[Interdisciplinary diagnostics and therapy of malignant mesothelioma]. / Interdisziplinäre Diagnostik und Therapie von malignen Mesotheliomen.

The asbestos-related malignant mesothelioma (MM) is one of the common occupational cancers in Germany with approximately 1000 new cases per year. Provided that the appropriate diagnostic criteria are fulfilled, MM can be diagnosed with high specificity from both histological and cytological specimens. However, many MM are detected cyto-/histologically only at advanced stages. Clinical/radiological aspects complement each other and enable interdisciplinary assessment of tumor stage and individualized decisions on the best possible therapeutic options. Diagnostically, video-assisted thoracoscopy (VATS) has the highest priority. Therapy planning is based on the MM subtype, tumor spread and stage, and the patient's clinical condition. MM has generally a very unfavorable prognosis. Accordingly, the standard therapy aims at a macroscopic radical tumor resection in terms of cytoreduction within the framework of a suitable multimodal therapy concept (chemotherapy, radiotherapy, psychooncology). The aim of palliative measures should be primarily symptom control. Overall, interdisciplinary diagnosis and therapy of MM is crucial for the best possible care of MM patients.

Recommendations for immunocytochemistry in lung cancer typing: An update on a resource-efficient approach with large-scale comparative Bayesian analysis.

OBJECTIVES: The majority of lung cancer cases are of advanced stage and diagnosis is usually made using minimally invasive small biopsies and cytological specimens. The WHO 2015 classification recommends limiting immunocytochemistry (ICC) to lung cancer typing and molecular testing drives for personalised therapies. An algorithm using Bayes' theorem could be useful for defining antibody profiles. This study aims to assess the impact of different antibody profiles for cytological samples on the accuracy of lung cancer typing with a large-scale Bayesian analysis. METHODS: A retrospective examination of 3419 consecutive smears and/or cytospins diagnosed over 2011-2016 found 1960 primary lung cancer tumours: 972 adenocarcinomas (ADC), 256 squamous carcinomas (SQC), 268 neuroendocrine tumours (NET), and 464 non-small cell cancer-not otherwise specified (NSCC-NOS). The a priori and a posteriori probabilities, before and after ICC using antibodies singly or in combination, were calculated for different lung cancer types. RESULTS: TTF-1 or CK7 alone improved the a posteriori probabilities of correct cytological typing for ADC to 86.5% and 95.8%, respectively. For SQC, using p40 (∆Np63) or CK5/6 together with CK5/14 led to comparable results (78.3% and 90.3%). With synaptophysin or CD56 alone, improvements in a posteriori probabilities to 87.5 and 90.3% for the correct recognition of NET could be achieved. CONCLUSIONS: Based on morphological and clinical data, the use of two antibodies appears sufficient for reliable detection of the different lung cancer types. This applies to diagnoses that were finalised following ICC both on a clinical or cytological basis and on a histological basis.

Cytological Diagnostic Procedures in Malignant Mesothelioma.

Malignant mesotheliomas (MM) are rare tumors with high mortality rates, whose incidence varies regionally and nationally, and the diagnosis is difficult. Histology-based diagnosis is considered the gold standard despite its low sensitivity of 57-84%. However, recent advances in cytological analysis offer promise for diagnostic advancements. In this study, we reappraised the current cytological guidelines for the MM diagnosis and concluded on their practicability and reliability. The study included 5731 consecutive specimens of pleural effusions from 4552 patients (3026 males of the average age of 67.5 years and 1526 females of the average age of 65.4 years) between December 2017 and January 2000. Out of these patients, 444 (9.8%) were diagnosed with MM. The effusions were examined by immunocytochemistry using routine Giemsa staining. Additionally, hyaluronic acid (HA) was assessed. Cytological findings confirmed 223 out of the 444 MM. The additional 88 cases with negative cytology were corroborated by supplemental assessments of HA above 30 mg/L. Cytological evaluation accomplished the sensitivity of 0.50, specificity of 0.99, and a positive predictive value (PPV) of 0.97 for MM diagnosis. The use of HA determination raised the sensitivity to 0.70 without affecting the specificity or PPV. We conclude that cytological evaluation of effusions aided by the assessment of HA demonstrates the diagnostic sensitivity and specificity for MM no less than the hitherto standard histological evaluation. The cytology-based MM diagnosis may thus be routinely considered when MM is suspected and may offer confirmatory advantages in difficult or doubtful diagnostic cases.

Frozen Section or Intraoperative Cytology: Are We Ready for a Paradigm Shift in Thoracic Surgery?

Intraoperative frozen sections of specimens taken during thoracic surgery are widely seen as the gold standard. However, the accuracy of intraoperative cytology remains contentious. The study aims to estimate the value of intraoperative cytology by analyzing feasibility, accuracy, time requirements, and possible limitations when compared to standard frozen sections. To this end, we examined a total of 532 intraoperatively harvested specimens out of the 518 resected thoracic tumors from 360 patients between August 2016 and August 2017. The specimens were subject to intraoperative rapid cytology that was later counter compared to the final histology results. The mean time between the intraoperative harvesting and arrival at the laboratory was 2.23 min, and it took a further 3.5 min until the results were communicated to the surgeon. Cytologically, 218 cases (41%) were classified as malignant, 291 (55%) as benign, and 23 (4%) remained unclear. In 55 malignant cases, we observed additional benign formations. The final histological examination performed later yielded 267 malignant and 265 benign cases. Therefore, the sensitivity and specificity of rapid intraoperative cytology were 82% and 99%, respectively, with a negative/positive predictive value of 86%/99%. We conclude that the intraoperative rapid cytology is a fast, accurate, sensitive, and specific procedure for intraoperative decision making and is a distinctly helpful alternative or adjunct for the thoracic surgeon, providing that one is aware of the plausible limitations of this technique.

Development and reliability of the histological THROMBEX-classification rule for thrombotic emboli of acute ischemic stroke patients.

BACKGROUND: Thrombus histology has become a potential diagnostic tool for the etiology assessment of patients with ischemic stroke caused by embolic proximal vessel occlusion. We validated a classification rule that differentiates between cardiac and arteriosclerotic emboli in individual stroke patients. We aim to describe in detail the development of this classification rule and disclose its reliability. METHODS: The classification rule is based on the hypothesis that cardiac emboli arise out of separation thrombi and arteriosclerotic emboli result from agglutinative thrombi. 125 emboli recovered by thrombectomy from stroke patients and 11 thrombi serving as references for cardiac (n = 5) and arteriosclerotic emboli (n = 6) were Hematoxylin and eosin, Elastica-van Gieson and CD61 stained and rated independently by two histopathologists blinded to the presumed etiology by several pre-defined criteria. Intra- and interobserver reliabilities of all criteria were determined. Out of the different criteria, three criteria with the most satisfactory reliability values were selected to compose the classification rule that was finally adjusted to the reference thrombi. Reliabilities of the classification rule were calculated by using the emboli of stroke patients. RESULTS: The classification rule reached intraobserver reliabilities for the two raters of 92.9% and 68.2%, respectively. Interobserver reliability was 69.9%. CONCLUSIONS: A new classification rule for emboli obtained from thrombectomy was established. Within the limitations of histological investigations, it is reliable and able to distinguish between cardioembolic and arteriosclerotic emboli.

Identification of molecular signatures associated with early relapse after complete resection of lung adenocarcinomas.

The only potentially curative treatment for lung adenocarcinoma patients remains complete resection of early-stage tumors. However, many patients develop recurrence and die of their disease despite curative surgery. Underlying mechanisms leading to establishment of systemic disease after complete resection are mostly unknown. We therefore aimed at identifying molecular signatures of resected lung adenocarcinomas associated with the risk of an early relapse. The study comprised 89 patients with totally resected stage IA-IIIA lung adenocarcinomas. Patients suffering from an early relapse within two years after surgery were compared to patients without a relapse in two years. Patients were clinically and molecular pathologically characterized. Tumor tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34, by Nanostring nCounter PanCancer Immune Profiling Panel as well as a comprehensive methylome profiling using the Infinium MethylationEPIC BeadChip. We detected differential DNA methylation patterns as well as significantly differentially expressed genes associated with an early relapse after complete resection. Especially, CD1A was identified as a potential biomarker, whose reduced expression is associated with an early relapse. These findings might help to develop biomarkers improving risk assessment and patient selection for adjuvant therapy as well as establish novel targeted therapeutic strategies.

First approach to distinguish between cardiac and arteriosclerotic emboli of individual stroke patients applying the histological THROMBEX-classification rule.

Endovascular treatment of strokes caused by large vessel occlusion enables the histopathological investigation of the retrieved embolus, possibly providing a novel opportunity to contribute to the diagnostic workup of etiology and to define secondary prevention measures in strokes with uncertain genesis. We aimed to develop a classification rule based on pathophysiological considerations and adjustment to reference thrombi for distinction between cardiac and arteriosclerotic emboli and to validate this classification rule on a patient cohort. From 125 patients with stroke due to large vessel occlusion and thrombectomy, 82 patients with known etiology (55 cardioembolic and 27 arterioembolic strokes) were included. The corresponding emboli were histologically evaluated by two raters blinded to the etiology of stroke by means of a novel classification rule. Presumed etiology and classification results were compared. Agreement concerning cardiac emboli was 72.2% (95% CI: 58.4-83.5) for rater I and 78.2% (95% CI: 65.0-88.2) for rater II. Agreement concerning arteriosclerotic emboli was 70.4% (95% CI: 49.8-86.3) for rater I and 74.1% (95% CI: 53.7-88.9) for rater II. Overall agreement reached 71.6% (95% CI: 60.5-81.1) for rater I and 76.8% (95% CI: 66.2-85.4) for rater II. Within the limits of generally restricted accuracy of histological evaluations, the classification rule differentiates between cardiac and arteriosclerotic emboli of acute ischemic stroke patients. Further improvement is needed to provide valuable complementary data for stroke etiology workup.

[Cryptogenic organizing pneumonia versus secondary organizing pneumonia]. / Kryptogen organisierende Pneumonie versus sekundäre organisierende Pneumonie.

Organizing pneumonia (OP) describes a histological pattern of acute or subacute lung damage. Clinically, patients present with cough, fever, and dyspnea. A distinction is made between idiopathic or cryptogenic organizing pneumonia (COP) and secondary organizing pneumonia (OP). In COP, neither clinical/radiological nor histological causes can be determined. It is classified as an interstitial idiopathic pneumonia (IIP) according to the criteria of the American Thoracic Society (ATS) and the European Respiratory Society (ERS). Secondary organizing pneumonia has a known triggering mechanism, such as infectious agents, certain medications, or concomitant symptoms of other primary pulmonary diseases and diseases of other organ systems. Common to both forms is the histological picture of intra-alveolar mesenchymal buds. These are myofibroblast proliferates that branch out along the alveolar spaces. They are usually accompanied by a moderate interstitial and alveolar, chronic, and macrophage-rich inflammatory cell infiltrate. The most important differential diagnosis is common interstitial pneumonia (UIP). It also shows fibroblast proliferates, which are, however, located in the interstitium. The correct classification of an IIP as a COP by means of clinical, radiological, and histological findings is essential, since the COP, in contrast to the UIP, responds very well to corticosteroids and therefore has an excellent prognosis compared to the UIP. The course of secondary organizing pneumonia depends on the respective underlying disease. Here it is important for the pathologist to correctly identify potential accompanying histological characteristics in order to be able to provide clues to a possible cause of OP.

[IgG4-associated lung disease with granulomatous lesions : Coexistence of two entities or the spectrum of one disease?] / IgG4-assoziierte Lungenerkrankung mit granulomatösen Läsionen : Koexistenz zweier Entitäten oder das Spektrum einer Erkrankung?

A 54-year-old patient with a history of pulmonary tuberculosis and occupational exposure to dust in early adulthood presented with symptoms of coughing with sputum, weight loss, occasional night sweats, and thoracic pain. Non-necrotizing granulomatosis in lung and lymph-node biopsies indicated sarcoidosis. Combined immunosuppressive therapy did not lead to an improvement. An atypical lung resectate with fibroinflammatory changes and obliterative endothelialitis may finally lead to the diagnosis of IgG4-associated lung disease with a bronchovascular pattern of involvement. The question discussed here is whether this is a coexistence of IgG4-associated lung disease with sarcoidosis or the spectrum of one disease.

[Mycobacterium szulgai as positive control helps to detect contaminations in the detection of Mycobacterium tuberculosis-complex in formalin fixed, paraffin embedded tissues by 16SrDNA PCR]. / Mycobacterium szulgai als Posititivkontrolle zur Detektion von Kontaminationen beim Nachweis des Mycobacterium tuberculosis-Komplexes durch eine spezifische 16S-rDNA-PCR an FFPE-Material.

The detection of Mycobacterium tuberculosis complex DNA by PCR using formalin-fixed paraffin-embedded material has become an integral part of molecular-pathological diagnostics. We describe an approach that enables the detection of contamination by using Mycobacterium szulgai as a positive control, contributing to the reduction of false-positive results.

[Overview: granulomatous diseases of the lung]. / Übersicht: Granulomatöse Erkrankungen der Lunge.

The spectrum of pulmonary granulomatoses is wide and includes infectious and noninfectious entities, each with very different therapeutic consequences. The first step of histological examination discriminates between necrotizing and non-necrotizing granulomatosis. After this, an infectious cause of the granulomatosis has to be excluded by special histological stains and molecular-pathologic methods, if necessary. Diagnosis also includes clinical, radiological, and microbiological findings. The process of pathological examination should be standardized as described.

[Histopathology of pulmonary tuberculosis]. / Histopathologie der Lungentuberkulose.

Although typical histological findings of tuberculosis are well known, the diagnosis of nonmicrobiologically proven tuberculosis with the instruments available to pathology is challenging. Indeed, necrotizing epithelioid cell granulomatosis is typical for tuberculosis, but it is also seen in a number of different infectious or noninfectious lung diseases. The tools of microscopy and molecular pathology are suitable for confirming the diagnosis or paving the way to a differential diagnosis, but molecular pathology applied to formalin-fixated and paraffin-embedded material is limited. This should be openly communicated to the referring clinician. After interdisciplinary re-evaluation of the findings, an alternative solution to confirm the diagnosis must therefore be found if the additional examinations are negative.

Quantification of liver iron overload disease with laser ablation inductively coupled plasma mass spectrometry.

BACKGROUND: Hereditary hemochromatosis is the most frequent, identified, genetic disorder in Caucasians affecting about 1 in 1000 people of Northern European ancestry, where the associated genetic defect (homozygosity for the p.Cys282Tyr polymorphism in the HFE gene) has a prevalence of approximately 1:200. The disorder is characterized by excess iron stores in the body. Due to the incomplete disease penetrance of disease-associated genotype, genetic testing and accurate quantification of hepatic iron content by histological grading of stainable iron, quantitative chemical determination of iron, or imaging procedures are important in the evaluation and staging of hereditary hemochromatosis. METHODS: We here established novel laser ablation inductively coupled plasma mass spectrometry protocols for hepatic metal bio-imaging for diagnosis of iron overload. RESULTS: We demonstrate that these protocols are a significant asset in the diagnosis of iron overload allowing iron measurements and simultaneous determination of various other metals and metalloids with high sensitivity, spatial resolution, and quantification ability. CONCLUSIONS: The simultaneous measurement of various metals and metalloids offers unique opportunities for deeper understanding of metal imbalances. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a highly powerful and sensitive technique for the analysis of a variety of solid samples with high spatial resolution. We conclude that this method is an important add-on to routine diagnosis of iron overload and associated hepatic metal dysbalances resulting thereof.

Development of a new clot formation protocol for standardized in vitro investigations of sonothrombolysis.

BACKGROUND: Agreement about the most suitable clot formation protocol for sonothrombolysis investigations is lacking. Lysis rates vary strongly owing to different test conditions and, thus, cannot be compared. We aim to establish a simple but physiologically grounded protocol for in vitro coagulation to enable standardized sonothrombolysis investigations. METHOD: Clots were generated from platelet-rich plasma (PRP) obtained by centrifugation (10 min, 180 × g) of human venous blood (VB). PRP was mixed with the boundary layer formed between the supernatant and the erythrocyte layer. To achieve clots with different platelet counts, PRP was gradually substituted with platelet-free plasma (PFP), harvested from the supernatant of VB after centrifugation (10 min, 2570 × g). Clot types were examined for histological appearance, hydrodynamic resistance under physiological flows, and lysis rate measured by weight loss after a 2-h treatment with recombinant tissue plasminogen activator (rt-PA) (60 kU/ml). Lysis rates of the most suitable clot were measured after a 1-h treatment with rt-PA (60 kU/ml), and combined treatment with rt-PA and 2-MHz transcranial color-coded sonography (TCCS) (0.179 W/cm(2)) or 2-MHz transcranial Doppler (TCD) (0.457 W/cm(2)). RESULTS: With increased platelet count, the hydrodynamic resistance of the artificial clots increased, their histological appearance became more physiological, and lysis rates decreased. The most suitable clots consisted of 1.5-ml PRP, 2.0-ml PFP, and 0.5-ml boundary layer. Their lysis rates were 36.7 ± 7.8% (rt-PA), 40.8 ± 8.6% (rt-PA+TCCS), and 40.4 ± 8.3% (rt-PA+TCD). COMPARISON WITH EXISTING METHODS: These systemic investigations were conducted for the first time. CONCLUSION: This protocol should be used for standardized sonothrombolysis investigations.

Blood pressure response to angiotensin II is enhanced in obese Zucker rats and is attributed to an aldosterone-dependent mechanism.

BACKGROUND AND PURPOSE: Plasma aldosterone levels correlate positively with obesity, suggesting a link between the hypertension associated with obesity and increased mineralocorticoid levels. We tested the hypothesis that aldosterone is involved in the BP response to angiotensin II (AngII) in obese rats. EXPERIMENTAL APPROACH: Lean (LZR) and obese (OZR) Zucker rats were treated with AngII (9 µg·h(-1) ; 4 weeks), and BP and plasma AngII and aldosterone were determined. KEY RESULTS: Chronic AngII increased the BP in OZR markedly more so than in LZR. Plasma AngII levels in LZR and OZR were similar after AngII treatment. The AngII stimulated a rise in plasma aldosterone that was sixfold more in OZR than in LZR. The thickness of the zona glomerulosa of the adrenal glands was selectively increased by AngII in OZR. Adrenal mRNA levels of CYP11B2 aldosterone synthase and the AT(1B) receptor were selectively increased in AngII-treated OZR. The BP response to chronic AngII stimulation was diminished in OZR after adrenalectomy when plasma aldosterone was absent. Acute bolus injections of AngII did not increase the BP response or aldosterone release in OZR. CONCLUSIONS AND IMPLICATIONS: The AngII-induced BP response is enhanced in obesity and this is associated with a specific increase in circulating aldosterone. Due to the AngII-induced growth of the zona glomerulosa in OZR, the AT(1B) receptors and aldosterone synthase may be selectively enhanced in obesity under concomitant AngII stimulation, increasing the adrenal synthesis of aldosterone. Our results confirm functionally that aldosterone plays a major role in obesity-related hypertension.

Introduction of a new model for time-continuous and non-contact investigations of in-vitro thrombolysis under physiological flow conditions.

BACKGROUND: Thrombolysis is a dynamic and time-dependent process influenced by the haemodynamic conditions. Currently there is no model that allows for time-continuous, non-contact measurements under physiological flow conditions. The aim of this work was to introduce such a model. METHODS: The model is based on a computer-controlled pump providing variable constant or pulsatile flows in a tube system filled with blood substitute. Clots can be fixed in a custom-built clot carrier within the tube system. The pressure decline at the clot carrier is measured as a novel way to measure lysis of the clot. With different experiments the hydrodynamic properties and reliability of the model were analyzed. Finally, the lysis rate of clots generated from human platelet rich plasma (PRP) was measured during a one hour combined application of diagnostic ultrasound (2 MHz, 0.179 W/cm2) and a thrombolytic agent (rt-PA) as it is commonly used for clinical sonothrombolysis treatments. RESULTS: All hydrodynamic parameters can be adjusted and measured with high accuracy. First experiments with sonothrombolysis demonstrated the feasibility of the model despite low lysis rates. CONCLUSIONS: The model allows to adjust accurately all hydrodynamic parameters affecting thrombolysis under physiological flow conditions and for non-contact, time-continuous measurements. Low lysis rates of first sonothrombolysis experiments are primarily attributable to the high stability of the used PRP-clots.

The platelet-rich plasma clot: a standardized in-vitro clot formation protocol for investigations of sonothrombolysis under physiological flows.

No agreement exists about which protocol for in-vitro clot formation is suitable for sonothrombolysis investigations. Lysis rates vary considerably because of different clotting processes and cannot be compared. We aim to establish a new protocol for in-vitro coagulation to permit standardized sonothrombolysis investigations. The proposed procedure is based upon clots prepared from platelet-rich plasma (PRP). This clot material (group A) was compared with the two most commonly used procedures, namely, recalcification of citrate-anticoagulated whole venous blood (group B) and spontaneous clotting of nonanticoagulated venous blood (group C). Histological examinations were performed and clot stability was tested under physiological flow conditions in vitro for all groups (each n = 10). Lysis rates measured by mass loss were compared using buffered plasma and recombinant tissue plasminogen activator (60 kU/ml), or buffered plasma alone. PRP clots displayed a high degree of similarity to emboli specimens in histological examinations and remained stable under pulsatile flow conditions. B and C clots were mechanically unstable and did not resist physiological flow and pressure. Measuring the lysis rate by weighing seems to be inaccurate, with lowest variability in PRP clots. PRP clots appeared more resistant to lysis. PRP clots should be used for standardized sonothrombolysis investigations.

On the significance of Surfactant Protein-A within the human lungs.

Surfactant Protein-A (SP-A) is the most prominent among four proteins in the pulmonary surfactant-system. SP-A is expressed by alveolar epithelial cells type II as well as by a portion of non small cell lung carcinomas (NSCLC).The expression of SP-A is complexly regulated on the transcriptional and the chromosomal level. SP-A is a major player in the pulmonary cytokine-network and moreover has been described to act in the pulmonary host defense.By the use of cell culture or animal models the functional properties have been repeatedly shown in many aspects, often bearing surprising properties which strongly indicate the physiological importance of SP-A. To date SP-A is recognized as a molecule essential for pulmonary development, structure and function. An upcoming number of reports deals with the role of SP-A for pulmonary pathology. This article gives an overview about the state of knowledge on SP-A focused in applications for human pulmonary disorders and points out the importance for pathology-orientated research approaches using immunohistochemistry or in situ hybridization as promising methods to further elucidate the role of this molecule in adult lung diseases.