Spatiotemporal analysis of tumour-infiltrating immune cells in biliary carcinogenesis.

BACKGROUND: Intraductal papillary neoplasms (IPN) and biliary epithelial neoplasia (BilIN) are well-defined precursor lesions of biliary tract carcinoma (BTC). The aim of this study was to provide a comprehensive characterisation of the inflammatory microenvironment in BTC precursor lesions. METHODS: Immunohistochemistry was employed to assess tumour-infiltrating immune cells in tissue samples from patients, for whom precursor lesions were identified alongside invasive BTC. The spatiotemporal evolution of the immune microenvironment during IPN-associated carcinogenesis was comprehensively analysed using triplet sample sets of non-neoplastic epithelium, precursor lesion and invasive BTC. Immune-cell dynamics during IPN- and BilIN-associated carcinogenesis were subsequently compared. RESULTS: Stromal CD3+ (P = 0.002), CD4+ (P = 0.007) and CD8+ (P < 0.001) T cells, CD20+ B cells (P = 0.008), MUM1+ plasma cells (P = 0.012) and CD163+ M2-like macrophages (P = 0.008) significantly decreased in IPN compared to non-tumorous biliary epithelium. Upon transition from IPN to invasive BTC, stromal CD68+ (P = 0.001) and CD163+ (P < 0.001) macrophages significantly increased. In contrast, BilIN-driven carcinogenesis was characterised by significant reduction of intraepithelial CD8+ T-lymphocytic infiltration from non-tumorous epithelium via BilIN (P = 0.008) to BTC (P = 0.004). CONCLUSION: IPN and BilIN are immunologically distinct entities that undergo different immune-cell variations during biliary carcinogenesis. Intraepithelial CD8+ T-lymphocytic infiltration of biliary tissue decreased already at the IPN-precursor stage, whereas BilIN-associated carcinogenesis showed a slowly progressing reduction towards invasive carcinoma.

Conventional and semi-automatic histopathological analysis of tumor cell content for multigene sequencing of lung adenocarcinoma.

BACKGROUND: Targeted genetic profiling of tissue samples is paramount to detect druggable genetic aberrations in patients with non-squamous non-small cell lung cancer (NSCLC). Accurate upfront estimation of tumor cell content (TCC) is a crucial pre-analytical step for reliable testing and to avoid false-negative results. As of now, TCC is usually estimated on hematoxylin-eosin (H&E) stained tissue sections by a pathologist, a methodology that may be prone to substantial intra- and interobserver variability. Here we the investigate suitability of digital pathology for TCC estimation in a clinical setting by evaluating the concordance between semi-automatic and conventional TCC quantification. METHODS: TCC was analyzed in 120 H&E and thyroid transcription factor 1 (TTF-1) stained high-resolution images by 19 participants with different levels of pathological expertise as well as by applying two semi-automatic digital pathology image analysis tools (HALO and QuPath). RESULTS: Agreement of TCC estimations [intra-class correlation coefficients (ICC)] between the two software tools (H&E: 0.87; TTF-1: 0.93) was higher compared to that between conventional observers (0.48; 0.47). Digital TCC estimations were in good agreement with the average of human TCC estimations (0.78; 0.96). Conventional TCC estimators tended to overestimate TCC, especially in H&E stainings, in tumors with solid patterns and in tumors with an actual TCC close to 50%. CONCLUSIONS: Our results determine factors that influence TCC estimation. Computer-assisted analysis can improve the accuracy of TCC estimates prior to molecular diagnostic workflows. In addition, we provide a free web application to support self-training and quality improvement initiatives at other institutions.

The Pathology of Severe COVID-19-Related Lung Damage.

BACKGROUND: The histomorphological changes of lung damage in severe coronavirus disease 2019 (COVID-19) have not yet been adequately characterized. In this article, we describe the sequence of pathological changes in COVID-19 and discuss the implications for approaches to treatment. METHODS: Standardized autopsies were performed on thirteen patients who had died of COVID-19. The findings were analyzed together with clinical data from the patients' medical records. RESULTS: Most (77%) of the deceased patients were men. Their median age at death was 78 years (range, 41-90). Most of them had major pre-existing chronic diseases, most commonly arterial hypertension. The autopsies revealed characteristic COVID-19-induced pathological changes in the lungs, which were regarded as the cause of death in most patients. The main histological finding was sequential alveolar damage, apparently due in large measure to focal capillary microthrombus formation. Alveolar damage leads to the death of the patient either directly or by the induction of pulmonary parenchymal fibrosis. Diffuse lung damage was seen exclusively in invasively ventilated patients. CONCLUSION: Autopsies are crucial for the systematic assessment of new diseases such as COVID-19: they provide a basis for further investigations of disease mechanisms and for the devising of potentially effective modes of treatment. The autopsy findings suggest that focal damage of the microvascular pulmonary circulation is a main mechanism of lethal lung disease due to the SARS-CoV-2 virus. It may also be a cause of persistent lung damage in patients who recover from severe COVID-19.

Primary pulmonary myxoid sarcoma with an unusual gene fusion between exon 7 of EWSR1 and exon 5 of CREB1.

Primary pulmonary mesenchymal tumors are rare, yet they compromise a variety of entities. A novel low-grade malignant neoplasm coined primary pulmonary myxoid sarcoma (PPMS) has been introduced in the WHO classification of lung tumors. Molecular analysis in PPMS revealed recurrent gene fusions between EWSR1 and CREB1, a member of the cAMP response element binding protein (CREB) family. However, only 23 PPMS have been reported in the literature reflecting their exceedingly low incidence. Here, we describe the case of a 41-year-old female patient with a lung tumor obstructing the right main bronchus. Histologically, the tumor was composed of spindle-shaped and epithelioid cells exhibiting a reticular growth pattern within a prominent myxoid matrix. Solid areas were also observed. Molecular analysis by next-generation sequencing identified a fusion transcript with an unusual gene fusion involving exon 7 of EWSR1 and exon 5 of CREB1. Together, the diagnosis PPMS was established.

In Silico Modeling of Immunotherapy and Stroma-Targeting Therapies in Human Colorectal Cancer.

Despite the fact that the local immunological microenvironment shapes the prognosis of colorectal cancer, immunotherapy has shown no benefit for the vast majority of colorectal cancer patients. A better understanding of the complex immunological interplay within the microenvironment is required. In this study, we utilized wet lab migration experiments and quantitative histological data of human colorectal cancer tissue samples (n = 20) including tumor cells, lymphocytes, stroma, and necrosis to generate a multiagent spatial model. The resulting data accurately reflected a wide range of situations of successful and failed immune surveillance. Validation of simulated tissue outcomes on an independent set of human colorectal cancer specimens (n = 37) revealed the model recapitulated the spatial layout typically found in human tumors. Stroma slowed down tumor growth in a lymphocyte-deprived environment but promoted immune escape in a lymphocyte-enriched environment. A subgroup of tumors with less stroma and high numbers of immune cells showed high rates of tumor control. These findings were validated using data from colorectal cancer patients (n = 261). Low-density stroma and high lymphocyte levels showed increased overall survival (hazard ratio 0.322, P = 0.0219) as compared with high stroma and high lymphocyte levels. To guide immunotherapy in colorectal cancer, simulation of immunotherapy in preestablished tumors showed that a complex landscape with optimal stroma permeabilization and immune cell activation is able to markedly increase therapy response in silico These results can help guide the rational design of complex therapeutic interventions, which target the colorectal cancer microenvironment. Cancer Res; 77(22); 6442-52. ©2017 AACR.

Genetic heterogeneity in synchronous colorectal cancers impacts genotyping approaches and therapeutic strategies.

Synchronous colorectal carcinomas (sCRC) are clinically challenging neoplasms. Although the epidemiological characteristics are quite well established, their biological basis is still poorly understood. Hence, we performed comprehensive molecular profiling of 23 sCRC cases comprising 50 synchronous primary tumors, 5 metastases, and corresponding normal tissue by targeted deep sequencing of 30 CRC-related genes, microsatellite analysis and analysis for methylated MLH1. We identified a striking inter- and intratumoral genetic heterogeneity of sCRC. Twenty (87%) cases showed genetic heterogeneity leaving only three cases with tumors that had an identical genetic make-up. Intertumoral heterogeneity was frequently observed for clinically actionable genes, including KRAS. Specifically, 44% of the cases harbored tumors of which at least one was KRAS mutated and the other KRAS wildtype. Moreover, 48% of the cases had at least double, sometimes even triple or quadruple mutations in KRAS, APC, TP53, PIK3CA, and TGFBR2, most of them being subclonal events. Lastly, we detected four cases (17%) with microsatellite instable (MSI) tumors with one case harboring one MSI- and a distinct microsatellite stable carcinoma. Our data demonstrate a striking genetic heterogeneity not only between different sCRC of a single case but also within a single tumor. These results contribute to the biological understanding of sCRC and directly impact genotyping strategies and oncological decision making. Testing one tumor or a single metastasis may not suffice in the sCRC setting as clinically relevant and tumor-specific genetic information may be left undetected compromising optimal oncological therapy. © 2015 Wiley Periodicals, Inc.

Differential diagnostic value of CD5 and CD117 expression in thoracic tumors: a large scale study of 1465 non-small cell lung cancer cases.

BACKGROUND: Thoracic pathologists are frequently faced with tissue specimens from intrathoracic/mediastinal tumors. Specifically the differentiation between thymic and pulmonary squamous cell carcinomas (SqCC) can be challenging. In order to clarify the differential diagnostic value of CD5 and CD117 in this setting, we performed a large scale expression study of both markers in 1465 non-small cell lung cancer (NSCLC) cases. METHODS: Tissue microarrays of formalin-fixed paraffin-embedded resection specimens of 1465 NSCLC were stained with antibodies against CD117 and CD5. Positivity of both markers was correlated with clinicopathological variables. RESULTS: CD117 was positive in 145 out of 1457 evaluable cases (9.9 %) and CD5 was positive in 133 out of 1427 evaluable cases (9.3 %). 28 cases (1.9 %) showed coexpression of CD117 and CD5. Among the 145 cases that were positive for CD117, 97 (66.8 %) were adenocarcinomas (ADC), 34 (23.4 %) were SqCC, 5 (3.4 %) were adenosquamous carcinomas (ADSqCC), 8 (5.5 %) were large cell carcinomas (LC), and one (0.6 %) was a pleomorphic carcinoma (PC). In the CD5 positive group consisting of 133 cases, 123 (92.4 %) were ADC, 0 (0 %) were SqCC, 4 (3.0 %) were ADSqCC, 3 (2.2 %) LC and 3 (2.2 %) were PC. None of the 586 SqCC showed expression of CD5. No association of CD117- or CD5 positivity to patients' age, pathological stages or to T-, N-, or M- categories was observed. CONCLUSIONS: A substantial subset of NSCLC exhibit positivity of CD117 and CD5. Since CD5 expression was not observed in pulmonary SqCC, but is expressed in the majority of thymic squamous cell carcinomas, the application of this immunomarker is a valuable tool in the differential diagnosis of thoracic neoplasms.