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Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1–4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin–IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.

After a diagnosis of unilateral breast cancer, increasing numbers of patients are requesting contralateral prophylactic mastectomy (CPM), the surgical removal of the healthy breast after diagnosis of unilateral breast cancer. It is important for the community of breast cancer specialists to provide meaningful guidance to women considering CPM. This manifesto discusses the issues and challenges of CPM and provides recommendations to improve oncological, surgical, physical and psychological outcomes for women presenting with unilateral breast cancer: (1) Communicate best available risks in manageable timeframes to prioritise actions; better risk stratification and implementation of risk-assessment tools combining family history, genetic and genomic information, and treatment and prognosis of the first breast cancer are required; (2) Reserve CPM for specific situations; in women not at high risk of contralateral breast cancer (CBC), ipsilateral breast-conserving surgery is the recommended option; (3) Encourage patients at low or intermediate risk of CBC to delay decisions on CPM until treatment for the primary cancer is complete, to focus on treating the existing disease first; (4) Provide patients with personalised information about the risk:benefit balance of CPM in manageable timeframes; (5) Ensure patients have an informed understanding of the competing risks for CBC and that there is a realistic plan for the patient; (6) Ensure patients understand the short- and long-term physical effects of CPM; (7) In patients considering CPM, offer psychological and surgical counselling before surgery; anxiety alone is not an indication for CPM; (8) Eliminate inequality between countries in reimbursement strategies; CPM should be reimbursed if it is considered a reasonable option resulting from multidisciplinary tumour board assessment; (9) Treat breast cancer patients at specialist breast units providing the entire patient-centred pathway.

Background: The HER2-specific tyrosine kinase inhibitor (TKI) tucatinib (Tuca) recently approved for advanced HER2+ breast cancer is making a move towards the early setting. Given its growing use, resistance is inevitable, as observed in the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on Tuca. Driven by the prevailing lack of knowledge about the mechanisms of resistance, in this study, we sought to define these mechanisms and identify treatment strategies to overcome them. We previously reported (SABCS 2021) that our BT474 TucaR models acquired EGFR amplification and showed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6. Since the HER pathway is activated by ligands, here we aim to assess if hyperactivation of EGFR via high levels of its ligands is an alternative mechanism of Tuca resistance. Materials and Methods: Our recently developed HER2+ BT474 (ATCC and AZ) cell models with acquired resistance to Tuca (TucaR) developed through long-term exposure to gradually increasing doses of Tuca and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (western blot) characterization were performed. Changes in cell growth and migration were assessed by methylene blue and Incucyte wound healing assays, respectively. Results: RNA-seq analysis demonstrated that the levels of TGFα was significantly higher in our BT474 TucaR models compared to P cells. Our results now demonstrate that exogenous supplementation of EGF to BT474-P cells rescues the Tuca-mediated inhibition of pEGFR, pHER2, and the downstream pAKT, pERK, and pS6 levels. Exogenous EGF was also found to reduce the levels of apoptosis, as assessed by cleaved PARP, mitigating the Tuca-induced cell death. Exogenous EGF or TGFα rendered naïve BT474 and SKBR3 cells resistant to Tuca while neratinib, a pan-HER TKI, effectively inhibited this ligand-driven cell growth. We previously showed that the HER signaling reactivation observed in our EGFR-amplified TucaR cells was inhibited by the EGFR-specific TKI gefitinib (Gef) (SABCS 2021) and that the TucaR cells displayed enhanced migratory capabilities (AACR 2022). Here, we demonstrate that in addition to curbing the growth of TucaR cells, Gef, either alone or together with Tuca, also markedly reverts the migration of the TucaR cells. Knockdown (KD) of EGFR but not HER2 selectively and substantially inhibited the migration of the TucaR cells. KD of EGFR also had a marked cell killing effect on only the TucaR cells, whereas HER2 KD inhibited the growth of P but not TucaR cells. Our findings are consistent with the notion that while the P cells are functionally dependent on HER2, in TucaR cells the survival dependence could be rewired to rely primarily on the hyperactive EGFR signaling. Genomic analysis further revealed that in addition to EGFR amplification, the AZ TucaR cells also acquired a gain of YES1, a src family receptor tyrosine kinase implicated in cancer cell growth, invasion, and metastasis. Functional studies using 2 siRNAs, however, showed that YES1 KD had no effect on the growth of TucaR cells, and the migration of both TucaR and P cells was equally affected by YES1 KD, precluding the potential role of YES1 in driving the resistant and enhanced migratory phenotypes. Conclusions: Hyperactivation of the EGFR pathway via amplification of EGFR or increased expression of its ligands confers resistance to Tuca, which may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Given the rapidly evolving treatment landscape of HER2+ breast cancer and biomarkers of resistance, our novel findings have potentially crucial therapeutic implications and suggest that rationally sequencing the currently available TKIs may be clinically important. Citation Format: Fu-Tien Liao, Tia Gordon, Chia Chia Liu, Pier Selenica, Yingjie Zhu, Juber Patel, Sarmistha Nanda, Lanfang Qin, Xiaoyong Fu, Andrea Gazzo, Antonio Marra, Juan Blanco-Heredia, Britta Weigelt, Jorge Reis-Filho, C. Kent Osborne, Mothaffar Rimawi, Rachel Schiff, Jamunarani Veeraraghavan. Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-17.

Background We sought to determine the prevalence of germline pathogenic variants (gPVs) in unselected patients with endometrial cancer (EC), define biallelic gPVs within tumors, and describe their associations with clinicopathologic features. Methods Germline assessment of at least 76 cancer predisposition genes was performed in patients with EC undergoing clinical tumor-normal Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing from January 1, 2015, to June 30, 2021. In patients with gPVs, biallelic alterations in ECs were identified through analysis of loss of heterozygosity and somatic PVs. Clinicopathologic variables were compared using nonparametric tests. Results Of 1625 patients with EC, 216 (13%) had gPVs, and 15 patients had 2 gPVs. There were 231 gPVs in 35 genes (75 [32%] high penetrance; 39 [17%] moderate penetrance; and 117 [51%] low, recessive, or uncertain penetrance). Compared with those without gPVs, patients with gPVs were younger (P = .002), more often White (P = .009), and less obese (P = .025) and had differences in distribution of tumor histology (P = .017) and molecular subtype (P Conclusions Of unselected patients with EC, 13% had gPVs, with 63% of gPVs in high-penetrance genes (MMR and homologous recombination) exhibiting biallelic inactivation, potentially driving cancer development. This supports germline assessment in EC given implications for treatment and cancer prevention.

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance. Significance: Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.

Mullerian adenosarcoma is an uncommon mesenchymal tumor of the gynecologic tract, usually of uterine origin. Tumors are generally low-grade and associated with good prognosis, whereas high-grade adenosarcomas are rare and less well studied. Herein, we sought to characterize the molecular features of 27 adenosarcomas (primary uterine, n = 19, cervical, n = 3, ovarian, n = 4, peritoneal, n = 1), enriched for high-grade tumors (n = 17) subjected to targeted panel sequencing. Recurrent genetic alterations in adenosarcomas included TP53 mutations (n = 4, 15%), restricted to high-grade cases, BAP1 homozygous deletions (n = 4, 15%), DICER1 mutations (n = 4, 15%), ARID1A mutations (n = 3), TERT promoter mutations (n = 2) and amplification (n = 1), ATRX frameshift mutation/homozygous deletions (n = 3), MDM2 (n = 2), CDK4 (n = 2) and CCNE1 (n = 2) amplifications, as well as alterations involving members of the PI3K (PTEN, n = 3; PIK3CA, n = 4; AKT1, n = 2) and MAPK (KRAS, n = 4, BRAF, n = 2) signaling pathways. One tumor harbored an ESR1-NCOA3 fusion and another had an MLH1 homozygous deletion, associated with loss of MLH1 and PMS2 protein expression. The fraction of genome altered was significantly higher in high-grade compared to low-grade adenosarcomas (P = 0.001). Somatic ATRX frameshift mutations were found in two patients with low-grade adenosarcoma with high-grade recurrences and one case of high-grade adenosarcoma with an adjacent low-grade component. Immunohistochemical analysis for BAP1 revealed loss of nuclear expression in 6/24 (25%) cases, including all 4 tumors with BAP1 deletions. Notably, out of 196 mesenchymal neoplasms of gynecologic origin, BAP1 homozygous deletion was only found in adenosarcomas (4/27,15% adenosarcomas vs 0/169, 0% other mesenchymal neoplasms, P = 0.0003). This study demonstrates that high-grade adenosarcomas are heterogeneous at the molecular level and are characterized by genomic instability and TP53 mutations; ATRX loss may be involved in high-grade transformation of low-grade adenosarcoma; and BAP1 inactivation appears to be a specific pathogenic driver in a subset of adenosarcomas.

INTRODUCTION: Small cell carcinoma of the bladder (SCCB) is a rare variant of bladder cancer with poor outcomes. We evaluated long-term outcomes of nonmetastatic (M0) and metastatic (M1) SCCB and correlated pathologic response with genomic alterations of patients treated with neoadjuvant chemotherapy (NAC). PATIENTS AND METHODS: Clinical history and pathology samples from SCCB patients diagnosed at our institution were reviewed. RESULTS: One hundred and ninety-nine SCCB patients were identified. (M0: 147 [74%]; M1: 52 [26%]). Among M0 patients, 108 underwent radical cystectomy (RC) (NAC: 71; RC only: 23; adjuvant chemotherapy: 14); 14 received chemoradiotherapy; the rest received chemotherapy alone or no cancer-directed therapy. RC-only patients had a median follow-up of 9.1 years, and median disease-free survival (DFS) and overall survival (OS) were 1.1 and 1.2 years, respectively. NAC patients had pathologic response (

Globale Ereignisse und geopolitische Spannungen fordern Demokratien zunehmend heraus. Individuelle und kollektive Grundrechte werden in Krisen eingeschränkt, die Gestaltungshoheit demokratisch gewählter Institutionen verlagert sich in Richtung Exekutive und Expert*innenkreise. Tiefgehende Auswirkungen der Digitalisierung erodieren zunehmend die Wirksamkeit traditioneller Institutionen. Die nachweisbare, ungleicher werdende Verteilung der Einkommen und Vermögen begünstigt Misstrauen und Frustration. Wir beleuchten drei Bereiche – Politik, Unternehmen, Schule – und skizzieren einen übergreifenden dialogischen Ansatz für Innovation und Erneuerung in gesellschaftlichen Krisenfeldern.

Abstract: This study further validates the sport-specific Mental Rotation Test – Basketball (MRT-BB) in which participants solve 24 items regarding basketball plays. The task of each item consists of comparing four alternative stimuli with a criterion stimulus and identifying the two “correct” alternatives. A total of 203 participants (101 females) took part in this experiment in which they solved the original MRT and the MRT-BB. The results replicate the findings of Weigelt and Memmert (2021 ). The number of items attempted declined toward the end of each set, with participants solving more items in the second set and men outperforming women. While participants solved more items on the MRT-BB, performance in both tests was positively correlated. Our replication of the previous results supports the validity of the MRT-BB. The correlation supports the notion that the mental rotation of the sport-specific stimuli is based on more general mental rotation skills.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

A complex relationship between adipose tissue and malignancy, involving an inflammatory response, has been reported. The goal of this work was to assess the prevalence of white adipose tissue (WAT) inflammation in patients with endometrial cancer (EC), and the association with circulating inflammation markers. Furthermore, the aim was to characterize the pathways activated in and the cell type composition of adipose tissue in patients with EC.Adipose tissue and blood samples were prospectively collected from 101 patients with EC at initial surgery. WAT inflammation was determined based on adipocytes surrounded by macrophages forming crown-like structures. Circulating levels of metabolic syndrome-associated and inflammatory markers were quantified. RNA-sequencing was performed on adipose samples (n = 55); differential gene expression, pathway, and cellular decomposition analyses were performed using state-of-the-art bioinformatics methods.WAT inflammation was identified in 46 (45.5%) of 101 EC patients. Dyslipidemia, hypertension, and diabetes mellitus were significantly associated with WAT inflammation (p .05). WAT inflammation was associated with greater body mass index (p .001) and higher circulating levels of leptin, high-sensitivity C-reactive protein, and interleukin-6, as well as lower levels of adiponectin and sex hormone-binding globulin (p .05). Transcriptomic analysis demonstrated increased levels of proinflammatory and pro-neoplastic-related gene expression in inflamed omental adipose tissue.WAT inflammation is associated with metabolic syndrome, obesity, and inflammatory markers, as well as increased expression of proinflammatory and proneoplastic genes.

Background: Clinical trials reported 25-30% pathologic complete response (pCR) rates in HER2+ breast cancer (BC) patients treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2 “addicted” tumors who may benefit from a chemotherapy-sparing strategy. Patients and Methods: Baseline HER2+ BC specimens from TBCRC023 and PAMELA trials of neoadjuvant lapatinib+trastuzumab (plus endocrine therapy in ER+ tumors) were used. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA. Results: In TBCRC023, 72 BCs had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio≥4.6 and %3+ IHC-staining≥97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (wt). For clinical implementation, the classifier was locked as HER2 ratio≥4.5 and %3+ IHC-staining≥90% and PIK3CA-wt and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier’s high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment de-escalation. Conclusions: Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable to chemotherapy plus dual anti-HER2 therapy in unselected patients.

Regularly updated information about states, trends and dynamics of water storage in different spatio-temporal scales has gained increasing importance, especially with a perspective on hydrological extreme events as well as water management issues. Monitoring these storage dynamics is challenging due to the spatial heterogeneity and the contribution of different storage compartments (e.g., near-surface soil moisture, deep unsaturated zone, groundwater). A promising monitoring technique is gravimetry, well suited for the integral observation of different storage compartments. While satellite gravimetry (GRACE, GRACE-FO) provides information on storage variations at a spatially large scale with low spatial and temporal resolution, the opposite is true for terrestrial gravimetry. Ways to combine both satellite and terrestrial gravimetry are addressed and evaluated within the German Collaborative Research Centre TerraQ. For the terrestrial approach, several gravimeters were deployed for continuous monitoring at different locations within Germany. The work presented here takes as an example a forest site within the TERENO observatory of north-eastern Germany, with continuous observations of a superconducting gravimeter (iGrav 033) since 2017.The signal footprint of such a gravimeter typically covers a radius of 0.5 to 2 km, depending on local topography, although most of the signal originates from the direct vicinity of the instrument. Also, the device can sense mass changes beyond this distance, depending on their magnitude (e.g., tides, atmosphere or global hydrological effects). In hydro-gravimetric studies, all non-desired signals are typically removed, resulting in residuals that are representative for the local hydrological effects only. Towards comparing and combining these terrestrial measurements with satellite products, one open question is how representative the terrestrial gravity residuals are in a regional context. With the goal to assess this spatial representativeness, we conducted seasonal relative gravity surveys with 2 CG-6 gravimeters in an extent of roughly 25 by 30 km around the iGrav installation. The survey data were combined with spatial information about topography and land-use. Water storage changes could thus be attributed to each survey point. A joint analysis with the continuous measurements of the superconducting gravimeter at the permanent installation site allowed for mapping the spatial patterns and similarities among all sites.This study is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 434617780 – SFB 1464

Air quality in urban cities with ports can significantly be impacted by shipping. For example Visschedijk and Denier van der Gon (2022) showed that, within Rotterdam area (the Netherlands), sea shipping is the main source of ultra-fine particles (UFP) with a contribution of 56% to total UFP emissions. Within EU-project SCIPPER (Shipping Contributions to Inland Pollution Push for the Enforcement of Regulations) a measurement campaign of six weeks was undertaken at the river Elbe near Hamburg (Germany). Sea going vessels pass by on the way to or from the port of Hamburg, which is located about 10 km upstream of the measurement site. Three different groups measured side-by side different gaseous compounds and aerosols on-shore. The aerosol measurements consisted of different equipment measuring different sizes ranging from 6 to 10 000 nm, also black carbon was measured by two groups. The emission factors (EF) of the different ships were calculated in #/kg fuel as well as mg/kg fuel from these measurements. This provides a unique dataset to better understand the emission characteristics of different ships as well as the comparability of different devices. This comparability is important if in the future legislation to decrease aerosol emissions of shipping is applied and needs to be verified. Results showed the relatively large contribution of mainly ultrafine particles, with 90% of the total amount of particles being smaller 80 nm and 75% of the total particle mass comes from particles smaller 200 nm. The comparison showed very promising results when the same equipment was tested side-by-side (R2 above 0.85 for all size ranges). The comparability of EFs of same sources that were measured by different devices was somewhat lower, but especially for smaller size ranges still promising (R2 of 0.69 for particle number ranging from 90 to 300 nm). For black carbon the experimental set-up proved to be too different (drying vs non-drying and different inlet cut-offs) to give comparable emission factors. Concluding the data showed promising results to be able to use on-shore monitoring in the future to monitor aerosol ship emissions, also when using different equipment.References:Visschedijk, A., Denier van der Gon, H. (2022). UFP emissie in de Rijnmond regio in 2019 (in Dutch). Utrecht: TNO-report R10616.

Pulmonary function is reduced in children after preterm birth. The variety of subgroups ranges from early to late preterm births. Limitations in pulmonary function can be observed even after late preterm birth without signs of bronchopulmonary dysplasia and/or history of mechanical ventilation. Whether this reduction in lung function is reflected in the cardiopulmonary capacity of these children is unclear. This study aims to investigate the impact of moderate to late premature birth on cardiopulmonary function. Cardiopulmonary exercise testing on a treadmill was performed by 33 former preterm infants between 8 and 10 years of age who were born between 32 + 0 and 36 + 6 weeks of gestation and compared with a control group of 19 children born in term of comparable age and sex. The former preterm children achieved comparable results to the term-born controls with respect to most of the cardiopulmonary exercise parameters $$(\dot{V}O_2peak 43.9\pm6.6\frac{\frac{ml}{kg}}{min}\min vs.41.9\pm8.8\frac{ml}{kg}/\min)$$ ( V ˙ O 2 p e a k 43.9 ± 6.6 ml kg min min v s . 41.9 ± 8.8 ml kg / min ) . The only differences were in a slightly higher oxygen uptake efficiency slope $$(OUES\;of\;1.6\;\pm0.4\;vs\;1.4\;\pm\;0.4)$$ ( O U E S o f 1.6 ± 0.4 v s 1.4 ± 0.4 ) and higher peak minute ventilation $$\dot VEpeak\;of\;55.2\pm 11.3 ml/min\;vs.\;49.1\;ml\;\pm\;8.8/min)$$ V ˙ E p e a k o f 55.2 ± 11.3 m l / m i n v s . 49.1 m l ± 8.8 / m i n ) in the group of children born preterm. With respect to heart rate recovery $$(-35.3\;\pm\;13.8\;bpm\;vs.\;-37.2\;bpm\;\pm\;14.0\;after\;1\;min)$$ ( - 35.3 ± 13.8 b p m v s . - 37.2 b p m ± 14.0 a f t e r 1 m i n ) and breathing efficiency $$\dot{(V}E/\dot VCO_2\;of\;35.9\;\pm\;4.1\;vs\;34.0\;\pm4.6)$$ ( V ˙ E / V ˙ C O 2 o f 35.9 ± 4.1 v s 34.0 ± 4.6 ) , there were no significant differences.Conclusion: Children born preterm did not show limitations in cardiopulmonary function in comparison with matched controls. What is Known:• Preterm birth is associated with reduced pulmonary function in later life, this is also true for former late preterms.• As a consequence of being born premature, the lungs have not finished their important embryological development. Cardiopulmonary fitness is an important parameter for overall mortality and morbidity in children and adults and a good pulmonary function is therefore paramount. What is New:• Children born prematurely were comparable to an age- and sex-matched control group with regards to almost all cardiopulmonary exercise variables.• A significantly higher OUES, a surrogate parameter for VO2peak was found for the group of former preterm children, most likely reflecting on more physical exercise in this group. Importantly, there were no signs of impaired cardiopulmonary function in the group of former preterm children.

Root-associated fungi could play a role in determining both the positive relationship between plant diversity and productivity in experimental grasslands, and its strengthening over time. This hypothesis assumes that specialized pathogenic and mutualistic fungal communities gradually assemble over time, enhancing plant growth more in species-rich than in species-poor plots. To test this hypothesis, we used high-throughput amplicon sequencing to characterize root-associated fungal communities in experimental grasslands of 1 and 15 years of age with varying levels of plant species richness. Specifically, we tested whether the relationship between fungal communities and plant richness and productivity becomes stronger with the age of the experimental plots. Our results showed that fungal diversity increased with plant diversity, but this relationship weakened rather than strengthened over the two time points. Contrastingly, fungal community composition showed increasing associations with plant diversity over time, suggesting a gradual build-up of specific fungal assemblages. Analyses of different fungal guilds showed that these changes were particularly marked in pathogenic fungi, whose shifts in relative abundance are consistent with the pathogen dilution hypothesis in diverse plant communities. Our results suggest that root-associated fungal pathogens play more specific roles in determining the diversity–productivity relationship than other root-associated plant symbionts.

The androgen/androgen receptor (AR)-signaling axis plays a central role in the development and growth of prostate cancer (PCa) cells. Upon androgen-binding the AR dimerizes with another AR, translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. In consequence treatments for locally advanced or metastatic PCa are commonly based on androgen deprivation therapies (ADT). Unfortunately, the clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. In the clinic failure of ADT is often characterized by the synthesis of a C-terminally truncated, constitutively active AR splice variant, termed AR-V7. In contrast to AR, the constitutively active AR-V7 does no longer need androgenic stimuli for nuclear entry and/or dimerization. The goal of the present study was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology (NanoBiT) structural complementation assay under androgen stimulation and deprivation conditions. Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 localize in the nucleus. However, after 15 min of androgen stimulation, all AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers localized in the nucleus. In this way, we can show an androgen-regulated interaction between AR-FL and AR-V7 at forming heterodimers that localize in the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus. Treatment with enzalutamide diminished the luminescence of AR-FL homodimers and AR-FL/AR-V7 heterodimers but not AR-V7/AR-V7 homodimers.

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers.Significance:Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring.See related commentary by Liggett and Sankaran, p. 889.This article is highlighted in the In This Issue feature, p. 873

C106 resistant cells drug withdrawal and Patient #12 MSK-Impact data

Patients Progression Cell Free DNA data

Patients Characteristics

C106 cell lines single cell sequencing analysis

RW7213 cell lines sequencing analysis

Supplementary Data from Cancer-Causative Mutations Occurring in Early Embryogenesis

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance.Significance:Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.This article is highlighted in the In This Issue feature, p. 1

Generation of acquired resistance PDX model

Characterization of acquired resistance in vitro models

Oncoprint of baseline alterations in CRC patients who developed resistance to KRAS G12C and EGFR inhibitors

Patients Longitudinal Cell Free DNA data

supplementary tables, figures, and methods

We report the emergence of the novel MEK1V211D gatekeeper mutation in a patient with BRAFK601E colon cancer treated with the allosteric MEK inhibitor binimetinib and the anti-EGFR antibody panitumumab. The MEK1V211D mutation concurrently occurs in the same cell with BRAFK601E and leads to RAF-independent activity but remains regulated by RAF. The V211D mutation causes resistance to binimetinib by both increasing the catalytic activity of MEK1 and reducing its affinity for the drug. Moreover, the mutant exhibits reduced sensitivity to all the allosteric MEK inhibitors tested. Thus, this mutation serves as a general resistance mutation for current MEK inhibitors; however, it is sensitive to a newly reported ATP-competitive MEK inhibitor, which therefore could be used to overcome drug resistance.Significance:We report a resistance mechanism to allosteric MEK inhibitors in the clinic. A MEK1V211D mutation developed in a patient with BRAFK601E colon cancer on MEK and EGFR inhibitors. This mutant increases the catalytic activity of MEK1 and reduces its affinity for binimetinib, but remains sensitive to ATP-competitive MEK inhibitors.This article is highlighted in the In This Issue feature, p. 1143

Supplementary Data from MAPK Pathway Genetic Alterations Are Associated with Prolonged Overall Survival in Low-Grade Serous Ovarian Carcinoma

Supplementary Data from Microsatellite Instability–High Endometrial Cancers with MLH1 Promoter Hypermethylation Have Distinct Molecular and Clinical Profiles

Purpose:To identify molecular subclasses of clear cell ovarian carcinoma (CCOC) and assess their impact on clinical presentation and outcomes.Experimental Design:We profiled 421 primary CCOCs that passed quality control using a targeted deep sequencing panel of 163 putative CCOC driver genes and whole transcriptome sequencing of 211 of these tumors. Molecularly defined subgroups were identified and tested for association with clinical characteristics and overall survival.Results:We detected a putative somatic driver mutation in at least one candidate gene in 95% (401/421) of CCOC tumors including ARID1A (in 49% of tumors), PIK3CA (49%), TERT (20%), and TP53 (16%). Clustering of cancer driver mutations and RNA expression converged upon two distinct subclasses of CCOC. The first was dominated by ARID1A-mutated tumors with enriched expression of canonical CCOC genes and markers of platinum resistance; the second was largely comprised of tumors with TP53 mutations and enriched for the expression of genes involved in extracellular matrix organization and mesenchymal differentiation. Compared with the ARID1A-mutated group, women with TP53-mutated tumors were more likely to have advanced-stage disease, no antecedent history of endometriosis, and poorer survival, driven by their advanced stage at presentation. In women with ARID1A-mutated tumors, there was a trend toward a lower rate of response to first-line platinum-based therapy.Conclusions:Our study suggests that CCOC consists of two distinct molecular subclasses with distinct clinical presentation and outcomes, with potential relevance to both traditional and experimental therapy responsiveness.See related commentary by Lheureux, p. 4838

Purpose:Microsatellite instability–high (MSI-H) endometrial carcinomas are underpinned by distinct mechanisms of DNA mismatch repair deficiency (MMR-D). We sought to characterize the clinical and genetic features of MSI-H endometrial cancers harboring germline or somatic mutations in MMR genes or MLH1 promoter hypermethylation (MLH1ph).Experimental Design:Of > 1,100 patients with endometrial cancer that underwent clinical tumor-normal sequencing, 184 had MSI-H endometrial cancers due to somatic MMR mutations or MLH1ph, or harbored pathogenic germline MMR mutations. Clinicopathologic features, mutational landscape, and tumor-infiltrating lymphocyte (TIL) scores were compared among MMR-D groups using nonparametric tests. Log-rank tests were used for categorical associations; Kaplan–Meier method and Wald test based on Cox proportional hazards models were employed for continuous variables and survival analyses.Results:Compared with patients with germline (n = 25) and somatic (n = 39) mutations, patients with MLH1ph endometrial cancers (n = 120) were older (P < 0.001), more obese (P = 0.001) and had more advanced disease at diagnosis (P = 0.025). MLH1ph endometrial cancers were enriched for JAK1 somatic mutations as opposed to germline MMR-D endometrial cancers which showed enrichment for pathogenic ERBB2 mutations. MLH1ph endometrial cancers exhibited lower tumor mutational burden and TIL scores compared with endometrial cancers harboring germline or somatic MMR mutations (P < 0.01). MLH1ph endometrial cancer patients had shorter progression-free survival (PFS) on univariate analysis, but in multivariable models, stage at diagnosis remained the only predictor of survival. For stage I/II endometrial cancer, two-year PFS was inferior for patients with MLH1ph endometrial cancers compared with germline and somatic MMR groups (70% vs. 100%, respectively).Conclusions:MLH1ph endometrial cancers likely constitute a distinct clinicopathologic entity compared with germline and somatic MMR-D ECs with potential treatment implications.

Purpose:To characterize the somatic mutational landscape, investigate associations between genetic alterations and clinical outcomes, and determine the prevalence of pathogenic germline mutations in low-grade serous ovarian carcinomas (LGSC).Experimental Design:Patients with LGSC tumors who underwent panel-based sequencing of up to 505 genes were identified. Data on somatic and germline mutations; copy-number alterations; and clinicopathologic features, including age at diagnosis, platinum sensitivity, and overall survival (OS), were collected.Results:Following central pathology rereview, 119 patients with LGSC were identified for analysis. Of these, 110 (92%) had advanced-stage disease (stages III/IV). Somatic KRAS (33%), NRAS (11%), EIF1AX (10%), and BRAF (11%) alterations were the most common; MAPK pathway alterations were found in 60% (n = 71) of LGSCs. KRAS mutations were significantly associated with age at diagnosis more than 50 years (P = 0.02) and platinum-sensitive disease (P = 0.03). On multivariate analysis, MAPK pathway alterations (P = 0.02) and platinum sensitivity (P = 0.005) were significantly associated with improved OS. Seventy-nine patients (66%) underwent germline genetic testing; seven pathogenic germline mutations were identified: MUTYH (n = 2), BAP1 (n = 1), RB1 (n = 1), CHEK2 (n = 1), APC (n = 1), and FANCA (n = 1). There were no germline BRCA1/2 mutations. One germline MUTYH-associated LGSC harbored loss-of-heterozygosity at the MUTYH locus, and the patient with the germline BAP1 mutation also harbored a somatic BAP1 frameshift mutation.Conclusions:This study showed that MAPK pathway alterations in LGSC, including KRAS mutations, are independently associated with platinum sensitivity and prolonged survival. Germline data, which were limited, identified few pathogenic germline mutations in patients with LGSC.See related commentary by Veneziani and Oza, p. 4357

Supplementary Data from Molecular Subclasses of Clear Cell Ovarian Carcinoma and Their Impact on Disease Behavior and Outcomes

Purpose:Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease.Experimental Design:Synchronous DCIS (n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS (n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome (n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes (n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods.Results:DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed.Conclusions:Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case.

Supplementary Fig. S3: Evolution of mutational signatures in treatment-naïve patients with de novo synchronous metastatic breast cancer. (A) Heatmap represents the similarity of the observed mutational signatures (blue, see color key) to those previously observed in human cancers (15, 36), separately for the trunk mutations (yellow), the mutations specific to the primary tumor (green) and the mutations specific to the metastatic lesion (pink). (B) Barplots illustrate the mutational signatures of the mutations specific to the primary tumor and the metastatic lesion of Cases 2, 4, 7 and 9. In each panel, the colored barplot illustrates each mutational signature according to the 96 substitution classification defined by the substitution classes (C>A, C>G, C>T, T>A, T>C and T>G bins) and the 5' and 3' sequence context, normalized using the observed trinucleotide frequency in the human exome to that in the human genome. The bars are ordered first by mutation class (C>A/G>T, C>G/G>C, C>T/G>A, T>A/A>T, T>C/A>G, T>G/A >C), then by the 5' flanking base (A, C, G, T) and then by the 3' flanking base (A, C, G, T). *: >20%.