FACILITATE: A real-world, multicenter, prospective study investigating the utility of a rapid, fully automated real-time PCR assay versus local reference methods for detecting epidermal growth factor receptor variants in NSCLC.

Accurate testing for epidermal growth factor receptor (EGFR) variants is essential for informing treatment decisions in non-small cell lung cancer (NSCLC). Automated diagnostic workflows may allow more streamlined initiation of targeted treatments, where appropriate, while comprehensive variant analysis is ongoing. FACILITATE, a real-world, prospective, multicenter, European study, evaluated performance and analytical turnaround time of the Idylla™ EGFR Mutation Test compared with local reference methods. Sixteen sites obtained formalin-fixed paraffin-embedded biopsy samples with ≥ 10% neoplastic cells from patients with NSCLC. Consecutive 5 µm sections from patient samples were tested for clinically relevant NSCLC-associated EGFR variants using the Idylla™ EGFR Mutation Test and local reference methods; performance (concordance) and analytical turnaround time were compared. Between January 2019 and November 2020, 1,474 parallel analyses were conducted. Overall percentage agreement was 97.7% [n = 1,418; 95% confidence interval (CI): 96.8-98.3], positive agreement, 87.4% (n = 182; 95% CI: 81.8-91.4) and negative agreement, 99.2% (n = 1,236; 95% CI: 98.5-99.6). There were 38 (2.6%) discordant cases. Ninety percent of results were returned with an analytical turnaround time of within 1 week using the Idylla™ EGFR Mutation Test versus ∼22 days using reference methods. The Idylla™ EGFR Mutation Test performed well versus local methods and had shorter analytical turnaround time. The Idylla™ EGFR Mutation Test can thus support application of personalized medicine in NSCLC.

Immune cell infiltration pattern in non-small cell lung cancer PDX models is a model immanent feature and correlates with a distinct molecular and phenotypic make-up.

BACKGROUND: The field of cancer immunology is rapidly moving towards innovative therapeutic strategies, resulting in the need for robust and predictive preclinical platforms reflecting the immunological response to cancer. Well characterized preclinical models are essential for the development of predictive biomarkers in the oncology as well as the immune-oncology space. In the current study, gold standard preclinical models are being refined and combined with novel image analysis tools to meet those requirements. METHODS: A panel of 14 non-small cell lung cancer patient-derived xenograft models (NSCLC PDX) was propagated in humanized NOD/Shi-scid/IL-2Rnull mice. The models were comprehensively characterized for relevant phenotypic and molecular features, including flow cytometry, immunohistochemistry, histology, whole exome sequencing and cytokine secretion. RESULTS: Models reflecting hot (>5% tumor-infiltrating lymphocytes/TILs) as opposed to cold tumors (<5% TILs) significantly differed regarding their cytokine profiles, molecular genetic aberrations, stroma content, and programmed cell death ligand-1 status. Treatment experiments including anti cytotoxic T-lymphocyte-associated protein 4, anti-programmed cell death 1 or the combination thereof across all 14 models in the single mouse trial format showed distinctive tumor growth response and spatial immune cell patterns as monitored by computerized analysis of digitized whole-slide images. Image analysis provided for the first time qualitative evaluation of the extent to which PDX models retain the histological features from their original human donors. CONCLUSIONS: Deep phenotyping of PDX models in a humanized setting by combinations of computational pathology, immunohistochemistry, flow cytometry and proteomics enables the exhaustive analysis of innovative preclinical models and paves the way towards the development of translational biomarkers for immuno-oncology drugs.

Status quo of ALK testing in lung cancer: results of an EQA scheme based on in-situ hybridization, immunohistochemistry, and RNA/DNA sequencing.

With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ' kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples.

Next generation sequencing of lung adenocarcinoma subtypes with intestinal differentiation reveals distinct molecular signatures associated with histomorphology and therapeutic options.

OBJECTIVES: We aim to provide a better understanding of the molecular landscape of primary lung adenocarcinomas with intestinal differentiation. MATERIAL AND METHODS: Five invasive mucinous adenocarcinomas (IMA) and seven pulmonary enteric adenocarcinomas (PEAD) were included in this study. Furthermore, we analyzed six pulmonary colloid adenocarcinomas (CAD), including one primary tumor, one metastasis, and two sample pairs consisting of the primary colloid lung tumor and a matching metastasis and an acinar component, respectively. All samples were characterized using immunohistochemistry (TTF-1, CK7, CK20, CDX2, Ki-67, ALK and PD-L1) and a next generation sequencing panel covering 404 cancer-related genes (FoundationOne® gene panel). RESULTS AND CONCLUSION: While Ki-67 expression was comparably low in IMA (range: 8-15%) and in primary CAD (range: 5-8%), we observed considerably higher proliferation rates in the non-colloid tumor compartment (16%) and metastases (72%) from CAD, as well as in the PEAD-group (36-71%). The overall tumor mutational burden was lowest in IMA (2.5 mutations per megabase), intermediate in CAD (5.8 mutations per megabase) and highest in PEAD (16.8 mutations per megabase). KRAS mutations were frequent in all three tumor subtypes, but TP53 mutations were mostly limited to PEAD. While chromosomal alterations were rare in IMA, we discovered MYC amplifications in three of four CAD. Comparing primary and metastatic CAD, we observed the acquisition of multiple mutations and chromosomal alterations. PEAD had a variety of chromosomal alterations, including two cases with RICTOR amplification. PD-L1 expression (20%, 50% and 80% of tumor cells) was limited to three PEAD samples, only. In conclusion, we provide a detailed insight into the molecular alterations across and within the different subtypes of pulmonary adenocarcinomas with intestinal differentiation. From a clinical perspective, we provide data on potential treatment strategies for patients with PEAD, including immunotherapy.

Sample pooling obscures diversity patterns in intertidal ciliate community composition and structure.

The aim of this study was to assess the effect of sample pooling on the portrayal of ciliate community structure and composition in intertidal sediment samples. Molecular ciliate community profiles were obtained from nine biological replicates distributed in three discrete sampling plots and from samples that were pooled prior to RNA extraction using terminal restriction fragment polymorphism (T-RFLP) analyses of SSU rRNA. Comparing the individual replicates of one sampling plot with each other, we found a differential variability among the individual biological replicates. T-RFLP profiles of pooled samples displayed a significantly different community composition compared with the cumulative individual biological replicate samples. We conclude that sample pooling obscures diversity patterns in ciliate and possibly also other microbial eukaryote studies. However, differences between pooled samples and replicates were less pronounced when community structure was analyzed. We found that the most abundant T-RFLP peaks were generally shared between biological replicates and pooled samples. Assuming that the most abundant taxa in an ecosystem under study are also the ones driving ecosystem processes, sample pooling may still be effective for the analyses of ecological key players.

Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions.

Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small-subunit rDNA) of an intertidal protistan model community, using the Roche GS-FLX and the most recent GS-FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2039 unique tags (from 3879 V4 GS-FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS-FLX). Pyrosequencing error rates were higher for V4 fragments compared with the V9 domain and for the GS-FLX Titanium compared with the GS-FLX system. Furthermore, this experiment revealed that error rates are taxon-specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data-processing pipelines.

Spatio-temporal variations in protistan communities along an O/HS gradient in the anoxic Framvaren Fjord (Norway).

Despite its relevance for ecology and biodiversity, the stability of spatial microeukaryote diversity patterns in time has received only little attention using gene-based strategies, and there is little knowledge about the relation of spatial vs. temporal variation. We addressed this subject by investigating seasonal fluctuations in protistan communities in three ecologically distinct marine habitats. We analyzed 3360 eukaryote small subunit rRNA gene sequences collected along an O(2)/H(2)S gradient in a Norwegian fjord in order to reveal shifts in protistan community composition and structure in three different seasons. In all nine clone libraries, ciliates and stramenopiles accounted for the largest proportion. Yet, as expected, at the phylotype level, the protistan communities from distinct habitats differed significantly, with the number of shared phylotypes between two habitats being as low as 18%. This confirmed previous notions that environmental factors along the stratification gradient shape biodiversity patterns. Surprisingly, the intrahabitat community composition and structure varied at a comparable order of magnitude over time, with only 18-28% phylotypes shared within the same habitat. Our study demonstrates that the consideration of local fluctuations in microeukaryote diversity over time offers additional information for diversity surveys and can significantly contribute to the revelation of spatial protistan community patterns.

Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation.

In order to study fungal diversity in oxygen minimum zones of the Arabian Sea, we analyzed 1440 cloned small subunit rRNA gene (18S rRNA gene) sequences obtained from environmental samples using three different PCR primer sets. Restriction fragment length polymorphism (RFLP) analyses yielded 549 distinct RFLP patterns, 268 of which could be assigned to fungi (Dikarya and zygomycetes) after sequence analyses. The remaining 281 RFLP patterns represented a variety of nonfungal taxa, even when using putatively fungal-specific primers. A substantial number of fungal sequences were closely related to environmental sequences from a range of other anoxic marine habitats, but distantly related to known sequences of described fungi. Community similarity analyses suggested distinctively different structures of fungal communities from normoxic sites, seasonally anoxic sites and permanently anoxic sites, suggesting different adaptation strategies of fungal communities to prevailing oxygen conditions. Additionally, we obtained 26 fungal cultures from the study sites, most of which were closely related (>97% sequence similarity) to well-described Dikarya. This indicates that standard cultivation mainly produces more of what is already known. However, two of these cultures were highly divergent to known sequences and seem to represent novel fungal groups on high taxonomic levels. Interestingly, none of the cultured isolates is identical to any of the environmental sequences obtained. Our study demonstrates the importance of a multiple-primer approach combined with cultivation to obtain deeper insights into the true fungal diversity in environmental samples and to enable adequate intersample comparisons of fungal communities.

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities.

BACKGROUND: Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets. RESULTS: The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs) far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i) BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA) gene sequences for taxonomic assignments of tags; (ii) a clustering of tags at k differences (Levenshtein distance) with a newly developed algorithm enabling very fast OTU clustering for large tag sequence data sets; and (iii) a novel parsing procedure to combine the data from individual analyses. CONCLUSION: Our data highlight the magnitude of the under-sampled 'protistan gap' in the eukaryotic tree of life. This study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.

Microbial eukaryotes in the hypersaline anoxic L'Atalante deep-sea basin.

The frontiers of eukaryote life in nature are still unidentified. In this study, we analysed protistan communities in the hypersaline (up to 365 g l(-1) NaCl) anoxic L'Atalante deep-sea basin located in the eastern Mediterranean Sea. Targeting 18S ribosomal RNA retrieved from the basin's lower halocline (3501 m depth) we detected 279 protistan sequences that grouped into 42 unique phylotypes (99% sequence similarity). Statistical analyses revealed that these phylotypes account only for a proportion of the protists inhabiting this harsh environment with as much as 50% missed by this survey. Most phylotypes were affiliated with ciliates (45%), dinoflagellates (21%), choanoflagelates (10%) and uncultured marine alveolates (6%). Sequences from other taxonomic groups like stramenopiles, Polycystinea, Acantharea and Euglenozoa, all of which are typically found in non-hypersaline deep-sea systems, are either missing or very rare in our cDNA clone library. Although many DHAB sequences fell within previously identified environmental clades, a large number branched relatively deeply. Phylotype richness, community membership and community structure differ significantly from a deep seawater reference community (3499 m depth). Also, the protistan community in the L'Atalante basin is distinctively different from any previously described hypersaline community. In conclusion, we hypothesize that extreme environments may exert a high selection pressure possibly resulting in the evolution of an exceptional and distinctive assemblage of protists. The deep hypersaline anoxic basins in the Mediterranean Sea provide an ideal platform to test for this hypothesis and are promising targets for the discovery of undescribed protists with unknown physiological capabilities.

Protistan community patterns within the brine and halocline of deep hypersaline anoxic basins in the eastern Mediterranean Sea.

Environmental factors restrict the distribution of microbial eukaryotes but the exact boundaries for eukaryotic life are not known. Here, we examine protistan communities at the extremes of salinity and osmotic pressure, and report rich assemblages inhabiting Bannock and Discovery, two deep-sea superhaline anoxic basins in the Mediterranean. Using a rRNA-based approach, we detected 1,538 protistan rRNA gene sequences from water samples with total salinity ranging from 39 to 280 g/Kg, and obtained evidence that this DNA was endogenous to the extreme habitat sampled. Statistical analyses indicate that the discovered phylotypes represent only a fraction of species actually inhabiting both the brine and the brine-seawater interface, with as much as 82% of the actual richness missed by our survey. Jaccard indices (e.g., for a comparison of community membership) suggest that the brine/interface protistan communities are unique to Bannock and Discovery basins, and share little (0.8-2.8%) in species composition with overlying waters with typical marine salinity and oxygen tension. The protistan communities from the basins' brine and brine/seawater interface appear to be particularly enriched with dinoflagellates, ciliates and other alveolates, as well as fungi, and are conspicuously poor in stramenopiles. The uniqueness and diversity of brine and brine-interface protistan communities make them promising targets for protistan discovery.

A molecular approach to identify active microbes in environmental eukaryote clone libraries.

A rapid method for the simultaneous extraction of RNA and DNA from eukaryote plankton samples was developed in order to discriminate between indigenous active cells and signals from inactive or even dead organisms. The method was tested using samples from below the chemocline of an anoxic Danish fjord. The simple protocol yielded RNA and DNA of a purity suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR) and PCR, respectively. We constructed an rRNA-derived and an rDNA-derived clone library to assess the composition of the microeukaryote assemblage under study and to identify physiologically active constituents of the community. We retrieved nearly 600 protistan target clones, which grouped into 84 different phylotypes (98% sequence similarity). Of these phylotypes, 27% occurred in both libraries, 25% exclusively in the rRNA library, and 48% exclusively in the rDNA library. Both libraries revealed good correspondence of the general community composition in terms of higher taxonomic ranks. They were dominated by anaerobic ciliates and heterotrophic stramenopile flagellates thriving below the fjord's chemocline. The high abundance of these bacterivore organisms points out their role as a major trophic link in anoxic marine systems. A comparison of the two libraries identified phototrophic dinoflagellates, "uncultured marine alveolates group I," and different parasites, which were exclusively detected with the rDNA-derived library, as nonindigenous members of the anoxic microeukaryote community under study.

Diversity estimates of microeukaryotes below the chemocline of the anoxic Mariager Fjord, Denmark.

Microbial communities of extreme environments have often been assumed to have low species richness. We analysed 18S rRNA gene signatures in a sample collected below the chemocline of the anoxic Mariager Fjord in Denmark, and from these data we computed novel parametric and standard nonparametric estimates of protistan phylotype richness. Our results indicate unexpectedly high richness in this environment: at the 99.5% phylotype definition, our most conservative estimate was 568 phylotypes (+/-114, standard error). Phylogenetic analyses revealed that the sequences collected cover the majority of described lineages in the eukaryotic domain. Out of 384 sequences analysed, 307 were identified as protistan targets, none of which was identical to known sequences. However, based on what is known about species that are phylogenetically related to the Mariager sequences, most of the latter seem to belong to strictly or facultative anaerobe organisms. We also found signatures that together with other environmental 18S rRNA gene sequences represent environmental clades of possibly high taxonomic levels (class to kingdom level). One of these clades, consisting exclusively of sequences from anoxic sampling sites, branches at the base of the eukaryotic evolutionary tree among the earliest eukaryotic lineages. Assuming eukaryotic evolution under oxygen-depleted conditions, these sequences may represent immediate descendants of early eukaryotic ancestors.

Microeukaryote community patterns along an O2/H2S gradient in a supersulfidic anoxic fjord (Framvaren, Norway).

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O(2)/H(2)S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O(2)/H(2)S gradient.

Cellular identity of an 18S rRNA gene sequence clade within the class Kinetoplastea: the novel genus Actuariola gen. nov. (Neobodonida) with description of the type species Actuariola framvarensis sp. nov.

Environmental molecular surveys of microbial diversity have uncovered a vast number of novel taxonomic units in the eukaryotic tree of life that are exclusively known by their small-subunit (SSU) rRNA gene signatures. In this study, we reveal the cellular and taxonomic identity of a novel eukaryote SSU rRNA gene sequence clade within the Kinetoplastea. Kinetoplastea are ubiquitously distributed flagellated protists of high ecological and medical importance. We isolated an organism from the oxic-anoxic interface of the anoxic Framvaren Fjord (Norway), which branches within an unidentified kinetoplastean sequence clade. Ultrastructural studies revealed a typical cellular organization that characterized the flagellated isolate as a member of the order Neobodonida Vickerman 2004, which contains five genera. The isolate differed in several distinctive characters from Dimastigella, Cruzella, Rhynchobodo and Rhynchomonas. The arrangement of the microtubular rod that supports the apical cytostome and the cytopharynx differed from the diagnosis of the fifth described genus (Neobodo Vickerman 2004) within the order Neobodonida. On the basis of both molecular and microscopical data, a novel genus within the order Neobodonida, Actuariola gen. nov., is proposed. Here, we characterize its type species, Actuariola framvarensis sp. nov., and provide an in situ tool to access the organism in nature and study its ecology.