis that embolization with subsequent hypoxia leads to the upregulation of both PD-L1 and HIF-2α in HNPGLs, and could thus facilitate targeted treatment with HIF-2α and checkpoint inhibitors in the case of inoperable, locally advanced, or metastatic disease.

Metastatic Pheochromocytoma and Paraganglioma: Somatostatin Receptor 2 Expression, Genetics, and Therapeutic Responses.

CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors. OBJECTIVE: Exploration of the relationship between SSTR2 immunoreactivity and SDHB immunoreactivity, mutational status, and clinical behavior of PPGLs. Evaluation of SSTR-based therapies in metastatic PPGLs. METHODS: Retrospective analysis of a multicenter cohort of PPGLs at 6 specialized Endocrine Tumor Centers in Germany, The Netherlands, and Switzerland. Patients with PPGLs participating in the ENSAT registry were included. Clinical data were extracted from medical records, and immunohistochemistry (IHC) for SDHB and SSTR2 was performed in patients with available tumor tissue. Immunoreactivity of SSTR2 was investigated using Volante scores. The main outcome measure was the association of SSTR2 IHC positivity with genetic and clinical-pathological features of PPGLs. RESULTS: Of 202 patients with PPGLs, 50% were SSTR2 positive. SSTR2 positivity was significantly associated with SDHB- and SDHx-related PPGLs, with the strongest SSTR2 staining intensity in SDHB-related PPGLs (P = .01). Moreover, SSTR2 expression was significantly associated with metastatic disease independent of SDHB/SDHx mutation status (P < .001). In metastatic PPGLs, the disease control rate with first-line SSTR-based radionuclide therapy was 67% (n = 22, n = 11 SDHx), and with first-line "cold" somatostatin analogs 100% (n = 6, n = 3 SDHx). CONCLUSION: SSTR2 expression was independently associated with SDHB/SDHx mutations and metastatic disease. We confirm a high disease control rate of somatostatin receptor-based therapies in metastatic PPGLs.

Lymphoid aggregates in bone marrow: a diagnostic pitfall.

Lymphoid aggregates in bone marrow specimens are a relatively frequent finding that may pose a diagnostic challenge for a pathologist. The distinction between reactive and neoplastic aggregates has significant clinical relevance. Although many testing modalities such as immunohistochemistry, flow cytometry and molecular studies are currently available in clinical laboratories, the appropriate utilisation of these modalities and the awareness of their potential pitfalls are important. When a neoplastic process is ruled out, the significance of benign lymphoid aggregates in bone marrow is often unclear, as they may be associated with a broad spectrum of conditions including infections, autoimmune disorders, medications, or may even be idiopathic.This review focuses on evidence-based criteria that can aid in making the distinction between benign and malignant lymphoid aggregates and discusses the advantages, disadvantages and limits of ancillary tests used for this purpose. Finally, the most common aetiologies of benign lymphoid aggregates and their associations with specific diseases are discussed.

Long-Term Persisting SARS-CoV-2 RNA and Pathological Findings: Lessons Learnt From a Series of 35 COVID-19 Autopsies.

BACKGROUND: Long-term sequelae of coronavirus disease 2019 (COVID-19), including the interaction between persisting viral-RNA and specific tissue involvement, pose a challenging issue. In this study, we addressed the chronological correlation (after first clinical diagnosis and postmortem) between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and organ involvement. METHODS: The presence of postmortem SARS-CoV-2 RNA from 35 complete COVID-19 autopsies was correlated with the time interval between the first diagnosis of COVID-19 and death and with its relationship to morphologic findings. RESULTS: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be evident up to 40 days after the first diagnosis and can persist to 94 hours after death. Postmortem SARS-CoV-2 RNA was mostly positive in lungs (70%) and trachea (69%), but all investigated organs were positive with variable frequency. Late-stage tissue damage was evident up to 65 days after initial diagnosis in several organs. Positivity for SARS-CoV-2 RNA in pulmonary swabs correlated with diffuse alveolar damage (p = 0.0009). No correlation between positive swabs and other morphologic findings was present. Cerebral (p = 0.0003) and systemic hemorrhages (p = 0.009), cardiac thrombi (p = 0.04), and ischemic events (p = 0.03) were more frequent in the first wave, whereas bacterial pneumonia (p = 0.03) was more prevalent in the second wave. No differences in biometric data, clinical comorbidities, and other autopsy findings were found. CONCLUSIONS: Our data provide evidence not only of long-term postmortem persisting SARS-CoV-2 RNA but also of tissue damage several weeks after the first diagnosis of SARS-CoV-2 infection. Additional conditions, such as concomitant bacterial pulmonary superinfection, lung aspergillosis, thromboembolic phenomena, and hemorrhages can further worsen tissue damage.

SARS-CoV-2 leads to a small vessel endotheliitis in the heart.

BACKGROUND: SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. METHODS: Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. FINDINGS: COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. INTERPRETATION: ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. FUNDING: No external funding was necessary for this study.

Higher prevalence of pulmonary macrothrombi in SARS-CoV-2 than in influenza A: autopsy results from 'Spanish flu' 1918/1919 in Switzerland to Coronavirus disease 2019.

Similar to the influenza A pandemic in 1918/1919, the new Coronavirus disease 2019 (COVID-19) has spread globally. The causes of death in COVID-19 are frequently compared to a seasonal influenza outbreak. Complete COVID-19 autopsy studies were almost non-existent in the first months of the outbreak and are still rare with respect to the number of deaths. It has been recently reported that capillary microthrombi are significantly more prevalent in patients with COVID-19 than in patients with influenza A. To date, the contribution of macrothrombi, i.e. visible thrombi in pulmonary arteries, to the death of patients with influenza A in comparison to COVID-19 remains unaddressed. Here, we report autopsy findings in 411 patients who died from the 'Spanish' influenza A pandemic between May 1918 and April 1919 at the University Hospital Zurich, Switzerland. We compare these results with influenza A autopsies from 2009 to 2020, other influenza A autopsy series and all COVID-19 autopsies published to date. No descriptions of any macroscopic thromboembolic events were mentioned in influenza A autopsy reports. In 75 published COVID-19 autopsies, pulmonary artery thrombosis/embolism was reported in 36%. The direct comparison of macroscopic autopsy findings suggests a significantly greater degree of grossly visible pulmonary macrothrombi in patients with COVID-19 in comparison to influenza A autopsies even though most patients received empiric thromboprophylaxis. This is consistent with the concept of a SARS-related de novo coagulopathy with generalised in situ clot formation, which could explain the high incidence of pulmonary thrombosis/embolism with or without underlying deep vein thrombosis and in the absence of a history of venous thromboembolic events.