Genetic subgroups inform on pathobiology in adult and pediatric Burkitt lymphoma.

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Fragment optimization and elaboration strategies - the discovery of two lead series of PRMT5/MTA inhibitors from five fragment hits.

Here we describe the early stages of a fragment-based lead discovery (FBLD) project for a recently elucidated synthetic lethal target, the PRMT5/MTA complex, for the treatment of MTAP-deleted cancers. Starting with five fragment/PRMT5/MTA X-ray co-crystal structures, we employed a two-phase fragment elaboration process encompassing optimization of fragment hits and subsequent fragment growth to increase potency, assess synthetic tractability, and enable structure-based drug design. Two lead series were identified, one of which led to the discovery of the clinical candidate MRTX1719.

Anti-tumor efficacy of a potent and selective non-covalent KRASG12D inhibitor.

Recent progress in targeting KRASG12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRASG12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRASG12D with KD and IC50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRASG12D as compared to KRASWT. MRTX1133 also demonstrated potent inhibition of activated KRASG12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRASG12D-mutant cell lines, with median IC50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRASWT cell lines. MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRASG12D-mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRASG12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival.

Design and evaluation of achiral, non-atropisomeric 4-(aminomethyl)phthalazin-1(2H)-one derivatives as novel PRMT5/MTA inhibitors.

MRTX1719 is an inhibitor of the PRMT5/MTA complex and recently entered clinical trials for the treatment of MTAP-deleted cancers. MRTX1719 is a class 3 atropisomeric compound that requires a chiral synthesis or a chiral separation step in its preparation. Here, we report the SAR and medicinal chemistry design strategy, supported by structural insights from X-ray crystallography, to discover a class 1 atropisomeric compound from the same series that does not require a chiral synthesis or a chiral separation step in its preparation.

Design and Discovery of MRTX0902, a Potent, Selective, Brain-Penetrant, and Orally Bioavailable Inhibitor of the SOS1:KRAS Protein-Protein Interaction.

SOS1 is one of the major guanine nucleotide exchange factors that regulates the ability of KRAS to cycle through its "on" and "off" states. Disrupting the SOS1:KRASG12C protein-protein interaction (PPI) can increase the proportion of GDP-loaded KRASG12C, providing a strong mechanistic rationale for combining inhibitors of the SOS1:KRAS complex with inhibitors like MRTX849 that target GDP-loaded KRASG12C. In this report, we detail the design and discovery of MRTX0902─a potent, selective, brain-penetrant, and orally bioavailable SOS1 binder that disrupts the SOS1:KRASG12C PPI. Oral administration of MRTX0902 in combination with MRTX849 results in a significant increase in antitumor activity relative to that of either single agent, including tumor regressions in a subset of animals in the MIA PaCa-2 tumor mouse xenograft model.

Molecular Biomarker Testing for the Diagnosis of Diffuse Gliomas.

CONTEXT.­: The diagnosis and clinical management of patients with diffuse gliomas (DGs) have evolved rapidly over the past decade with the emergence of molecular biomarkers that are used to classify, stratify risk, and predict treatment response for optimal clinical care. OBJECTIVE.­: To develop evidence-based recommendations for informing molecular biomarker testing for pediatric and adult patients with DGs and provide guidance for appropriate laboratory test and biomarker selection for optimal diagnosis, risk stratification, and prediction. DESIGN.­: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. A systematic review of literature was conducted to address the overarching question, "What ancillary tests are needed to classify DGs and sufficiently inform the clinical management of patients?" Recommendations were derived from quality of evidence, open comment feedback, and expert panel consensus. RESULTS.­: Thirteen recommendations and 3 good practice statements were established to guide pathologists and treating physicians on the most appropriate methods and molecular biomarkers to include in laboratory testing to inform clinical management of patients with DGs. CONCLUSIONS.­: Evidence-based incorporation of laboratory results from molecular biomarker testing into integrated diagnoses of DGs provides reproducible and clinically meaningful information for patient management.

Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers.

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)phthalazin-1(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.

Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor.

KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of solid tumors. However, when compared to KRASG12C, selective inhibition of KRASG12D presents a significant challenge due to the requirement of inhibitors to bind KRASG12D with high enough affinity to obviate the need for covalent interactions with the mutant KRAS protein. Here, we report the discovery and characterization of the first noncovalent, potent, and selective KRASG12D inhibitor, MRTX1133, which was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model.

Validating Whole Slide Imaging Systems for Diagnostic Purposes in Pathology.

CONTEXT.­: The original guideline, "Validating Whole Slide Imaging for Diagnostic Purposes in Pathology," was published in 2013 and included 12 guideline statements. The College of American Pathologists convened an expert panel to update the guideline following standards established by the National Academies of Medicine for developing trustworthy clinical practice guidelines. OBJECTIVE.­: To assess evidence published since the release of the original guideline and provide updated recommendations for validating whole slide imaging (WSI) systems used for diagnostic purposes. DESIGN.­: An expert panel performed a systematic review of the literature. Frozen sections, anatomic pathology specimens (biopsies, curettings, and resections), and hematopathology cases were included. Cytology cases were excluded. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, the panel reassessed and updated the original guideline recommendations. RESULTS.­: Three strong recommendations and 9 good practice statements are offered to assist laboratories with validating WSI digital pathology systems. CONCLUSIONS.­: Systematic review of literature following release of the 2013 guideline reaffirms the use of a validation set of at least 60 cases, establishing intraobserver diagnostic concordance between WSI and glass slides and the use of a 2-week washout period between modalities. Although all discordances between WSI and glass slide diagnoses discovered during validation need to be reconciled, laboratories should be particularly concerned if their overall WSI-glass slide concordance is less than 95%.

The environments of the radio galaxy population in simba.

We investigate the environmental properties of the z = 0 radio galaxy population using the simba cosmological hydrodynamic simulation. We identify centrals and satellites from a population of high and low excitation radio galaxies (HERGs and LERGs) in simba, and study their global properties. We find that [Formula: see text] of radio galaxies are satellites, and that there are insignificant differences in the global properties of LERGs based on their central/satellite classification. HERG satellites display lower values of star formation, 1.4 GHz radio luminosity, and Eddington fractions than HERG centrals. We further investigate the environments of radio galaxies and show that HERGs typically live in less dense environments, similar to star-forming galaxies. The environments of high-mass LERGs are similar to non-radio galaxies, but low-mass LERGs live in underdense environments similar to HERGs. LERGs with overmassive black holes reside in the most dense environments, while HERGs with overmassive black holes reside in underdense environments. The richness of a LERG's environment decreases with increasing Eddington fraction, and the environments of all radio galaxies do not depend on radio luminosity for [Formula: see text]. Complementing these results, we find that LERGs cluster on the same scale as the total galaxy population, while multiple HERGs are not found within the same dark matter halo. Finally, we show that high density environments support the growth of HERGs rather than LERGs at z = 2. Simba predicts that with more sensitive surveys, we will find populations of radio galaxies in environments much similar to the total galaxy population.

A Broad-Based Approach to Social Needs Screening in a Pediatric Primary Care Network.

OBJECTIVE: In 2016, the American Academy of Pediatrics recommended universally screening patients for social needs, and in 2018, a quality measure for social needs screening was included in some Massachusetts Medicaid contracts. However, exact guidelines for screening were not provided. We describe the results and implications from a broad-based health-related social needs (HRSN or "social needs") screening program within our large, pediatric primary care network. METHODS: We adapted items from The Health Leads toolkit to create our network's screening tool: The Health Needs Assessment (HNA). We trained staff to use the tool and provided staff with resources to assist families with their needs. All patients with a primary care physician in the network were eligible to complete an HNA. We calculated descriptive statistics and estimated the risk of identifying a social need using multivariable regression analyses. RESULTS: Between June 2018 and May 2019, 100,097 patients completed an HNA; 8% of patients identified a social need, and 33% of those patients requested assistance with the need(s). The multivariate analysis revealed an association between several patient characteristics-health insurance type, age, median household income by zip code, complex chronic conditions, race/ethnicity-and identifying a social need. CONCLUSIONS: Our large, pediatric primary care network successfully instituted a broad-based HRSN screening program in response to state and national screening recommendations. We observed a low prevalence of reported social needs and a propensity to forego assistance. Additional research is needed to understand the barriers around the disclosure of social needs and requests for assistance.

Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.

Collection and Handling of Thoracic Small Biopsy and Cytology Specimens for Ancillary Studies: Guideline From the College of American Pathologists in Collaboration With the American College of Chest Physicians, Association for Molecular Pathology, American Society of Cytopathology, American Thoracic Society, Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, Society of Interventional Radiology, and Society of Thoracic Radiology.

CONTEXT.­: The need for appropriate specimen use for ancillary testing has become more commonplace in the practice of pathology. This, coupled with improvements in technology, often provides less invasive methods of testing, but presents new challenges to appropriate specimen collection and handling of these small specimens, including thoracic small biopsy and cytology samples. OBJECTIVE.­: To develop a clinical practice guideline including recommendations on how to obtain, handle, and process thoracic small biopsy and cytology tissue specimens for diagnostic testing and ancillary studies. METHODS.­: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Core needle biopsy, touch preparation, fine-needle aspiration, and effusion specimens with thoracic diseases including malignancy, granulomatous process/sarcoidosis, and infection (eg, tuberculosis) were deemed within scope. Ancillary studies included immunohistochemistry and immunocytochemistry, fluorescence in situ hybridization, mutational analysis, flow cytometry, cytogenetics, and microbiologic studies routinely performed in the clinical pathology laboratory. The use of rapid on-site evaluation was also covered. RESULTS.­: Sixteen guideline statements were developed to assist clinicians and pathologists in collecting and processing thoracic small biopsy and cytology tissue samples. CONCLUSIONS.­: Based on the systematic review and expert panel consensus, thoracic small specimens can be handled and processed to perform downstream testing (eg, molecular markers, immunohistochemical biomarkers), core needle and fine-needle techniques can provide appropriate cytologic and histologic specimens for ancillary studies, and rapid on-site cytologic evaluation remains helpful in appropriate triage, handling, and processing of specimens.

Single-Organ and Multisystem Hypereosinophilic Syndrome Patients with Gastrointestinal Manifestations Share Common Characteristics.

BACKGROUND: Eosinophilic gastrointestinal diseases (EGIDs) are defined by marked eosinophilia in the gastrointestinal (GI) tract resulting in a wide variety of GI symptoms. When accompanied by blood hypereosinophilia (HE; absolute eosinophil count ≥1500/mm3), EGID can occur as an isolated GI disorder (hypereosinophilic syndrome [HES]/EGID overlap) or as part of a multisystem hypereosinophilic syndrome (Multisystem HES). OBJECTIVE: To describe the GI disease of patients categorized as those with HES/EGID overlap versus those with Multisystem HES. METHODS: Consecutively enrolled patients on a natural history protocol to study eosinophilia with biopsy-proven EGID involving the esophagus, stomach, small-bowel, and/or colon were evaluated for clinical, histopathologic, and endoscopic features by retrospective chart review. RESULTS: Among the 56 patients with EGID and HE, 34 were categorized as HES/EGID overlap and 22 as Multisystem HES. Demographics, GI symptoms, and associated comorbidities were similar between the 2 groups. Multisegment GI eosinophilia was present in 20 of 30 (67%) patients who underwent tissue sampling of all 4 GI segments. Tissue eosinophilia in all 4 GI segments was found in 5 of 30 (17%) patients. Dietary therapy was more common in patients with HES/EGID overlap (65% vs 23%, P = .0028). Patients with Multisystem HES were more likely to receive glucocorticoids (100% vs 79%, P = .0349) and nonglucocorticoid systemic therapies (77% vs 38%, P = .0061). One-third (8 of 22) of patients with Multisystem HES presented with isolated GI symptoms before developing extraintestinal manifestations at a median of 1 year (range, 0.25-15 years). CONCLUSION: There are striking clinical similarities between patients with Multisystem HES and those with HES/EGID overlap, despite differing treatment approaches. Moreover, Multisystem HES can present with isolated GI involvement. Larger prospective studies are needed to confirm these findings.

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

Mechanisms of glucocorticoid resistance in hypereosinophilic syndromes.

BACKGROUND: Glucocorticoids (GC) are considered first-line therapy for most patients with hypereosinophilic syndrome (HES). Although response rates are generally high, many patients require moderate to high doses for control of eosinophilia and symptoms, and up to 15% of patients do not respond at all. Despite this, little is known about the mechanisms of GC resistance in patients with HES. OBJECTIVE: To explore the aetiology of GC resistance in HES. METHODS: Clinical data and samples from 26 patients with HES enrolled on a prospective study of GC responsiveness and 23 patients with HES enrolled on a natural history study of eosinophilia for whom response to GC was known were analysed retrospectively. Expression of GC receptor isoforms was assessed by quantitative RT-PCR in purified eosinophils. Serum cytokine levels were quantified by suspension array assay in multiplex. RESULTS: Despite an impaired eosinophil response to GC after 7 days of treatment, the expected rise in absolute neutrophil count was seen in 7/7 GC-resistant patients, suggesting that GC resistance in HES is not a global phenomenon. Eosinophil mRNA expression of glucocorticoid receptor (GR) isoforms (α, ß, and P) was similar between GC-sensitive (n = 20) and GC-resistant (n = 9) patients with HES. Whereas geometric mean serum levels were also comparable between GC-r (n = 11) and GC-s (n = 19) for all cytokines tested, serum IL-5 levels were >100 pg/mL only in GC-r patients. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the mechanism of GC resistance in HES is not due to a global phenomenon affecting all lineages, but may be due, at least in some patients, to impairment of eosinophil apoptosis by increased levels of IL-5.

Quantitative Image Analysis of Human Epidermal Growth Factor Receptor 2 Immunohistochemistry for Breast Cancer: Guideline From the College of American Pathologists.

CONTEXT.­: Advancements in genomic, computing, and imaging technology have spurred new opportunities to use quantitative image analysis (QIA) for diagnostic testing. OBJECTIVE.­: To develop evidence-based recommendations to improve accuracy, precision, and reproducibility in the interpretation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) for breast cancer where QIA is used. DESIGN.­: The College of American Pathologists (CAP) convened a panel of pathologists, histotechnologists, and computer scientists with expertise in image analysis, immunohistochemistry, quality management, and breast pathology to develop recommendations for QIA of HER2 IHC in breast cancer. A systematic review of the literature was conducted to address 5 key questions. Final recommendations were derived from strength of evidence, open comment feedback, expert panel consensus, and advisory panel review. RESULTS.­: Eleven recommendations were drafted: 7 based on CAP laboratory accreditation requirements and 4 based on expert consensus opinions. A 3-week open comment period received 180 comments from more than 150 participants. CONCLUSIONS.­: To improve accurate, precise, and reproducible interpretation of HER2 IHC results for breast cancer, QIA and procedures must be validated before implementation, followed by regular maintenance and ongoing evaluation of quality control and quality assurance. HER2 QIA performance, interpretation, and reporting should be supervised by pathologists with expertise in QIA.