Tumor mutational burden (TMB) represents a new determinant of clinical benefit from immune checkpoint blockade that identifies responders independent of PD‐L1 expression levels and is currently being ...explored in clinical trials. Although TMB can be measured directly by comprehensive genomic approaches such as whole‐genome and exome sequencing, broad availability, short turnaround times, costs and amenability to formalin‐fixed and paraffin‐embedded tissue support the use of gene panel sequencing for approximating TMB in routine diagnostics. However, data on the parameters influencing panel‐based TMB estimation are limited. Here, we report an extensive in silico analysis of the TCGA data set that simulates various panel sizes and compositions. We demonstrate that panel size is a critical parameter that influences confidence intervals (CIs) and cutoff values as well as important test parameters including sensitivity, specificity, and positive predictive value. Moreover, we evaluate the Illumina TSO500 panel, which will be made available for TMB estimation, and propose dynamic, entity‐specific cutoff values based on current clinical trial data. Optimizing the cost–benefit ratio, our data suggest that panels between 1.5 and 3 Mbp are ideally suited to estimate TMB with small CIs, whereas smaller panels tend to deliver imprecise TMB estimates for low to moderate TMB (0–30 muts/Mbp), connected with insufficient separation of hypermutated tumors from non‐hypermutated tumors.
What's new?
While the immune system can fight cancer, tumor clones may hijack the body's own mechanisms of dampening immune responses by expressing immune‐checkpoint proteins. Tumor mutational burden (TMB) is an emerging selection biomarker for patients who would benefit from immune checkpoint inhibitor therapy. Unification of TMB estimation and driver mutation detection in a single panel sequencing assay is highly desirable but challenging, however. Combinatorial calculations and simulations in the TCGA dataset show that panel sizes greater than 1.5 Mbp are recommendable, and–although not immunogenic–synonymous and nonsense mutations can be included in TMB estimation together with cancer‐type specific correction factors.
Assessment of Tumor Mutational Burden (TMB) for response stratification of cancer patients treated with immune checkpoint inhibitors is emerging as a new biomarker. Commonly defined as the total ...number of exonic somatic mutations, TMB approximates the amount of neoantigens that potentially are recognized by the immune system. While whole exome sequencing (WES) is an unbiased approach to quantify TMB, implementation in diagnostics is hampered by tissue availability as well as time and cost constrains. Conversely, panel‐based targeted sequencing is nowadays widely used in routine molecular diagnostics, but only very limited data are available on its performance for TMB estimation. Here, we evaluated three commercially available larger gene panels with covered genomic regions of 0.39 Megabase pairs (Mbp), 0.53 Mbp and 1.7 Mbp using i) in silico analysis of TCGA (The Cancer Genome Atlas) data and ii) wet‐lab sequencing of a total of 92 formalin‐fixed and paraffin‐embedded (FFPE) cancer samples grouped in three independent cohorts (non‐small cell lung cancer, NSCLC; colorectal cancer, CRC; and mixed cancer types) for which matching WES data were available. We observed a strong correlation of the panel data with WES mutation counts especially for the gene panel >1Mbp. Sensitivity and specificity related to TMB cutpoints for checkpoint inhibitor response in NSCLC determined by wet‐lab experiments well reflected the in silico data. Additionally, we highlight potential pitfalls in bioinformatics pipelines and provide recommendations for variant filtering. In summary, our study is a valuable data source for researchers working in the field of immuno‐oncology as well as for diagnostic laboratories planning TMB testing.
What's new?
Tumor mutational burden (TMB) is an emerging biomarker to predict response to immune checkpoint inhibitors. While TMB can be measured by whole exome sequencing (WES), costs, turn‐around time, and tissue availability currently favor a panel sequencing approach using FFPE tissue for routine diagnostics. However, performance remains to be clarified. Our study is the first worldwide that analyses three major commercial gene panels by comparing TMB approximation across panels as well as against the technical reference standard WES. The data suggest that TMB approximation using gene panel sequencing of FFPE tumor tissue is feasible and can be implemented in routine diagnostics.
In contrast to male rats, aggression in virgin female rats has been rarely studied. Here, we established a rat model of enhanced aggression in females using a combination of social isolation and ...aggression-training to specifically investigate the involvement of the oxytocin (OXT) and arginine vasopressin (AVP) systems within the lateral septum (LS). Using neuropharmacological, optogenetic, chemogenetic as well as microdialysis approaches, we revealed that enhanced OXT release within the ventral LS (vLS), combined with reduced AVP release within the dorsal LS (dLS), is required for aggression in female rats. Accordingly, increased activity of putative OXT receptor-positive neurons in the vLS, and decreased activity of putative AVP receptor-positive neurons in the dLS, are likely to underly aggression in female rats. Finally, in vitro activation of OXT receptors in the vLS increased tonic GABAergic inhibition of dLS neurons. Overall, our data suggest a model showing that septal release of OXT and AVP differentially affects aggression in females by modulating the inhibitory tone within LS sub-networks.
Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of ...targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS‐based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin‐fixed, paraffin‐embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR‐based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR‐/BRAF‐/ALK‐ and ROS1‐directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (<5%), minimal tissue requirements, fast turnaround times (6 days on average) and lower costs of the diagnostic approach presented here compared to sequential single‐gene testing, highlight its practicability in order to support individualized decisions in routine patient care, enrollment in molecularly stratified clinical trials, as well as translational research.
What's new?
Next‐generation sequencing (NGS) is an attractive option in the molecular workup of non‐small cell lung cancer (NSCLC). However, large‐scale implementation for routine diagnostics is a non‐trivial task. Here, the authors present the largest cohort of advanced NSCLC tested for mutations and translocations by combined targeted RNA‐ and DNA‐sequencing in routine diagnostics to date. The integrated approach exceeds current international guidelines and illustrates the performance and clinical impact of one‐stop shop NGS profiling. Due to amplification‐based sequencing, dropout rates are minimal and turnaround times low. By facilitating better patient stratification, such an approach can improve oncologic management and boost translational research.
Variant classification in precision oncology Leichsenring, Jonas; Horak, Peter; Kreutzfeldt, Simon ...
International journal of cancer,
1 December 2019, 2019-12-01, 2019-12-00, 20191201, Volume:
145, Issue:
11
Journal Article
Peer reviewed
Next‐generation sequencing has become a cornerstone of therapy guidance in cancer precision medicine and an indispensable research tool in translational oncology. Its rapidly increasing use during ...the last decade has expanded the options for targeted tumor therapies, and molecular tumor boards have grown accordingly. However, with increasing detection of genetic alterations, their interpretation has become more complex and error‐prone, potentially introducing biases and reducing benefits in clinical practice. To facilitate interdisciplinary discussions of genetic alterations for treatment stratification between pathologists, oncologists, bioinformaticians, genetic counselors and medical scientists in specialized molecular tumor boards, several systems for the classification of variants detected by large‐scale sequencing have been proposed. We review three recent and commonly applied classifications and discuss their individual strengths and weaknesses. Comparison of the classifications underlines the need for a clinically useful and universally applicable variant reporting system, which will be instrumental for efficient decision making based on sequencing analysis in oncology. Integrating these data, we propose a generalizable classification concept featuring a conservative and a more progressive scheme, which can be readily applied in a clinical setting.
What's new?
With increasingly comprehensive molecular profiling of cancers, the clinical interpretation of genetic alterations is becoming more and more challenging. Here the authors review several classifications for gene variant interpretation that were recently introduced to guide clinical management. They highlight shared features and differences and point out major influencing factors and unresolved issues that still need to be addressed. Based on this analysis, they propose a unified classification concept that may become broadly implemented when remaining issues are solved.
Cancer of unknown primary (CUP) denotes cancer cases where metastatic spread is histologically confirmed, but no respective primary tumor can be identified. The challenging diagnosis of CUP is ...further complicated in cases with previously identified malignancies or with dubious clonal relationship between metastatic sites due to ambiguous histology. Our study aims at elucidating clonal relationships by comparing the respective mutational spectra. Targeted next‐generation sequencing (NGS) employing formalin‐fixed and paraffin‐embedded (FFPE) tumor tissue was performed on 174 consecutive CUP patients. Among these, 43/174 (24.7%) patients had a documented prior malignancy. Data on pairwise targeted NGS testing to address clonal relationships between the previous malignancy and the presumed CUP (n = 11) or between different CUP metastatic sites (n = 7) was available in 18 patients. NGS could clarify clonal relationships in 16/18 cases. Among the 11 CUP patients with antecedent malignancies, four cases were clonally independent of the previous malignancy but harbored deleterious germline mutations in BRCA/BAP1/ATM genes. Seven CUP cases were clonally related to the antecedent malignancy, changing the CUP diagnosis to relapse of the prior malignancy. In the seven CUP cases, with doubtfully related metastatic sites, NGS confirmed clonal relationship in five cases and was inconclusive in two. In conclusion, NGS proved an efficient tool to elucidate clonal relationships in clinically challenging CUP cases. Our study cautions against a premature diagnosis of CUP. Relapses of antecedent malignancies should be carefully considered. CUPs clonally independent from the antecedent malignancy should raise a red flag of a potential cancer‐predisposing germline mutation.
What's new?
Cancer of unknown primary (CUP) often presents a difficult challenge. Is it a recurrence of a previous cancer or metastasis of a new malignancy? In this study, the authors used targeted next‐generation sequencing (NGS) to compare the genetic signatures of CUPs with signatures of the patients’ previous cancers. This pair‐wise testing revealed that many CUPs are clonally related to previous cancers. In cases where CUPs are clonally independent from a previous malignancy, the authors note that this should raise a red flag of a potential cancer‐predisposing germline mutation.
Compared to the innate immune system, the contribution of the adaptive immune response during obesity and insulin resistance is still not completely understood. Here we demonstrate that high fat diet ...(HFD) increases the frequencies of activated CD4+ and CD8+ T cells and frequencies of T cells positive for IFN-γ and IL-17 in the adipose tissue. The adipocyte-derived soluble factor adiponectin reduces IFN-γ and IL-17 positive CD4+ T cells from HFD mice and dampens the differentiation of naïve T cells into Th1 cells and Th17 cells. Adiponectin reduces Th17 cell differentiation and restrains glycolysis in an AMPK dependent fashion. Treatment with adult worm extracts of the rodent filarial nematode
(LsAg) reduces adipose tissue Th1 and Th17 cell frequencies during HFD and increases adiponectin levels. Stimulation of T cells in the presence of adipocyte-conditioned media (ACM) from LsAg-treated mice reduces Th1 and Th17 frequencies and this effect was abolished when ACM was treated with an adiponectin neutralizing antibody. Collectively, these data reveal a novel role of adiponectin in controlling pro-inflammatory CD4+ T cells during obesity and suggest that the beneficial role of helminth infections and helminth-derived products on obesity and insulin resistance may be in part mediated by adiponectin.
Pancreatic cysts or dilated pancreatic ducts are often found by cross‐sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non‐mucinous ...lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell‐free DNA in the diagnostic endoscopic ultrasound (EUS)‐guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS‐guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow‐up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty‐six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty‐seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS‐ and/or GNAS‐mutation was diagnosed by NGS. 27.0% of the KRAS‐mutated and 10.0% of the GNAS‐mutated lesions harbored multiple mutations. KRAS/GNAS‐testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS‐testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS‐testing by deep targeted NGS is a suitable method to distinguish mucinous from non‐mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.
Tumor mutational burden (TMB) is a new biomarker for prediction of response to PD-(L)1 treatment. Comprehensive sequencing approaches (i.e., whole exome and whole genome sequencing) are ideally ...suited to measure TMB directly. However, as their applicability in routine diagnostics is currently limited by high costs, long turnaround times and poor availability of fresh tissue, targeted next-generation sequencing (NGS) of formalin-fixed and paraffin-embedded (FFPE) samples appears to be a more feasible and straight-forward approach for TMB approximation, which can be seamlessly integrated in already existing diagnostic workflows and pipelines. In this work, we provide an overview of the clinical implications of TMB testing and highlight key parameters including pre-analysis, analysis and post-analytical steps that influence and shape TMB approximation by panel sequencing. Collectively, the data will not only serve as a field guide and state of the art knowledge source for molecular pathologists who consider implementation of TMB measurement in their lab, but also enable clinicians in understanding the specific parameters influencing TMB test results and reporting.
Cancer of unknown primary (CUP) denotes a malignancy with histologically confirmed metastatic spread while the primary tumor remains elusive. Here, we address prognostic and therapeutic implications ...of mutations and copy number variations (CNVs) detected in tumor tissue in the context of a comprehensive clinical risk assessment. Targeted panel sequencing was performed in 252 CUP patients. 71.8% of patients had unfavorable CUP according to ESMO guidelines. 74.7% were adeno‐ and 13.7% squamous cell carcinomas. DNA was extracted from microdissected formalin‐fixed, paraffin‐embedded tissues. For library preparation, mostly multiplex PCR‐based Ion Torrent AmpliSeq™ technology with Oncomine comprehensive assays was used. Most frequent genetic alterations were mutations/deletions of TP53 (49.6%), CDKN2A (19.0%) and NOTCH1 (14.1%) as well as oncogenic activation of KRAS (23.4%), FGFR4 (14.9%) and PIK3CA (10.7%). KRAS activation was predominantly found in adenocarcinomas (p = 0.01), PIK3CA activation in squamous cell carcinomas (p = 0.03). Male sex, high ECOG score, unfavorable CUP, higher number of involved organs and RAS activation predicted decreased event‐free and overall survival in multivariate analysis. Deletions of CDKN2A were prognostically adverse regarding overall survival. TP53 mutations did not significantly influence prognosis in the overall cohort, but worsened prognosis in otherwise favorable CUP subtypes. Although not standard in CUP, for 17/198 (8.6%) patients molecularly targeted treatment was recommended and 10 patients (5.1%) were treated accordingly. In conclusion, besides the identification of drug targets, panel sequencing in CUP is prognostically relevant, with RAS activation and CDKN2A deletion emerging as novel independent risk factors in a comprehensive assessment with clinicopathological data.
What's new?
While some cancers of unknown primary (CUP) respond to treatment, most have poor survival rates. In this study, the authors used next‐generation sequencing (NGS) to identify specific genetic mutations that indicate a poorer prognosis in CUP. These included CDKN2A deletion and RAS activation, while TP53 mutations indicated poor prognosis mainly in squamous cell carcinoma CUP and in clinically favorable CUP subtypes. NGS results were also able to guide targeted treatment in some CUPs, demonstrating that routine NGS‐based sequencing can enable access to individualized treatment in clinical practice, outside clinical trials.
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