Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

Difficulties in diagnostics of lung tumours in biopsies: an interpathologist concordance study evaluating the international diagnostic guidelines.

AIMS: Accurate and reliable diagnosis is essential for lung cancer treatment. The study aim was to investigate interpathologist diagnostic concordance for pulmonary tumours according to WHO diagnostic criteria. METHODS: Fifty-two unselected lung and bronchial biopsies were diagnosed by a thoracic pathologist based on a broad spectrum of immunohistochemical (IHC) stainings, molecular data and clinical/radiological information. Slides stained with H&E, thyroid transcription factor-1 (TTF-1) clone SPT24 and p40 were scanned and provided digitally to 20 pathologists unaware of reference diagnoses. The pathologists independently diagnosed the cases and stated if further diagnostic markers were deemed necessary. RESULTS: In 31 (60%) of the cases, ≥80% of the pathologists agreed with each other and with the reference diagnosis. Lower agreement was seen in non-small cell neuroendocrine tumours and in squamous cell carcinoma with diffuse TTF-1 positivity. Agreement with the reference diagnosis ranged from 26 to 45 (50%-87%) for the individual pathologists. The pathologists requested additional IHC staining in 15-44 (29%-85%) of the 52 cases. In nearly half (17 of 36) of the malignant cases, one or more pathologist advocated for a different final diagnosis than the reference without need of additional IHC markers, potentially leading to different clinical treatment. CONCLUSIONS: Interpathologist diagnostic agreement is moderate for small unselected bronchial and lung biopsies based on a minimal panel of markers. Neuroendocrine morphology is sometimes missed and TTF-1 clone SPT24 should be interpreted with caution. Our results suggest an intensified education need for thoracic pathologists and a more generous use of diagnostic IHC markers.

Induction of alveolar and bronchiolar phenotypes in human lung organoids.

Patient-derived organoids have revolutionized biomedical research and therapies by "transferring the patient into the Petri dish". In vitro access to human lung organoids representing distal lung tissue, i.e. alveolar organoids, would facilitate research pertaining to a wide range of medical conditions and might open for a future approach to individualized treatment.We propose a protocol to derive a single human lung biopsy towards both alveolar and bronchiolar organoids. By modulating Wnt pathway, we obtained a differential gene expression of the main markers for both subtypes, such as a higher expression of surfactant protein C in alveolar organoids or a higher expression of mucine 5AC in bronchiolar organoids. Although the specific cell enrichment was not complete, the differentiation was observed as early as passage 1 based on morphology, and confirmed by QPCR and histology at passage 2. These results are consistent with a functional specification of lung epithelium towards both alveoli- and bronchi-enriched organoids from first passages.

Low expression of SerpinB2 is associated with reduced survival in lung adenocarcinomas.

Lung cancer is a leading cause of cancer deaths worldwide and new biomarkers are of utmost importance. Studies have indicated that the anti-plasminogen activators SerpinB2 and Neuroserpin, and the adhesion molecule L1CAM, have a coordinated impact on development of metastasis. Here, we examined whether expression of these markers was associated with clinico-pathologic characteristics and prognosis in resected non-small cell lung cancer (NSCLC). Surgical specimens from 438 NSCLC patients treated at Haukeland University Hospital, Bergen, Norway (1993-2010) were included (median age 68 years; 213 adenocarcinomas, 135 squamous cell carcinomas, 90 others). Representative tumor sections were stained for SerpinB2, Neuroserpin, and L1CAM. Low expression of SerpinB2 was associated with reduced lung cancer specific survival (LCSS) in adenocarcinomas (p = 0.017), also in stage I (p = 0.031). In contrast, high SerpinB2 was associated with reduced LCSS in stage I squamous cell carcinomas (p = 0.022). Although Neuroserpin and L1CAM showed some associations with clinico-pathologic phenotype, there were no associations with survival. In multivariate survival analysis of adenocarcinomas, low SerpinB2 demonstrated independent prognostic value (HR 1.8, p = 0.008). In summary, low expression of SerpinB2 in lung adenocarcinomas was an independent prognostic factor. In contrast to findings by others, we found no impact of L1CAM on survival.

Microvascular proliferation is associated with aggressive tumour features and reduced survival in lung adenocarcinoma.

Despite new treatment options in lung cancer, there is still a need for better biomarkers to assist in therapy decisions. Angiogenesis has been associated with tumour growth and dissemination, and the vascular proliferation index (VPI) is a valuable prognostic marker in other tumours. Nestin, a marker of immature endothelium, was previously applied in combination with Ki67 for proliferating endothelium as a novel marker (Nestin-Ki67) of ongoing angiogenesis. Here, the prevalence and prognostic impact of vascular proliferation on lung cancer-specific survival (LCSS) in lung adenocarcinomas was studied. Selected tumour slides from a cohort of 210 patients treated surgically for adenocarcinoma at Haukeland University Hospital (Norway) from 1993 to 2010 were stained for Nestin-Ki67. VPI, the ratio between the density of proliferating vessels and the overall microvessel density were used, and the cut-off value was set at 4.4% (upper quartile). High VPI was associated with the presence of blood vessel invasion (p = 0.007) and tumour necrosis (p = 0.007). Further, high VPI was significantly associated with reduced LCSS (p = 0.020). By multivariate analysis, VPI remained an independent prognostic factor for reduced LCSS (HR 1.7; 95% CI 1.04-2.68; p = 0.033) when adjusted for other prognostic clinico-pathological features. In conclusion, microvessel proliferation assessed using the VPI was associated with aggressive tumour features such as blood vessel invasion and tumour necrosis and, independently, decreased LCSS. This marker should be further explored in separate cohorts, and in trials of anti-angiogenesis therapy.

Vascular invasion is an adverse prognostic factor in resected non-small-cell lung cancer.

Lung cancer is a leading cause of death, and there is a need for better prognostic factors in treatment decisions. Vascular invasion is a known negative prognosticator, but it is not clear how to evaluate this feature. Here, we studied the prevalence and prognostic impact of blood and lymphatic vascular invasion (BVI, LVI), tumour grade, necrosis, inflammation and pleural invasion on cancer-specific survival (LCSS) and time to recurrence (TTR) in non-small-cell lung cancer (NSCLC). A total of 438 patients surgically treated for NSCLC (1993-2010) were examined, including 213 adenocarcinomas (AC), 135 squamous cell carcinomas (SCC) and 90 other NSCLC. BVI and LVI were found in 25% and 21% of the cases, with reduced LCSS and TTR for both markers in AC and SCC (p < 0.005 for all). BVI and LVI remained independent prognostic factors for LCSS and TTR in separate multivariate models for AC and SCC. Combined BVI/LVI (7%) showed significantly reduced LCSS and TTR (p < 0.001), also by multivariate analysis. Our results support that BVI and LVI are valuable for prognostic staging. Vascular invasion identifies a group of patients at higher risk of recurrence and lung cancer-related death, and this could influence stratification of patients for treatment and follow-up.

Post-cholecystectomy symptoms were caused by persistence of a functional gastrointestinal disorder.

AIM: To classify gallstone disease as a basis for assessment of post-cholecystectomy symptoms. METHODS: One hundred and fifty three patients with a clinical and ultrasonographic diagnosis of gallstones filled out a structured questionnaire on abdominal pain symptoms and functional gastrointestinal disorder (FGID) before and at six months after cholecystectomy. Symptom frequency groups (SFG) were categorized according to frequency of pain attacks. According to certain pain characteristics in gallstone patients, a gallstone symptom score was accorded on a scale from one to ten. A visual analogue scale was used to quantify pain. Operative specimens were examined for size and magnitude of stone contents as well as presence of bacteria. Follow-up took place after six months with either a consultation or via a mailed questionnaire. Results were compared with those obtained pre-operatively to describe and analyze symptomatic outcome. RESULTS: SFG groups were categorized as severe (24.2%), moderate (38.6%), and mild (22.2%) attack frequency, and a chronic pain condition (15%). Pain was cured or improved in about 90% of patients and two-thirds of patients obtained complete symptom relief. Patients with the most frequent pain episodes were less likely to obtain symptom relief. FGID was present in 88% of patients pre-operatively and in 57% post-operatively (P = 0.244). Those that became asymptomatic or improved with regard to pain also had most relief from FGID (P = 0.001). No pre-operative FGID meant almost complete cure. CONCLUSION: Only one third of patients with FGID experienced postoperative relief, indicating that FGID was a dominant cause of post-cholecystectomy symptoms.

Effective Hemostasis in Severe Mesenteric Vein Laceration with Tachosil(®), using a Low- or Non-thrombogenic Patch to Prevent Tachosil(®)-induced Thrombosis.

Fibrinogen- and thrombin-coated collagen fleece (FTCC) facilitates surgical hemostasis, and is of particular value during resection of parenchymatous organs. Since thrombosis may ensue if the preparation is unintentionally applied intravascularly, it has not been recommended for treating lacerations of large veins, and no previous reports describe its use in vein repair. Our observations in two patients suggest, however, that FTCC might be indicated for hemostasis in vein injury where vascular suture is difficult or not possible, provided a low- or non-thrombogenic patch is interposed to prevent FTCC-induced vein thrombosis. Our two patients had severe lacerations of the proximal superior mesenteric vein (SMV) not amenable to conventional vein repair. Rapid hemostasis was obtained without suturing using Tachosil(®), an FTCC preparation, covered with omentum. In the first patient hemostasis was obtained at the expense of vein thrombosis, apparently due to contact between the coagulant-containing side of Tachosil(®) and the inside of the vein wall. In our second patient we therefore put a small patch of parietal peritoneum on the section of the Tachosil(®) targeted to cover the vein tear to avoid direct contact between Tachosil(®) and the vein lumen. Ultrasound examination 3 days postoperatively, and autopsy 11.5 months later showed that the vein was widely patent with no stenosis or thrombus. Our observations in these two patients were that an FTCC-omentum pack alone secured rapid hemostasis in severe SMV laceration, and when a peritoneal patch was interposed between FTCC and a lacerated SMV, FTCC-induced vein thrombosis did not occur.

[Decline in hospital autopsies]. / Farre sykehusobduksjoner--lang svartid.

BACKGROUND: According to the new autopsy regulation from April 2004 the next to kin must be informed of an autopsy and the right to deny consent. The present study aims at documenting the impact of this regulation on the number of autopsies performed and to determine whether regular reminders and changed autopsy routines could reduce the time needed to complete autopsy reports. MATERIAL AND METHODS: The following information was gathered; the number of autopsies before and after implementation of the regulation, the number of next to kin who had been informed and the number that had denied consent. The consequence of monthly reminders to the pathologists for the time needed to complete the autopsy report was examined for the years 2000 to 2004. The autopsy routine was altered for six weeks in 2005; the doctors performed autopsies for two successive weeks instead of one. The time to complete the report in this period was compared with that of the same period the year before. The chi-squared test, the Mann-Whitney test and the Kruskal-Wallis test H test were used. RESULTS: After the autopsy regulation was introduced the number of autopsies/year decreased from 432 (39%) to 332 (31%) of all who had died in the hospital (p < 0.001). For 211 (20%) of the deaths, the next to kin had not been informed. The number who denied consent increased from 258 (23%) to 373 (35%). Monthly reminders reduced the median time for completing the autopsy report from 58 to 38 days (p < 0.001). Altering the autopsy routine reduced the median time needed to complete the report from 46 days the year before to 14 days after the changed routine (p < 0.001). INTERPRETATION: Better information to next to kin will probably increase the number of autopsies. The time needed for completing the autopsy report can be reduced by simple means.