Methods in cancer research: Assessing therapy response of spheroid cultures by life cell imaging using a cost-effective live-dead staining protocol.

Spheroid cultures of cancer cell lines or primary cells represent a more clinically relevant model for predicting therapy response compared to two-dimensional cell culture. However, current live-dead staining protocols used for treatment response in spheroid cultures are often expensive, toxic to the cells, or limited in their ability to monitor therapy response over an extended period due to reduced stability. In our study, we have developed a cost-effective method utilizing calcein-AM and Helix NP™ Blue for live-dead staining, enabling the monitoring of therapy response of spheroid cultures for up to 10 days. Additionally, we used ICY BioImage Analysis and Z-stacks projection to calculate viability, which is a more accurate method for assessing treatment response compared to traditional methods on spheroid size. Using the example of glioblastoma cell lines and primary glioblastoma cells, we show that spheroid cultures typically exhibit a green outer layer of viable cells, a turquoise mantle of hypoxic quiescent cells, and a blue core of necrotic cells when visualized using confocal microscopy. Upon treatment of spheroids with the alkylating agent temozolomide, we observed a reduction in the viability of glioblastoma cells after an incubation period of 7 days. This method can also be adapted for monitoring therapy response in different cancer systems, offering a versatile and cost-effective approach for assessing therapy efficacy in three-dimensional culture models.

Somatic RIT1 delins in arteriovenous malformations hyperactivate RAS-MAPK signaling amenable to MEK inhibition.

Arteriovenous malformations (AVM) are benign vascular anomalies prone to pain, bleeding, and progressive growth. AVM are mainly caused by mosaic pathogenic variants of the RAS-MAPK pathway. However, a causative variant is not identified in all patients. Using ultra-deep sequencing, we identified novel somatic RIT1 delins variants in lesional tissue of three AVM patients. RIT1 encodes a RAS-like protein that can modulate RAS-MAPK signaling. We expressed RIT1 variants in HEK293T cells, which led to a strong increase in ERK1/2 phosphorylation. Endothelial-specific mosaic overexpression of RIT1 delins in zebrafish embryos induced AVM formation, highlighting their functional importance in vascular development. Both ERK1/2 hyperactivation in vitro and AVM formation in vivo could be suppressed by pharmacological MEK inhibition. Treatment with the MEK inhibitor trametinib led to a significant decrease in bleeding episodes and AVM size in one patient. Our findings implicate RIT1 in AVM formation and provide a rationale for clinical trials with targeted treatments.

Characterization of Polarimetric Properties in Various Brain Tumor Types Using Wide-Field Imaging Mueller Polarimetry.

Neuro-oncological surgery is the primary brain cancer treatment, yet it faces challenges with gliomas due to their invasiveness and the need to preserve neurological function. Hence, radical resection is often unfeasible, highlighting the importance of precise tumor margin delineation to prevent neurological deficits and improve prognosis. Imaging Mueller polarimetry, an effective modality in various organ tissues, seems a promising approach for tumor delineation in neurosurgery. To further assess its use, we characterized the polarimetric properties by analysing 45 polarimetric measurements of 27 fresh brain tumor samples, including different tumor types with a strong focus on gliomas. Our study integrates a wide-field imaging Mueller polarimetric system and a novel neuropathology protocol, correlating polarimetric and histological data for accurate tissue identification. An image processing pipeline facilitated the alignment and overlay of polarimetric images and histological masks. Variations in depolarization values were observed for grey and white matter of brain tumor tissue, while differences in linear retardance were seen only within white matter of brain tumor tissue. Notably, we identified pronounced optical axis azimuth randomization within tumor regions. This study lays the foundation for machine learning-based brain tumor segmentation algorithms using polarimetric data, facilitating intraoperative diagnosis and decision making.

Cribriform Morular Thyroid Carcinoma - Ultimobranchial Pouch-Related? Deep Molecular Insights of a Unique Case.

A 44-year-old female patient with a familial adenomatous polyposis (FAP) was diagnosed with a cribriform morular thyroid carcinoma (CMTC). We observed within the very necrotic tumor a small but distinct poorly differentiated carcinomatous component. As expected, next generation sequencing of both components revealed a homozygous APC mutation and in addition, a TERT promoter mutation. A TP53 mutation was found exclusively in the CMTC part, while the poorly differentiated component showed a clonal evolution, harboring an activating PIK3CA mutation and copy number gains of BRCA2, FGF23, FGFR1, and PIK3CB-alterations which are typically seen in squamous cell carcinoma. The mutational burden in both components was low, and there was no evidence for microsatellite instability. No mutations involving the mitogen-activated protein kinase (MAPK) pathway, typically seen in papillary thyroid carcinomas, were detected. Immunohistochemically, all tumor parts were negative for thyroglobulin, providing further evidence that this entity does not belong to the follicular epithelial cell-derived thyroid carcinoma group. CD5 was negative in the poorly differentiated component, making a relation to intrathyroidal thymic carcinoma rather unlikely. However, since this marker was seen in the morules, a loss in the poorly differentiated component and a relation to the ultimobranchial body cannot be excluded either. After total thyroidectomy and radioiodine ablation, the patient was disease-free with no residual tumor burden on 2-year follow-up.

Effects of formalin fixation on polarimetric properties of brain tissue: fresh or fixed?

Significance: Imaging Mueller polarimetry (IMP) appears as a promising technique for real-time delineation of healthy and neoplastic tissue during neurosurgery. The training of machine learning algorithms used for the image post-processing requires large data sets typically derived from the measurements of formalin-fixed brain sections. However, the success of the transfer of such algorithms from fixed to fresh brain tissue depends on the degree of alterations of polarimetric properties induced by formalin fixation (FF). Aim: Comprehensive studies were performed on the FF induced changes in fresh pig brain tissue polarimetric properties. Approach: Polarimetric properties of pig brain were assessed in 30 coronal thick sections before and after FF using a wide-field IMP system. The width of the uncertainty region between gray and white matter was also estimated. Results: The depolarization increased by 5% in gray matter and remained constant in white matter following FF, whereas the linear retardance decreased by 27% in gray matter and by 28% in white matter after FF. The visual contrast between gray and white matter and fiber tracking remained preserved after FF. Tissue shrinkage induced by FF did not have a significant effect on the uncertainty region width. Conclusions: Similar polarimetric properties were observed in both fresh and fixed brain tissues, indicating a high potential for transfer learning.

Evaluation of MTAP and p16 immunohistochemical deficiency as surrogate marker for CDKN2A/B homozygous deletion in gliomas.

Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for CDKN2A/B deletions can be performed by a variety of approaches, including copy number variation (CNV) analysis based on gene array analysis, next generation sequencing (NGS) or fluorescence in situ hybridisation (FISH), but questions remain regarding the accuracy of testing modalities. In this study, we assessed: (1) the utility of S-methyl-5'-thioadenosine phosphorylase (MTAP) and cellular tumour suppressor protein pl61NK4a (p16) immunostainings as surrogate markers for CDKN2A/B HD in gliomas, and (2) the prognostic value of MTAP, across different histological tumour grades and IDH mutation status. One hundred consecutive cases of diffuse and circumscribed gliomas (Cohort 1) were collected, in order to correlate MTAP and p16 expression with the CDKN2A/B status in the CNV plot of each tumour. IDH1 R132H, ATRX and MTAP immunohistochemistry was performed on next generation tissue microarrays (ngTMAs) of 251 diffuse gliomas (Cohort 2) for implementing survival analysis. Complete loss of MTAP and p16 by immunohistochemistry was 100% and 90% sensitive as well as 97% and 89% specific for CDKN2A/B HD, respectively, as identified on CNV plot. Only two cases (2/100) with MTAP and p16 loss of expression did not demonstrate CDKN2A/B HD in CNV plot; however, FISH analysis confirmed the HD for CDKN2A/B. Moreover, MTAP deficiency was associated with shortened survival in IDH-mutant astrocytomas (n=75; median survival 61 vs 137 months; p<0.0001), IDH-mutant oligodendrogliomas (n=59; median survival 41 vs 147 months; p<0.0001) and IDH-wild-type gliomas (n=117; median survival 13 vs 16 months; p=0.011). In conclusion, MTAP immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with CDKN2A/B status, robustness, rapid turnaround time and low costs, and provides significant prognostic value in IDH-mutant astrocytomas and oligodendrogliomas, while p16 should be used cautiously.

Integrated longitudinal analysis of adult grade 4 diffuse gliomas with long-term relapse interval revealed upregulation of TGF-ß signaling in recurrent tumors.

BACKGROUND: Adult-type diffuse gliomas, CNS WHO grade 4 are the most aggressive primary brain tumors and represent a particular challenge for therapeutic intervention. METHODS: In a single-center retrospective study of matched pairs of initial and post-therapeutic glioma cases with a recurrence period greater than 1 year, we performed whole exome sequencing combined with mRNA and microRNA expression profiling to identify processes that are altered in recurrent gliomas. RESULTS: Mutational analysis of recurrent gliomas revealed early branching evolution in 75% of the patients. High plasticity was confirmed at the mRNA and miRNA levels. SBS1 signature was reduced and SBS11 was elevated, demonstrating the effect of alkylating agent therapy on the mutational landscape. There was no evidence for secondary genomic alterations driving therapy resistance. ALK7/ACVR1C and LTBP1 were upregulated, whereas LEFTY2 was downregulated, pointing towards enhanced Tumor Growth Factor ß (TGF-ß) signaling in recurrent gliomas. Consistently, altered microRNA expression profiles pointed towards enhanced Nuclear Factor Kappa B and Wnt signaling that, cooperatively with TGF-ß, induces epithelial to mesenchymal transition (EMT), migration, and stemness. TGF-ß-induced expression of pro-apoptotic proteins and repression of antiapoptotic proteins were uncoupled in the recurrent tumor. CONCLUSIONS: Our results suggest an important role of TGF-ß signaling in recurrent gliomas. This may have clinical implications since TGF-ß inhibitors have entered clinical phase studies and may potentially be used in combination therapy to interfere with chemoradiation resistance. Recurrent gliomas show high incidence of early branching evolution. High tumor plasticity is confirmed at the level of microRNA and mRNA expression profiles.

Pleiotropy of PP2A Phosphatases in Cancer with a Focus on Glioblastoma IDH Wildtype.

Serine/Threonine protein phosphatase 2A (PP2A) is a heterotrimeric (or occasionally, heterodimeric) phosphatase with pleiotropic functions and ubiquitous expression. Despite the fact that they all contribute to protein dephosphorylation, multiple PP2A complexes exist which differ considerably by their subcellular localization and their substrate specificity, suggesting diverse PP2A functions. PP2A complex formation is tightly regulated by means of gene expression regulation by transcription factors, microRNAs, and post-translational modifications. Furthermore, a constant competition between PP2A regulatory subunits is taking place dynamically and depending on the spatiotemporal circumstance; many of the integral subunits can outcompete the rest, subjecting them to proteolysis. PP2A modulation is especially important in the context of brain tumors due to its ability to modulate distinct glioma-promoting signal transduction pathways, such as PI3K/Akt, Wnt, Ras, NF-κb, etc. Furthermore, PP2A is also implicated in DNA repair and survival pathways that are activated upon treatment of glioma cells with chemo-radiation. Depending on the cancer cell type, preclinical studies have shown some promise in utilising PP2A activator or PP2A inhibitors to overcome therapy resistance. This review has a special focus on "glioblastoma, IDH wild-type" (GBM) tumors, for which the therapy options have limited efficacy, and tumor relapse is inevitable.

Programmed Death-Ligand 1 Expression in Lung Cancer and Paired Brain Metastases-a Single-Center Study in 190 Patients.

Introduction: Expression of programmed death-ligand 1 (PD-L1) is the only routinely used tissue biomarker for predicting response to programmed cell death protein 1/PD-L1 inhibitors. It is to date unclear whether PD-L1 expression is preserved in brain metastases (BMs). Methods: In this single-center, retrospective study, we evaluated PD-L1 expression using the SP263 assay in consecutively resected BMs of lung carcinomas and paired primary tumors, diagnosed from 2000 to 2015, with correlation to clinicopathological and molecular tumor and patient characteristics. Results: PD-L1 tumor proportional score (TPS) could be evaluated on whole tissue slides in 191 BMs and 84 paired primary lung carcinomas. PD-L1 TPS was less than 1% in 113 of 191 (59.2%), 1% to 49% in 34 of 191 (17.8%), and greater than or equal to 50% in 44 of 191 (23.0%) BMs. TPS was concordant between BMs and paired primary lung carcinomas in most cases, with discordance regarding the clinically relevant cutoffs at 1% and 50% in 18 of 84 patients (21.4%). Four of 18 discordant cases had no shared mutations between the primary lung carcinoma and BM. Intratumoral heterogeneity, as assessed using tissue microarray cores, was only significant at the primary site (p Wilcoxon signed rank = 0.002) with higher PD-L1 TPS at the infiltration front (mean = 40.4%, interquartile range: 0%-90%). Neither TPS greater than or equal to 1% nor TPS greater than or equal to 50% nor discordance between the primary lung carcinoma and BMs had prognostic significance regarding overall survival or BM-specific overall survival. Conclusions: PD-L1 expression was mostly concordant between primary lung carcinoma and its BM and between resections of BM and stereotactic biopsies, mirrored by tissue microarray cores. Differences in PD-L1 TPS existed primarily in cases with TPS greater than 10%, for which also human assessment tends to be most error prone.

Quantitative Analysis of the MGMT Methylation Status of Glioblastomas in Light of the 2021 WHO Classification.

Background: Glioblastomas with methylation of the promoter region of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene exhibit increased sensitivity to alkylating chemotherapy. Quantitative assessment of the MGMT promoter methylation status might provide additional prognostic information. The aim of our study was to determine a quantitative methylation threshold for better survival among patients with glioblastomas. Methods: We included consecutive patients ≥18 years treated at our department between 11/2010 and 08/2018 for a glioblastoma, IDH wildtype, undergoing quantitative MGMT promoter methylation analysis. The primary endpoint was overall survival. Results: A total of 321 patients were included. Median overall survival was 12.6 months. Kaplan−Meier and adjusted Cox regression analysis showed better survival for the groups with 16−30%, 31−60%, and 61−100% methylation. In contrast, survival in the group with 1−15% methylation was similar to those with unmethylated promoter regions. A secondary analysis confirmed this threshold. Conclusions: Better survival is observed in patients with glioblastomas with ≥16% methylation of the MGMT promoter region than with <16% methylation. Survival with tumors with 1−15% methylation is similar to with unmethylated tumors. Above 16% methylation, we found no additional benefit with increasing methylation.

CNS Antigen-Specific Neuroinflammation Attenuates Ischemic Stroke With Involvement of Polarized Myeloid Cells.

BACKGROUND AND OBJECTIVES: Experimental studies indicate shared molecular pathomechanisms in cerebral hypoxia-ischemia and autoimmune neuroinflammation. This has led to clinical studies investigating the effects of immunomodulatory therapies approved in multiple sclerosis on inflammatory damage in stroke. So far, mutual and combined interactions of autoimmune, CNS antigen-specific inflammatory reactions and cerebral ischemia have not been investigated so far. METHODS: Active MOG35-55 experimental autoimmune encephalomyelitis (EAE) was induced in male C57Bl/6J mice. During different phases of EAE, transient middle cerebral artery occlusion (tMCAO, 60 minutes) was induced. Brain tissue was analyzed for infarct size and immune cell infiltration. Multiplex gene expression analysis was performed for 186 genes associated with neuroinflammation and hypoxic-ischemic damage. RESULTS: Mice with severe EAE disease showed a substantial reduction in infarct size after tMCAO. Histopathologic analysis showed less infiltration of CD45+ hematopoietic cells in the infarct core of severely diseased acute EAE mice; this was accompanied by an accumulation of Arginase1-positive/Iba1-positive cells. Gene expression analysis indicated an involvement of myeloid cell-driven anti-inflammatory mechanisms in the attenuation of ischemic injury in severely diseased mice exposed to tMCAO in the acute EAE phase. DISCUSSION: CNS autoantigen-specific autoimmunity has a protective influence on primary tissue damage after experimental stroke, indicating a very early involvement of CNS antigen-specific, myeloid cell-associated anti-inflammatory immune mechanisms that mitigate ischemic injury in the acute EAE phase.

Alterations in homologous recombination repair genes in prostate cancer brain metastases.

Improved survival rates for prostate cancer through more effective therapies have also led to an increase in the diagnosis of metastases to infrequent locations such as the brain. Here we investigate the repertoire of somatic genetic alterations present in brain metastases from 51 patients with prostate cancer brain metastases (PCBM). We highlight the clonal evolution occurring in PCBM and demonstrate an increased mutational burden, concomitant with an enrichment of the homologous recombination deficiency mutational signature in PCBM compared to non-brain metastases. Focusing on known pathogenic alterations within homologous recombination repair genes, we find 10 patients (19.6%) fulfilling the inclusion criteria used in the PROfound clinical trial, which assessed the efficacy of PARP inhibitors (PARPi) in homologous recombination deficient prostate cancer. Eight (15.7%) patients show biallelic loss of one of the 15 genes included in the trial, while 5 patients (9.8%) harbor pathogenic alterations in BRCA1/2 specifically. Uncovering these molecular features of PCBM may have therapeutic implications, suggesting the need of clinical trial enrollment of PCBM patients when evaluating potential benefit from PARPi.

Mutational profiles of primary pulmonary adenocarcinoma and paired brain metastases disclose the importance of KRAS mutations.

BACKGROUND: Brain metastases present a significant complication in lung cancer with an unmet therapeutic need. METHODS: In this single-centre, retrospective study, we genotyped a clinico-pathologically well-annotated cohort of consecutively resected brain metastases of lung adenocarcinomas and paired primary tumours, diagnosed from 2000 to 2015, using the Ion Torrent Oncomine Comprehensive Cancer Panel v3. RESULTS: Among 444 consecutive brain metastases, 210 (49%) originated from lung cancer. Analysis was successful in 111 samples, including 54 pairs of brain metastasis and primary tumour. Most driver alterations were preserved in brain metastases. Private alterations exclusive to primary tumours, brain metastases or both sites (intersecting cases) were present in 22%, 26% and 26% of cases, respectively. Seven percent had no shared mutations. KRAS mutations were more frequent in primary tumours metastasised to the brain (32/55, 58%) compared to TCGA (33%, p < 0.005) and own data from routine diagnostics, independent from clinical or pathological characteristics. Fourteen cases showed alterations in the EGFR signalling pathway including additional KRAS alterations that were private to brain metastases. KRAS G12C was detected most frequently (26% of patients) and KRAS G12C and G13C variants were significantly enriched in brain metastases. Synchronous and metachronous cases had a similar mutation profile. CONCLUSIONS: Our results suggest an important role of KRAS alterations in the pathobiology of brain metastases from lung adenocarcinomas. This has direct therapeutic implications as inhibitors selectively targeting KRAS G12C are entering the clinics.

Epigenetic landscape of pancreatic neuroendocrine tumours reveals distinct cells of origin and means of tumour progression.

Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α- or ß-cells of the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type specific hormones. In the current study we examine whether tumour DNA methylation (DNAme) profiling combined with genomic data is able to identify cell of origin and to reveal pathways involved in PanNET progression. We analyse genome-wide DNAme data of 125 PanNETs and sorted α- and ß-cells. To confirm cell identity, we investigate ARX and PDX1 expression. Based on epigenetic similarities, PanNETs cluster in α-like, ß-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreases in the intermediate tumours, which present unclear differentiation. Specific transcription factor methylation and expression vary in the respective α/ß-tumour groups. Depending on DNAme similarity to α/ß-cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential means of PanNET progression.

TERT Promoter Mutation Analysis to Distinguish Glioma From Gliosis.

Among the most challenging diagnostic issues in surgical neuropathology is the distinction between scant infiltration by diffuse gliomas and reactive gliosis. The best documented ancillary marker to establish a definitive diagnosis of glioma in this setting is the identification of hotspot mutations in the isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) genes, which is limited, however, by the low prevalence of these mutations in gliomas of elderly adults. Since telomerase reverse transcriptase (TERT) promoter mutations are present in the vast majority of IDH-wildtype diffuse gliomas, we hypothesized that combined analysis of IDH and TERT might overcome these limitations. For this purpose, we analyzed a series of non-neoplastic and neoplastic CNS samples for the prevalence of TERT hotspot mutations. TERT mutations were identified in none out of 58 (0%) reactive gliosis samples, and in 91 out of 117 (78%) IDH-wildtype gliomas. Based on a series of 200 consecutive diffuse gliomas, we found that IDH mutation analysis alone had a sensitivity of 28% (63% and 12%, respectively, in patients below and above age of 50) for detection of gliomas, whereas a combined analysis of IDH and TERT was 85% sensitive (87% and 84%, respectively, below and above age of 50). In sum, our findings suggest that TERT promoter mutation analysis contributes favorably to a molecular panel in cases equivocal for glioma versus gliosis on morphological grounds, especially in patients above age of 50, in which IDH analysis alone performs poorly.

mTOR mediates a mechanism of resistance to chemotherapy and defines a rational combination strategy to treat KRAS-mutant lung cancer.

Oncogenic KRAS mutations comprise the largest subset of lung cancer defined by genetic alterations, but in the clinic no targeted therapies are available that effectively control mutational KRAS activation. Consequently, patients with KRAS-driven tumors are routinely treated with cytotoxic chemotherapy, which is often transiently effective owing to development of drug resistance. In this study, we show that hyperactivated mammalian target of rapamycin (mTOR) pathway is a characteristic hallmark of KRAS-mutant lung adenocarcinoma after chemotherapy treatment, and that KRAS-mutant lung cancer cells rely on persistent mTOR signaling to resist chemotherapeutic drugs. Coherently, mTOR inhibition circumvents the refractory phenotype and restores sensitivity of resistant KRAS-mutant lung cancer cells to chemotherapy. Importantly, drug combinations of clinically approved mTOR inhibitors and chemotherapy drugs synergize in inhibiting cell proliferation of KRAS-mutant cancer cells in vitro and in vivo, and the efficacy of this combination treatment correlates with the magnitude of mTOR activity induced by chemotherapy alone. These results pinpoint mTOR as a mechanism of resistance to chemotherapy in KRAS-mutant lung cancer and validate a rational and readily translatable strategy that combines mTOR inhibitors with standard chemotherapy to treat KRAS-mutant adenocarcinoma, the most common and deadliest lung cancer subset.

Tumor Necrosis Factor-α Initiates miRNA-mRNA Signaling Cascades in Obstruction-Induced Bladder Dysfunction.

Bladder outlet obstruction (BOO) and the ensuing clinical lower urinary tract dysfunction are common in elderly patients. BOO is accompanied by urodynamic changes in bladder function and leads to organ fibrosis and ultimately loss of contractility. Comprehensive transcriptome analysis of bladder samples from human patients with different urodynamically defined phenotypes of BOO revealed tumor necrosis factor (TNF)-α as the top upstream signaling pathway regulator. Herein, we validated next-generation sequencing and pathway analysis in cell-based models using bladder smooth muscle and urothelial cells exposed to TNF-α. miRNA profiling and transcriptome analysis of TNF-α-treated bladder smooth muscle cells revealed striking similarities with human BOO. Using a comparative approach, TNF-specific and TNF-independent pathways were delineated in human biopsy specimens. Concomitant down-regulation of smooth muscle cell-specific miRNAs and smooth muscle markers after TNF-α treatment was in accordance with the loss of contractility in humans in advanced obstruction-induced bladder remodeling. The expression levels of four abundant TNF-regulated miRNAs were modulated; the compensatory up-regulation of miR-199a-5p reduced NF-κB signaling. Essential hubs of TNF-α signaling pathways mitogen-activated protein kinase kinase kinase (apoptosis signal-regulating kinase 1) and inhibitor of nuclear factor κ B kinase subunit ß (IκB kinase ß) were targeted by miR-199a-5p. miR-199a-5p might be part of a negative feedback loop, reducing the impact of TNF, whereas its down-regulation in acontractile bladders from BOO patients advances the disease. The compensatory up-regulation of miR-199a-5p together with TNF-α inhibition may be therapeutically beneficial.

Multi-laboratory proficiency testing of clinical cancer genomic profiling by next-generation sequencing.

Next-generation sequencing (NGS) enables parallel analysis of multiple genomic targets. The increasing demand for NGS-based multiplexed molecular diagnostics requires standardized protocols and recommendations to ensure reproducibility and accuracy of test results for routine clinical decision making. However, the lack of clinical NGS data from multi-laboratory tests and the absence of inter-laboratory comparisons have hampered the establishment of instructive clinical NGS standards. To fill the gap, we set up Proficiency Testing (PT) for inter-laboratory comparison, in which formalin-fixed paraffin-embedded specimens from eight lung and eight colon cancers were analyzed by 15 European molecular diagnostic laboratories on three different platforms using multiple target enrichment systems. We first performed platform, test, and informatics pipeline validation and conducted sensitivity and specificity analysis by random in silico down-sampling. We then implemented a multi-level filtering strategy based on performance tests of base substitution, replicate runs, and Sanger sequencing verified variants. We finally applied the filter criteria to the NGS data from the respective PT participants and obtained high inter-laboratory agreement. We demonstrated accuracy, scalability, and robustness of NGS by means of PT, serving as a benchmark for detecting clinically actionable molecular alterations in research and diagnostic laboratories. In conclusion, this study strongly highlights the importance of establishing standards for NGS-based testing, particularly when the test results impact on clinical decisions, and systematically provides data sets from multiple different labs to infer such standards.

Integrated Genomic and Immunophenotypic Classification of Pancreatic Cancer Reveals Three Distinct Subtypes with Prognostic/Predictive Significance.

Purpose: Current clinical classification of pancreatic ductal adenocarcinoma (PDAC) is unable to predict prognosis or response to chemo- or immunotherapy and does not take into account the host reaction to PDAC cells. Our aim is to classify PDAC according to host- and tumor-related factors into clinically/biologically relevant subtypes by integrating molecular and microenvironmental findings.Experimental Design: A well-characterized PDAC cohort (n = 110) underwent next-generation sequencing with a hot spot cancer panel while next-generation tissue microarrays were immunostained for CD3, CD4, CD8, CD20, PD-L1, p63, hyaluronan-mediated motility receptor (RHAMM), and DNA mismatch repair proteins. Previous data on FOXP3 were integrated. Immune cell counts and protein expression were correlated with tumor-derived driver mutations, clinicopathologic features (TNM 8th edition, 2017), survival, and epithelial-mesenchymal transition (EMT)-like tumor budding.Results: Three PDAC subtypes were identified: the "immune escape" (54%), poor in T and B cells and enriched in FOXP3+ regulatory T cells (Treg), with high-grade budding, frequent CDKN2A, SMAD4, and PIK3CA mutations, and poor outcome; the "immune rich" (35%), rich in T and B cells and poorer in FOXP3+ Tregs, with infrequent budding, lower CDKN2A and PIK3CA mutation rate, and better outcome and a subpopulation with tertiary lymphoid tissue (TLT), mutations in DNA damage response genes (STK11 and ATM), and the best outcome; and the "immune exhausted" (11%), with immunogenic microenvironment and two subpopulations-one with PD-L1 expression and a high PIK3CA mutation rate and a microsatellite-unstable subpopulation with a high prevalence of JAK3 mutations. The combination of low budding, low stromal FOXP3 counts, presence of TLTs, and absence of CDKN2A mutations confers significant survival advantage in patients with PDAC.Conclusions: Immune host responses correlate with tumor characteristics, leading to morphologically recognizable PDAC subtypes with prognostic/predictive significance. Clin Cancer Res; 24(18); 4444-54. ©2018 AACRSee related commentary by Khalil and O'Reilly, p. 4355.