During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies ...identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5 mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 (R132)) and IDH2 (IDH2 (R172)) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.
Immunohistochemistry of the PD-L1 protein may be predictive for anti-PD-1 and anti-PD-L1 immunotherapy in pulmonary adenocarcinoma and in clinically unselected cohorts of so-called non-small-cell ...lung cancer. Several PD-L1 immunohistochemistry assays with custom reagents and scoring-criteria are developed in parallel. Biomarker testing and clinical decision making would profit from harmonized PD-L1 diagnostics. To assess interobserver concordance and PD-L1 immunohistochemistry staining patterns, 15 pulmonary carcinoma resection specimens (adenocarcinoma: n=11, squamous-cell carcinoma: n=4) were centrally stained with the assays 28-8, 22C3, SP142, and SP263 according to clinical trial protocols. The slides were evaluated independently by nine pathologists. Proportions of PD-L1-positive carcinoma cells and immune cells were scored according to a 6-step system that integrates the criteria employed by the four PD-L1 immunohistochemistry assays. Proportion scoring of PD-L1-positive carcinoma cells showed moderate interobserver concordance coefficients for the 6-step scoring system (Light's kappa=0.47–0.50). The integrated dichotomous proportion cut-offs (≥1, ≥5, ≥10, ≥50%) showed good concordance coefficients (κ=0.6–0.8). Proportion scoring of PD-L1-positive immune cells yielded low interobserver concordance coefficients both for the 6-step-score (κ<0.2) and the dichotomous cut-offs (κ=0.12–0.25). The assays 28-8 and 22C3 stained similar proportions of carcinoma cells in 12 of 15 cases. SP142 stained fewer carcinoma cells compared to 28-8, 22C3, and SP263 in four cases, whereas SP263 stained more carcinoma cells in nine cases. SP142 and SP263 stained immune cells more intensely. The data indicate that carcinoma cells can be reproducibly scored in PD-L1 immunohistochemistry for pulmonary adenocarcinoma and squamous-cell carcinoma. No differences in interobserver concordance were noticed among the tested assays. The scoring of immune cells yielded low concordance rates and might require specific standardization. The four tested PD-L1 assays did not show comparable staining patterns in all cases. Thus, studies that correlate staining patterns and response to immunotherapy are required to test the significance of the observed differences.
ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the ...efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.
By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.
Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.
Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.
Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and ...TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non‐small cell tumors and secondary transitions from non‐small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of “small cell‐ness” based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT‐signaling in the context of mutual bi‐allelic RB1 and TP53 lesions. Additionaly, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH‐ASCL1‐RB‐p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon‐based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.
What's new?
Using next generation sequencing and establishing features of ‘small cell‐ness’, we identified a NOTCH‐ASCL1‐RB1‐TP53 signaling axis driving small cell cancers. In contrast to the previously described bi‐allelic RB1/TP53 loss in neuroendocrine stem cells as origin of primary small cell neuroendocrine cancers, the NOTCH‐ASCL1 mediated signaling defines an alternative pathway driving secondary small cell neuroendocrine cancers arising from non‐small cell cancers. Moreover, we show a preclinical rational for therapeutically testing WNT‐inhibitors in small cell cancers.
•The CRISP registry collects representative, nationwide data on NSCLC in Germany.•CRISP mirrors the treatment and disease trajectory of patients with advanced NSCLC.•Biomarker testing rates increased ...over time, yet are still improvable.•17.7 % of the patients presented with a druggable alteration.•Median PFS was longer for patients with a druggable EGFR or ALK alteration.
An increasing number of treatment-determining biomarkers has been identified in non-small cell lung cancer (NSCLC) and molecular testing is recommended to enable optimal individualized treatment. However, data on implementation of these recommendations in the “real-world” setting are scarce. This study presents comprehensive details on the frequency, methodology and results of biomarker testing of advanced NSCLC in Germany.
This analysis included 3,717 patients with advanced NSCLC (2,921 non-squamous; 796 squamous), recruited into the CRISP registry at start of systemic therapy by 150 German sites between December 2015 and June 2019. Evaluated were the molecular biomarkers EGFR, ALK, ROS1, BRAF, KRAS, MET, TP53, RET, HER2, as well as expression of PD-L1.
In total, 90.5 % of the patients were tested for biomarkers. Testing rates were 92.2 % (non-squamous), 70.7 % (squamous) and increased from 83.2 % in 2015/16 to 94.2% in 2019. Overall testing rates for EGFR, ALK, ROS1, and BRAF were 72.5 %, 74.5 %, 66.1 %, and 53.0 %, respectively (non-squamous). Testing rates for PD-L1 expression were 64.5 % (non-squamous), and 58.5 % (squamous). The most common testing methods were immunohistochemistry (68.5 % non-squamous, 58.3 % squamous), and next-generation sequencing (38.7 % non-squamous, 14.4 % squamous). Reasons for not testing were insufficient tumor material or lack of guideline recommendations (squamous). No alteration was found in 37.8 % (non-squamous), and 57.9 % (squamous), respectively. Most common alterations in non-squamous tumors (all patients/all patients tested for the respective biomarker): KRAS (17.3 %/39.2 %), TP53 (14.1 %/51.4 %), and EGFR (11.0 %/15.1 %); in squamous tumors: TP53 (7.0 %/69.1 %), MET (1.5 %/11.1 %), and EGFR (1.1 %/4.4 %). Median PFS (non-squamous) was 8.7 months (95 % CI 7.4–10.4) with druggable EGFR mutation, and 8.0 months (95 % CI 3.9–9.2) with druggable ALK alterations.
Testing rates in Germany are high nationwide and acceptable in international comparison, but still leave out a significant portion of patients, who could potentially benefit. Thus, specific measures are needed to increase implementation.
Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal ...cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.
The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics ...with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.
The present prospective study aimed at determining the impact of cell-free tumor DNA (ct-DNA), CA125 and HE4 from blood and ascites for quantification of tumor burden in patients with advanced ...high-grade serous epithelial ovarian cancer (EOC).
Genomic DNA was extracted from tumor FFPE and ct-DNA from plasma before surgery and on subsequent post-surgical days. Extracted DNA was subjected to hybrid-capture based next generation sequencing. Blood and ascites were sampled before surgery and on subsequent post-surgical days. 20 patients (10 undergoing complete resection (TR0), 10 undergoing incomplete resection (TR>0)) were included.
The minor allele frequency (MAF) of TP53 mutations in ct-DNA of all patients with TR0 decreased significantly, compared to only one patient with TR>0. It was not possible to distinguish between patients with TR0 and patients with TR>0, using CA125 and HE4 from blood and ascites.
Based upon the present findings, ct-DNA assessment in patients with high-grade serous EOC might help to better determine disease burden compared to standard tumor markers. Further studies should prospectively evaluate whether this enhancement of accuracy can help to optimize management of patients with EOC.
Objectives To analyze circulating microRNAs (miRNA) in serum as non-invasive biomarker in patients with localized prostate cancer (PCA), benign prostate hyperplasia (BPH) and healthy individuals ...(HI). Methods Total RNA was isolated from serum samples and the circulating levels of different RNA species (miRNA, miR-16; small nuclear RNA, RNU1A-1 ; messenger RNA, HPRT1 ), as well as of 4 oncogenic miRNAs ( miR-26a , miR-32 , miR-195 , miR-let7i ), were determined using a quantitative real-time polymerase chain reaction. We also evaluated miRNA levels in a second cohort of 10 PCA patients in cancer/nonmalignant tissue, and pre- and post-prostatectomy serum samples. Results The levels of miR-16 and RNU1A - 1 were reliably measured, whereas HPRT1 levels were often below the detection limit of our assay. Circulating oncogenic miRNA levels were different, and especially the miR-26a level allowed sensitive (89%) discrimination of PCA and BPH patients at a moderate specificity (56%; area under the curve AUC: 0.703); the analysis of oncogenic miRNAs in combination increased the diagnostic accuracy (sensitivity: 78.4%; specificity: 66.7%; AUC: 0.758). Despite the low number of patients limiting the statistical power of the study, we observed correlations with clinical-pathologic parameters: miR-16 , miR - 195 , and miR-26a were significantly correlated with surgical margin positivity; miR - 195 and miR-let7i were significantly correlated with the Gleason score. Tissue miRNA levels were correlated with preprostatectomy miRNA levels in serum, and serum miRNA decreased after prostatectomy, thereby indicating tumor-associated release of miRNA. Conclusions Tumor-associated miRNAs in serum allow noninvasive discrimination of PCA and BPH.
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