Large cell neuroendocrine lung carcinoma: consensus statement from The British Thoracic Oncology Group and the Association of Pulmonary Pathologists.

Over the past 10 years, lung cancer clinical and translational research has been characterised by exponential progress, exemplified by the introduction of molecularly targeted therapies, immunotherapy and chemo-immunotherapy combinations to stage III and IV non-small cell lung cancer. Along with squamous and small cell lung cancers, large cell neuroendocrine carcinoma (LCNEC) now represents an area of unmet need, particularly hampered by the lack of an encompassing pathological definition that can facilitate real-world and clinical trial progress. The steps we have proposed in this article represent an iterative and rational path forward towards clinical breakthroughs that can be modelled on success in other lung cancer pathologies.

Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting.

Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.

Crowdsourcing for translational research: analysis of biomarker expression using cancer microarrays.

BACKGROUND: Academic pathology suffers from an acute and growing lack of workforce resource. This especially impacts on translational elements of clinical trials, which can require detailed analysis of thousands of tissue samples. We tested whether crowdsourcing - enlisting help from the public - is a sufficiently accurate method to score such samples. METHODS: We developed a novel online interface to train and test lay participants on cancer detection and immunohistochemistry scoring in tissue microarrays. Lay participants initially performed cancer detection on lung cancer images stained for CD8, and we measured how extending a basic tutorial by annotated example images and feedback-based training affected cancer detection accuracy. We then applied this tutorial to additional cancer types and immunohistochemistry markers - bladder/ki67, lung/EGFR, and oesophageal/CD8 - to establish accuracy compared with experts. Using this optimised tutorial, we then tested lay participants' accuracy on immunohistochemistry scoring of lung/EGFR and bladder/p53 samples. RESULTS: We observed that for cancer detection, annotated example images and feedback-based training both improved accuracy compared with a basic tutorial only. Using this optimised tutorial, we demonstrate highly accurate (>0.90 area under curve) detection of cancer in samples stained with nuclear, cytoplasmic and membrane cell markers. We also observed high Spearman correlations between lay participants and experts for immunohistochemistry scoring (0.91 (0.78, 0.96) and 0.97 (0.91, 0.99) for lung/EGFR and bladder/p53 samples, respectively). CONCLUSIONS: These results establish crowdsourcing as a promising method to screen large data sets for biomarkers in cancer pathology research across a range of cancers and immunohistochemical stains.

Evidence of mycobacterial disease in COPD patients with lung volume reduction surgery; the importance of histological assessment of specimens: a cohort study.

BACKGROUND: Patients with COPD are at risk of non-tuberculous mycobacterial infection (NTM). This study examined the histology of lung tissue from COPD patients following lung volume reduction with particular focus on evidence of mycobacterial infection. METHODS: Retrospective histological study of 142 consecutive lung volume reduction surgical specimens (126 separate patients) at Royal Brompton Hospital between 2000 - 2013, with prospectively collected preoperative data on exacerbation rate, lung function and body mass index. RESULTS: 92% of patients had at least one other histological diagnosis in addition to emphysema. 10% of specimens had histological evidence of mycobacterial infection, one with co-existent aspergilloma. Mycobacteria were only identified in those patients with granulomas that were necrotising. These patients had higher exacerbation rates, lower TLCO and FEV1. CONCLUSION: A proportion of severe COPD patients will have evidence of mycobacterial infection despite lack of clinical and radiological suspicion. This may have implications for long-term management of these patients.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

The evolving role of image-guided biopsy and specimen fixation-update from an oncology setting.

Image-guided biopsy is well established in clinical practice, however a recent shift towards "personalized medicine" and genomic research, particularly in the oncology setting, has resulted in a greater demand for tissue, not only at preliminary diagnosis but at multiple time points in the patient's journey. Research into the molecular pathobiology underpinning cancer development and progression continues to identify diagnostic, predictive and prognostic biomarkers that help determine and guide treatment both at the outset, and as patient's progress or recur. This extensive tissue analysis however, necessitates larger tissue cores and a greater number of biopsies with correct fixation of the specimens obtained. We discuss the impact that this shift towards genomic medicine has taken on both radiologists and histopathologists and stress the importance of correct specimen preparation as well as biopsy technique to maximize diagnostic yield, by reviewing different methods of specimen fixation that are now required in clinical practice dependent on the clinical question posed.

Somatic cancer genetics in the UK: real-world data from phase I of the Cancer Research UK Stratified Medicine Programme.

INTRODUCTION: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. METHODS: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. RESULTS: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. CONCLUSION: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx.

Comparability of slack water and Lagrangian flow respirometry methods for community metabolic measurements.

Coral reef calcification is predicted to decline as a result of ocean acidification and other anthropogenic stressors. The majority of studies predicting declines based on in situ relationships between environmental parameters and net community calcification rate have been location-specific, preventing accurate predictions for coral reefs globally. In this study, net community calcification and production were measured on a coral reef flat at One Tree Island, Great Barrier Reef, using Lagrangian flow respirometry and slack water methods. Net community calcification, daytime net photosynthesis and nighttime respiration were higher under the flow respirometry method, likely due to increased water flow relative to the slack water method. The two methods also varied in the degrees to which they were influenced by potential measurement uncertainties. The difference in the results from these two commonly used methods implies that some of the location-specific differences in coral reef community metabolism may be due to differences in measurement methods.

Anthropogenic changes to seawater buffer capacity combined with natural reef metabolism induce extreme future coral reef CO2 conditions.

Ocean acidification, via an anthropogenic increase in seawater carbon dioxide (CO2 ), is potentially a major threat to coral reefs and other marine ecosystems. However, our understanding of how natural short-term diurnal CO2 variability in coral reefs influences longer term anthropogenic ocean acidification remains unclear. Here, we combine observed natural carbonate chemistry variability with future carbonate chemistry predictions for a coral reef flat in the Great Barrier Reef based on the RCP8.5 CO2 emissions scenario. Rather than observing a linear increase in reef flat partial pressure of CO2 (pCO2 ) in concert with rising atmospheric concentrations, the inclusion of in situ diurnal variability results in a highly nonlinear threefold amplification of the pCO2 signal by the end of the century. This significant nonlinear amplification of diurnal pCO2 variability occurs as a result of combining natural diurnal biological CO2 metabolism with long-term decreases in seawater buffer capacity, which occurs via increasing anthropogenic CO2 absorption by the ocean. Under the same benthic community composition, the amplification in the variability in pCO2 is likely to lead to exposure to mean maximum daily pCO2 levels of ca. 2100 µatm, with corrosive conditions with respect to aragonite by end-century at our study site. Minimum pCO2 levels will become lower relative to the mean offshore value (ca. threefold increase in the difference between offshore and minimum reef flat pCO2 ) by end-century, leading to a further increase in the pCO2 range that organisms are exposed to. The biological consequences of short-term exposure to these extreme CO2 conditions, coupled with elevated long-term mean CO2 conditions are currently unknown and future laboratory experiments will need to incorporate natural variability to test this. The amplification of pCO2 that we describe here is not unique to our study location, but will occur in all shallow coastal environments where high biological productivity drives large natural variability in carbonate chemistry.

Stratified medicine for cancer therapy.

As knowledge of the biological processes underlying malignant transformation becomes increasingly sophisticated, apparently similar diseases can be redefined according to the critical disrupted biological pathways and networks. The key genetic changes in most cancers can be mapped to one of a relatively few pathways, making it possible to classify tumours by their abnormal pathways and to identify potentially treatable--'druggable'--targets within these. The aim of the stratified approach to cancer therapy is to improve the effectiveness, tolerability and affordability of novel therapeutic agents.