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Background: Comprehensive genomic profiling (CGP) is frequently adopted to direct the clinical care of myeloid neoplasms and solid tumors, but its utility in the care of lymphoid and histiocytic cancers is less well defined., Methods: In this study, we aimed to evaluate the frequency at which mutations identified by CGP altered management in non-myeloid hematologic malignancies. We retrospectively examined the CGP results of 105 samples from 101 patients with non-myeloid hematologic malignancies treated at an academic medical center who had CGP testing between 2014 and 2021., Results: CGP revealed one or more pathogenic or likely pathogenic variant in 92 (88%) of samples and 73 (72%) of tested patients had one or more mutations with diagnostic, prognostic, or therapeutic significance. The identification of a resistance variant resulted in the suspension of the active treatment or affected subsequent treatment choice in 9 (69%) out of 13 patients. However, the presence of a therapy sensitizing variant only led to consideration of a biomarker-directed therapy in 6 (10%) out of 61 patients., Conclusions: Overall, CGP of non-myeloid hematologic malignancies identified clinically significant variants in 72% of patients and resulted in a change in management in 22% of patients., Competing Interests: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. C.L. has performed consulting for Rigel and ad board for Autolus and ADC Therapeutics. B.A.C. has received honoraria from Foundation Medicine and served on an ad board for Guardant. M.F.G is employed by Labcorp. J.S. performs consulting for Abbvie, Astellas, AstraZeneca, Bayer, Beigene, Daiichi-Sankyo, Eli Lilly, GE Healthcare, GSK, Ipsen, Johnson and Johnson, Jazz Pharmaceuticals, Merck, Natera, Pfizer, Roche/Genentech, Regeneron, Sanofi, Taiho, Takeda, and Xilio Therapeutics, owns stock options in Triumvira Immunologics, and receives research funding from Abbvie, Amgen, Apollo Therapeutics, AStar D3, Bayer, Beigene, Curegenix, Daiichi-Sankyo, Eli Lilly, Erasca, GSK, Leap Therapeutics, Novartis, Pfizer, Quanta Therapeutics, Revolution Medicines, and Roche/ Genentech. The remaining authors have no relevant disclosures.

The molecular mechanisms that regulate breast cancer cell (BCC) metastasis and proliferation within the leptomeninges (LM) are poorly understood, which limits the development of effective therapies. In this work, we show that BCCs in mice can invade the LM by abluminal migration along blood vessels that connect vertebral or calvarial bone marrow and meninges, bypassing the blood-brain barrier. This process is dependent on BCC engagement with vascular basement membrane laminin through expression of the neuronal pathfinding molecule integrin α6. Once in the LM, BCCs colocalize with perivascular meningeal macrophages and induce their expression of the prosurvival neurotrophin glial-derived neurotrophic factor (GDNF). Intrathecal GDNF blockade, macrophage-specific GDNF ablation, or deletion of the GDNF receptor neural cell adhesion molecule (NCAM) from BCCs inhibits breast cancer growth within the LM. These data suggest integrin α6 and the GDNF signaling axis as new therapeutic targets against breast cancer LM metastasis.

Background: Gastroesophageal cancer is a major cause of cancer-related mortality worldwide. Treatment of both early stage and advanced disease remains highly reliant on cytotoxic chemotherapy. About 4-24% of gastroesophageal cancers are microsatellite instability high (MSI-H). The MSI-H subtype is associated with favorable prognosis, resistance to cytotoxic chemotherapy, and sensitivity to immune checkpoint inhibitors (ICI). Recent studies have demonstrated promising activity of ICIs in the MSI-H subtype, resulting in fundamental changes in the management of MSI-H gastroesophageal adenocarcinoma., Purpose: In this review, we discuss the prevalence, characteristics, prognosis, and management of MSI-H gastroesophageal adenocarcinoma, with a focus on recent and ongoing studies that have changed the landscape of treatment for the MSI-H subtype. We also discuss current challenges in the management of resectable and advanced MSI-H gastroesophageal cancer, including the need for more accurate biomarkers of response to ICI therapy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA 2'- O -methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs has been demonstrated to impact cellular processes, including translation and splicing. In addition, Nm modifications at the 5' cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self-mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field., (© 2024 Zhou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Introduction: The KRAS G12C inhibitor sotorasib was approved for treating advanced NSCLC in the second line or later on the basis of the CodeBreaK100 trial. Nevertheless, data on the real-world efficacy and safety of sotorasib, and to its optimal dose, remain limited., Methods: Patients treated with sotorasib for NSCLC through the Veterans Health Administration were retrospectively identified from the Corporate Data Warehouse. Survival, response, and toxicity data were obtained from chart review., Results: Among the 128 patients treated with sotorasib through the Veterans Health Administration, objective response rate was 34%, progression-free survival (PFS) six months, and overall survival 12 months. Similar PFS was observed among the 16 patients who received frontline sotorasib without any prior systemic therapy for NSCLC. Toxicity leading to sotorasib interruption or dose reduction occurred in 37% of patients, whereas sotorasib discontinuation for toxicity occurred in 25%. Notably, sotorasib dose reduction was associated with substantially improved PFS and OS., Conclusions: In this real-world study, the observed efficacy of sotorasib was similar to the results of CodeBreaK100. Patients who received frontline sotorasib had similar PFS to our overall cohort, suggesting that first-line sotorasib monotherapy may benefit patients who are not eligible for chemotherapy. Toxicities leading to sotorasib interruption, dose reduction, or discontinuation were common. Sotorasib dose reduction was associated with improved survival, suggesting that sotorasib dose reduction may not compromise efficacy., Competing Interests: Dr. Lin reported serving as a consultant for Rigel and on the advisory board of Autolus. Dr. Dowell participated in advisory boards for Mirati, Astra Zeneca, Catalyst, and Takeda. Dr. Kelley reported research funding (paid to his institution) from 10.13039/100004336Novartis, 10.13039/100002491Bristol-Myers Squibb, 10.13039/100009857Regeneron, and Mirati Therapeutics. The remaining authors declare no conflict of interest.

Acquired von Willebrand syndrome (AVWS) is a rare disorder that is characterised by an acquired deficiency of von Willebrand factor. AVWS was suspected in a patient with type III von Willebrand disease (VWD) who did not respond to factor replacement therapy. Given the crucial implications for management, we describe this patient's clinical presentation, diagnosis and periprocedural management. To facilitate pericardiocentesis, periprocedural management included steroids, intravenous immunoglobulin and factor replacement therapy. In other patients with suspected immune-mediated AVWS, a similar approach may be effective. This case also highlights the importance of distinguishing AVWS from inherited VWD., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Immune checkpoint inhibitors (ICIs) are used for an increasing number of indications across various tumor types, as well as several tumor-agnostic indications in patients with advanced cancer. Although many patients benefit from ICI therapy, others do not, highlighting a need for better predictive biomarkers. Tumor mutational burden (TMB) reflects the global number of mutations within a tumor and has been widely explored as a predictive biomarker of ICI response. The current tumor type-agnostic US Food and Drug Administration approval of pembrolizumab for metastatic solid tumors defines high TMB (TMB-H) as ≥10 mut/Mb as measured by FoundationOne CDx. This fixed cutoff may not be the ideal value across all solid tumors., Methods: We performed a retrospective analysis of the association of survival outcomes with TMB in patients treated with ICI for five major cancer types, using real-world data from the VA. Survival was measured from initiation of ICI, and Kaplan-Meier survival curves were compared by log-rank test., Results: Overall survival (OS) was significantly longer for patients with TMB-H versus TMB low tumors in non-small-cell lung cancer (NSCLC; n = 1,593), head and neck (H&N) cancer (n = 222), and urothelial cancer (n = 332). OS was not significantly different based on TMB status in melanoma (n = 207) or esophageal/gastric cancer (n = 248)., Conclusion: Consistent with previous studies, a predictive value of TMB ≥10 mut/Mb for ICI response was found in NSCLC and H&N, but not in esophageal/gastric cancer. Although inconclusive in the literature, significant association was found in urothelial cancer. The predictive value of TMB in melanoma was inconclusive. Our analysis does not support the use of a fixed threshold for TMB as a standalone predictive biomarker for ICI across all solid tumors.

The tyrosine kinase inhibitor dasatinib is approved for the treatment of chronic myeloid leukaemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL). Patients on dasatinib can rarely develop a form of benign reversible reactive lymphadenopathy termed follicular lymphoid hyperplasia (FLH). Here, we describe a patient with Ph+ ALL who developed follicular lymphoma (FL) after prolonged treatment with dasatinib and who had complete remission of FL after discontinuation of dasatinib. This case suggests that dasatinib-associated FLH could be a premalignant condition that can transform into FL. Moreover, withdrawal of dasatinib may be sufficient for remission of dasatinib-associated FL., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Neurotrophic tyrosine receptor kinase 1-3 ( NTRK1-3 ) gene fusions are found in a broad range of tumor types. Clinical trials demonstrated high response rates to tropomyosin receptor kinase (TRK) inhibitors in NTRK fusion-positive cancers, but few reports have described real-world experience with these targeted agents. We evaluated the prevalence of NTRK fusions and the outcomes with TRK inhibitor therapy in a real-world population of patients in the Veterans Health Administration., Methods: Patients with NTRK fusions or rearrangements were identified from the Veterans Affairs (VA) National Precision Oncology Program (NPOP), and patients who were prescribed TRK inhibitors were identified from the Corporate Data Warehouse. Baseline data and clinical outcomes were obtained by retrospective review of medical records., Results: A total of 33 patients with NTRK fusions or rearrangements were identified, including 25 patients comprising 0.12% of all patients with solid tumors sequenced through VA NPOP. Twelve patients with NTRK fusions or rearrangements were treated with TRK inhibitors, none of whom had objective responses. Eight patients experienced toxicities leading to drug interruption, dose reduction, or discontinuation., Conclusion: In this retrospective study of VA patients, NTRK fusions and rearrangements were less common than in previous studies, and objective responses to TRK inhibitors were not observed. Real-world experience with TRK inhibitors differs markedly from clinical trial findings, possibly due to differences in patient demographics, tumor types, and sequencing methods. Our findings highlight the need to study TRK inhibitors in the real-world setting and in populations underrepresented in clinical trials.

Purpose: Intrapatient heterogeneity of programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB) in gastroesophageal adenocarcinoma (GEA) could influence their roles as predictive biomarkers for response to immune checkpoint inhibitors (ICI). In this retrospective analysis, we evaluated the spatiotemporal heterogeneity and prognostic relevance of PD-L1 expression and TMB in GEA., Experimental Design: A cohort of 211 patients with stage II-IV GEA was retrospectively reviewed for a total of 407 tumor samples with PD-L1 expression data and 319 tumor samples with TMB data. PD-L1 status was defined as positive if combined positive score (CPS) ≥1 using the 22C3 pharmDx assay. TMB levels were categorized as low, intermediate, or high (≤5, 5-15, or >15 mutations/Mb), or using a single threshold (<10 or ≥10 mutation/Mb), determined by next-generation sequencing using a targeted gene panel., Results: Of 407 tumors, 56% were PD-L1 negative and 44% PD-L1 positive. Of 319 tumors, 50% were TMB-low, 45% TMB-intermediate, and 5% TMB-high; 86% had <10 and 14% ≥10 mutations/Mb. TMB level was significantly associated with MSI-status. PD-L1 expression and TMB exhibited marked spatial heterogeneity between baseline primary and metastatic tumors (61% and 69% concordance), and temporal heterogeneity between tumors before and after chemotherapy (57%-63% and 73%-75% concordance). PD-L1 expression and TMB were not significantly associated with overall survival., Conclusions: PD-L1 expression and TMB exhibit marked spatial and temporal heterogeneity in GEA. This heterogeneity should be considered when obtaining tumor samples for molecular testing and when deciding whether ICI therapy is appropriate. See related commentary by Klempner et al., p. 6401 ., (©2020 American Association for Cancer Research.)

N 6 -methyladenosine (m 6 A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m 6 A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m 6 A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m 6 A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m 6 A-mediated splicing regulation, in which an m 6 A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m 6 A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pseudouridine (Ψ) is the most abundant RNA modification in cellular RNA present in tRNA/rRNA/snRNA and also in mRNA and long noncoding RNA (lncRNA). Elucidation of Ψ function in mRNA/lncRNA requires mapping and quantitative assessment of its modification fraction at single-base resolution. The most widely used Ψ mapping method for mRNA/lncRNA relies on its reaction with N -Cyclohexyl- N '-(2-morpholinoethyl)carbodiimide (CMC), forming an adduct with the Ψ base in RNA that is detectable by reverse transcription (RT) stops. However, this method has not produced consistent Ψ maps in mRNAs; furthermore, available protocols do not lend confidence to the estimation of Ψ fraction at specific sites, which is a crucial parameter for investigating the biological relevance of mRNA modifications. Here we develop a quantitative RT-PCR based method that can detect and quantify the modification fraction of target Ψ sites in mRNA/lncRNA, termed C MC-RT and l igation a ssisted P CR analysis of Ψ modification (CLAP). The method still relies on RT stop at a CMC-Ψ site, but uses site-specific ligation and PCR to generate two distinct PCR products in the same sample, corresponding to the modified and unmodified site, that are visualized by gel electrophoresis. CLAP not only requires a small amount of cellular RNA to validate Ψ sites but also determines the Ψ fraction semiquantitatively at target sites in mRNA/lncRNA. We determined the Ψ status of four mRNA sites and one lncRNA site whose modification fractions range from 30% to 84% in three human cell lines. Our method enables precise mapping and assessment of Ψ modification levels in low abundance cellular RNAs., (© 2019 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

The reversible N 6 -methyladenosine (m 6 A) modification of eukaryotic messenger RNAs (mRNAs) is a widespread regulatory mechanism that impacts every step in the mRNA life cycle. The effect of m 6 A on mRNA fate depends on the binding of "m 6 A reader" proteins - RNA binding proteins that specifically bind to RNAs containing m 6 A. Here, we describe an RNA pull-down method that can be used to identify novel m 6 A reader proteins starting from a known m 6 A-modified site in cellular or viral RNA. We further describe how a combination of immunoprecipitation-based sequencing methods can be used to identify m 6 A-modified sites bound by an m 6 A reader protein on a transcriptome-wide level. The discovery of new m 6 A reader proteins and their m 6 A-modified targets would provide further insight into the mechanisms and functions of m 6 A in the cell., (Copyright © 2017 Elsevier Inc. All rights reserved.)

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and affects almost every stage of the mRNA life cycle. The YTH-domain proteins can specifically recognize m6A modification to control mRNA maturation, translation and decay. m6A can also alter RNA structures to affect RNA-protein interactions in cells. Here, we show that m6A increases the accessibility of its surrounding RNA sequence to bind heterogeneous nuclear ribonucleoprotein G (HNRNPG). Furthermore, HNRNPG binds m6A-methylated RNAs through its C-terminal low-complexity region, which self-assembles into large particles in vitro. The Arg-Gly-Gly repeats within the low-complexity region are required for binding to the RNA motif exposed by m6A methylation. We identified 13,191 m6A sites in the transcriptome that regulate RNA-HNRNPG interaction and thereby alter the expression and alternative splicing pattern of target mRNAs. Low-complexity regions are pervasive among mRNA binding proteins. Our results show that m6A-dependent RNA structural alterations can promote direct binding of m6A-modified RNAs to low-complexity regions in RNA binding proteins., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this issue of Molecular Cell, Wang et al. (2016a) report crystal structures of the core of the METTL3/METTL14 m(6)A methyltransferase complex and propose how the two subunits interact and cooperate to bind and methylate RNA., (Copyright © 2016. Published by Elsevier Inc.)

N(6)-Methyladenosine (m(6)A) is a reversible and abundant internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) with roles in RNA processing, transport, and stability. Although m(6)A does not preclude Watson-Crick base pairing, the N(6)-methyl group alters the stability of RNA secondary structure. Since changes in RNA structure can affect diverse cellular processes, the influence of m(6)A on mRNA and lncRNA structure has the potential to be an important mechanism for m(6)A function in the cell. Indeed, an m(6)A site in the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently shown to induce a local change in structure that increases the accessibility of a U5-tract for recognition and binding by heterogeneous nuclear ribonucleoprotein C (HNRNPC). This m(6)A-dependent regulation of protein binding through a change in RNA structure, termed "m(6)A-switch", affects transcriptome-wide mRNA abundance and alternative splicing. To further characterize this first example of an m(6)A-switch in a cellular RNA, we used nuclear magnetic resonance and Förster resonance energy transfer to demonstrate the effect of m(6)A on a 32-nucleotide RNA hairpin derived from the m(6)A-switch in MALAT1. The observed imino proton nuclear magnetic resonance resonances and Förster resonance energy transfer efficiencies suggest that m(6)A selectively destabilizes the portion of the hairpin stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure. The m(6)A-modified hairpin has a predisposed conformation that resembles the hairpin conformation in the RNA-HNRNPC complex more closely than the unmodified hairpin. The m(6)A-induced structural changes in the MALAT1 hairpin can serve as a model for a large family of m(6)A-switches that mediate the influence of m(6)A on cellular processes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Group II introns are self-splicing, retrotransposable ribozymes that contribute to gene expression and evolution in most organisms. The ongoing identification of new group II introns and recent bioinformatic analyses have suggested that there are novel lineages, which include the group IIE and IIF introns. Because the function and biochemical activity of group IIE and IIF introns have never been experimentally tested and because these introns appear to have features that distinguish them from other introns, we set out to determine if they were indeed self-splicing, catalytically active RNA molecules. To this end, we transcribed and studied a set of diverse group IIE and IIF introns, quantitatively characterizing their in vitro self-splicing reactivity, ionic requirements, and reaction products. In addition, we used mutational analysis to determine the relative role of the EBS-IBS 1 and 2 recognition elements during splicing by these introns. We show that group IIE and IIF introns are indeed distinct active intron families, with different reactivities and structures. We show that the group IIE introns self-splice exclusively through the hydrolytic pathway, while group IIF introns can also catalyze transesterifications. Intriguingly, we observe one group IIF intron that forms circular intron. Finally, despite an apparent EBS2-IBS2 duplex in the sequences of these introns, we find that this interaction plays no role during self-splicing in vitro. It is now clear that the group IIE and IIF introns are functional ribozymes, with distinctive properties that may be useful for biotechnological applications, and which may contribute to the biology of host organisms.

It has long been understood that many of the same manipulations that increase longevity in Caenorhabditis elegans also increase resistance to various acute stressors, and vice-versa; moreover these findings hold in more complex organisms as well. Nevertheless, the mechanistic relationship between these phenotypes remains unclear, and in many cases the overlap between stress resistance and longevity is inexact. Here we review the known connections between stress resistance and longevity, discuss instances in which these connections are absent, and summarize the theoretical explanations that have been posited for these phenomena.