Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma.

BACKGROUND & AIMS: Single-agent anti-PD1 checkpoint inhibitors convey outstanding clinical benefits in a small fraction (∼20%) of patients with advanced hepatocellular carcinoma (aHCC) but the molecular mechanisms determining response are unknown. To fill this gap, we herein analyze the molecular and immune traits of aHCC in patients treated with anti-PD1. METHODS: Overall, 111 tumor samples from patients with aHCC were obtained from 13 centers before systemic therapies. We performed molecular analysis and immune deconvolution using whole-genome expression data (n = 83), mutational analysis (n = 72), and histologic evaluation with an endpoint of objective response. RESULTS: Among 83 patients with transcriptomic data, 28 were treated in frontline, whereas 55 patients were treated after tyrosine kinase inhibitors (TKI) either in second or third line. Responders treated in frontline showed upregulated interferon-γ signaling and major histocompatibility complex II-related antigen presentation. We generated an 11-gene signature (IFNAP), capturing these molecular features, which predicts response and survival in patients treated with anti-PD1 in frontline. The signature was validated in a separate cohort of aHCC and >240 patients with other solid cancer types where it also predicted response and survival. Of note, the same signature was unable to predict response in archival tissue of patients treated with frontline TKIs, highlighting the need for fresh biopsies before immunotherapy. CONCLUSION: Interferon signaling and major histocompatibility complex-related genes are key molecular features of HCCs responding to anti-PD1. A novel 11-gene signature predicts response in frontline aHCC, but not in patients pretreated with TKIs. These results must be confirmed in prospective studies and highlights the need for biopsies before immunotherapy to identify biomarkers of response.

maxATAC: Genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks.

Transcription factors read the genome, fundamentally connecting DNA sequence to gene expression across diverse cell types. Determining how, where, and when TFs bind chromatin will advance our understanding of gene regulatory networks and cellular behavior. The 2017 ENCODE-DREAM in vivo Transcription-Factor Binding Site (TFBS) Prediction Challenge highlighted the value of chromatin accessibility data to TFBS prediction, establishing state-of-the-art methods for TFBS prediction from DNase-seq. However, the more recent Assay-for-Transposase-Accessible-Chromatin (ATAC)-seq has surpassed DNase-seq as the most widely-used chromatin accessibility profiling method. Furthermore, ATAC-seq is the only such technique available at single-cell resolution from standard commercial platforms. While ATAC-seq datasets grow exponentially, suboptimal motif scanning is unfortunately the most common method for TFBS prediction from ATAC-seq. To enable community access to state-of-the-art TFBS prediction from ATAC-seq, we (1) curated an extensive benchmark dataset (127 TFs) for ATAC-seq model training and (2) built "maxATAC", a suite of user-friendly, deep neural network models for genome-wide TFBS prediction from ATAC-seq in any cell type. With models available for 127 human TFs, maxATAC is the largest collection of high-performance TFBS prediction models for ATAC-seq. maxATAC performance extends to primary cells and single-cell ATAC-seq, enabling improved TFBS prediction in vivo. We demonstrate maxATAC's capabilities by identifying TFBS associated with allele-dependent chromatin accessibility at atopic dermatitis genetic risk loci.

Spatiotemporal Dynamics of Immune Cells in Early Left Ventricular Remodeling After Acute Myocardial Infarction in Mice.

Myocardial infarction remains a leading cause of morbidity and death. Insufficient delivery of oxygen to the myocardium sets into play a complicated process of repair that involves the temporal recruitment of different immune cells so as to remove debris and necrotic cells expeditiously and to form effective scar tissue. Clearly defined and overlapping phases have been identified in the process, which transitions from an overall proinflammatory to anti-inflammatory phenotype with time. Variations in the strength of the phases as well as in the co-ordination among them have profound consequences. Too strong of an inflammatory phase can result in left ventricular wall thinning and eventual rupture, whereas too strong of an anti-inflammatory phase can lead to cardiac stiffening, arrhythmias, or ventricular aneurisms. In both cases, heart failure is an intermediate consequence with death being the likely outcome. Here, we summarize the role of key immune cells in the repair process of the heart after left ventricular myocardial infarction, along with the associated cytokines and chemokines. A better understanding of the immune response ought to lead hopefully to improved therapies that exploit the natural repair process for mending the infarcted heart.

Platelet response to direct thrombin inhibitor or fondaparinux treatment in patients with suspected heparin-induced thrombocytopenia.

Making a definitive diagnosis of heparin-induced thrombocytopenia (HIT) can be problematic. A prompt platelet rise following treatment has been proposed as a "post-test" criterion for diagnosis. However, the platelet response following discontinuation of heparin and initiation of a recommended alternative anticoagulant remains largely undefined and unstudied. This study aimed to characterize platelet response to initial treatment in patients with a low, intermediate, or high likelihood of having HIT. This was a multicenter retrospective cohort study. Patients were over 18 years in age, underwent serologic testing for HIT, and received alternative anticoagulation treatment for HIT. Classification of each patient's likelihood of having HIT was based on an empiric, pre-hoc combination of the 4T score and serology results. The primary outcome for this study was a platelet count response after initiation of direct thrombin inhibitor (DTI) or fondaparinux therapy within 48 h. 124 patients were analyzed. The sensitivity and specificity of having an immediate platelet rise of at least 10,000/µL by day 2 after starting treatment among high-likelihood for HIT patients were 0.71 (95% CI 0.55-0.84) and 0.64 (95% CI 0.5-0.76), respectively. The negative predictive value of no platelet rise was 75.5% (95% CI 0.61-0.86). A prompt platelet count rise may be appropriate to consider along with other known criteria for the clinical diagnosis of HIT. The rise should be immediate following discontinuation of heparin and initiation of recommended treatment, with an upward rise within 48 h.

Evolution of Systemic Therapy in Advanced Hepatocellular Carcinoma.

The recognition that hepatocellular carcinoma (HCC) is a rising problem globally dates back decades; however, the development of effective medical treatment for the disease has only led to robust improvements in patient outcomes in the recent past. As knowledge evolves and regimens are proven to be more active, the importance of multidisciplinary management in patients with all stages of HCC will become more important to optimize patient outcomes. Key to optimizing patient outcomes is an understanding of the evolution and current role of these therapies in the HCC landscape.

Accessible chromatin maps of inflammatory bowel disease intestine nominate cell-type mediators of genetic disease risk.

Inflammatory Bowel Disease ( IBD ) is a chronic and often debilitating autoinflammatory condition, with an increasing incidence in children. Standard-of-care therapies lead to sustained transmural healing and clinical remission in fewer than one-third of patients. For children, TNFα inhibition remains the only FDA-approved biologic therapy, providing an even greater urgency to understanding mechanisms of response. Genome-wide association studies ( GWAS ) have identified 418 independent genetic risk loci contributing to IBD, yet the majority are noncoding and their mechanisms of action are difficult to decipher. If causal, they likely alter transcription factor ( TF ) binding and downstream gene expression in particular cell types and contexts. To bridge this knowledge gap, we built a novel resource: multiome-seq (tandem single-nuclei ( sn )RNA-seq and chromatin accessibility ( snATAC )-seq) of intestinal tissue from pediatric IBD patients, where anti-TNF response was defined by endoscopic healing. From the snATAC-seq data, we generated a first-time atlas of chromatin accessibility (putative regulatory elements) for diverse intestinal cell types in the context of IBD. For cell types/contexts mediating genetic risk, we reasoned that accessible chromatin will co-localize with genetic disease risk loci. We systematically tested for significant co-localization of our chromatin accessibility maps and risk variants for 758 GWAS traits. Globally, genetic risk variants for IBD, autoimmune and inflammatory diseases are enriched in accessible chromatin of immune populations, while other traits (e.g., colorectal cancer, metabolic) are enriched in epithelial and stromal populations. This resource opens new avenues to uncover the complex molecular and cellular mechanisms mediating genetic disease risk.

Elevated CD153 Expression on Aged T Follicular Helper Cells is Vital for B cell Responses.

Our recent data showed that an aberrant IL-10-producing T follicular helper population (Tfh10) accumulates dramatically with age and is associated with age-related declines in vaccine responsiveness. Through single cell gene expression and chromatin accessibility analysis of IL-10+ and IL-10- memory CD4+ T cells from young and aged mice, we identified increased expression of CD153 on aged Tfh and Tfh10 cells. Mechanistically, we linked inflammaging (increased IL-6 levels) to elevated CD153 expression of Tfh cells through c-Maf. Surprisingly, blockade of CD153 in aged mice significantly reduced their vaccine-driven antibody response, which was associated with decreased expression of ICOS on antigen-specific Tfh cells. Combined, these data show that an IL-6/c-Maf/CD153 circuit is critical for maintaining ICOS expression. Thus, although overall Tfh-mediated B cell responses are reduced in the context of vaccines and aging, our data suggest that elevated expression of CD153 on Tfh cells potentiates the remaining Tfh function in aged mice.

An atlas of gene regulatory networks for memory CD4 + T cells in youth and old age.

Aging profoundly affects immune-system function, promoting susceptibility to pathogens, cancers and chronic inflammation. We previously identified a population of IL-10-producing, T follicular helper-like cells (" Tfh10 "), linked to suppressed vaccine responses in aged mice. Here, we integrate single-cell ( sc )RNA-seq, scATAC-seq and genome-scale modeling to characterize Tfh10 - and the full CD4 + memory T cell ( CD4 + TM ) compartment - in young and old mice. We identified 13 CD4 + TM populations, which we validated through cross-comparison to prior scRNA-seq studies. We built gene regulatory networks ( GRNs ) that predict transcription-factor control of gene expression in each T-cell population and how these circuits change with age. Through integration with pan-cell aging atlases, we identified intercellular-signaling networks driving age-dependent changes in CD4 + TM. Our atlas of finely resolved CD4 + TM subsets, GRNs and cell-cell communication networks is a comprehensive resource of predicted regulatory mechanisms operative in memory T cells, presenting new opportunities to improve immune responses in the elderly.

RB116: an RB1+ retinoblastoma cell line expressing primitive markers.

PURPOSE: Retinoblastoma (RB), an intraocular tumor of childhood, is commonly associated with mutations in the RB1 gene. RB116 is a novel, early passage RB cell line that has not been previously characterized. In this study, we examined RB116 for the expression of RB1 and tested the hypothesis that RB116 cells would express stem cell markers as well as retinal progenitor cell markers. We compared RB116 cells with other well known RB cell lines, including Y79 and WERI-RB27. METHODS: We evaluated expression of RB1 in RB116 cells by sequencing, multiplex ligation-dependent probe amplification, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), western immunoblot, and immunocytochemistry. Next, RB116 cells, along with Y79 and WERI-RB27 cells, were examined for expression of stem cell markers (ABCG2, Nanog, Oct3/4, ALDH1A1) and retinal progenitor markers (PAX6, CHX10) by quantitative immunocytochemistry. Immunocytochemical findings were accompanied by PCR analysis. RESULTS: RB116 cells expressed RB1 at the mRNA and protein levels, with no mutations detected by either sequencing analysis, or gene dosage abnormalities detected by multiplex ligation-dependent probe amplification. The RB1 protein was immunoreactive in RB116 cells with an atypical perinuclear localization. RB116 cells also expressed stem cell markers, with 3%-5% of cells immunopositive for ABCG2, Oct3/4 and ALDH1A1, with at least 18% of cells immunoreactive to Nanog. These findings were confirmed by RT-PCR. Small percentages of RB116 cells also exhibited immunoreactivity to retinal progenitor markers PAX6 (9.8%) and CHX10 (1.2%). Expression of mRNAs for these markers was confirmed by qRT-PCR. CONCLUSIONS: RB116 cells demonstrate RB1 expression accompanied by atypical perinuclear localization. RB116 cells also express primitive stem cell and retinal progenitor cell markers. Further studies on the phenotypes of both RB1-positive and RB1-negative human RB cells may be important in assessing differentiation potential of these cells, as well as designing targeted differentiation therapies.

Hepatocellular Carcinoma: Pick the Winner-Tyrosine Kinase Inhibitor Versus Immuno-oncology Agent-Based Combinations.

The treatment landscape for advanced hepatocellular carcinoma has changed dramatically over the past 4 years. We now have numerous options for patients in frontline, second-line, and beyond. The most significant impact has been the introduction of immunotherapy into our treatment paradigms. We now have regimens that induce consistent double-digit objective response rates and markedly improve overall survival (OS) with favorable side effect profiles. The combination of atezolizumab and bevacizumab has demonstrated that the combination of targeting programmed death-ligand 1 and the vascular endothelial growth factor axis can improve outcomes versus sorafenib in the IMBrave150 study. Results from the COSMIC-312 study evaluating the multikinase vascular endothelial growth factor receptor, hepatocyte growth factor receptor, and AXL tyrosine kinase receptor inhibitor cabozantinib in combination with atezolizumab improved progression-free survival versus sorafenib, but at this time, there is no improvement in OS and response rates were lower than expected. Additional data with similar combinations are awaited on the basis of encouraging early-phase data. In addition, the combination of cytotoxic T-lymphocyte-associated protein 4 and programmed cell death-1/programmed death-ligand 1 targeting is yielding similar promising early results, and the phase III HIMALAYA study met its primary end points of improving OS versus sorafenib for durvalumab plus tremelimumab and demonstrated noninferiority for single-agent durvalumab as well. However, this combination did not improve progression-free survival and objective response rates with this combination did not seem significantly different from that with single-agent durvalumab. Although there are still knowledge gaps in this rapidly changing landscape, we will address some of the important questions relevant to making therapeutic decisions in the management of advanced hepatocellular carcinoma in the modern era on the basis of our current knowledge of the safety and efficacy of these evolving regimens. The goal is to provide clinicians with the knowledge needed to optimize outcomes for their patients.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

Making National Cancer Institute-Designated Comprehensive Cancer Center Knowledge Accessible to Community Oncologists via an Online Tumor Board: Longitudinal Observational Study.

BACKGROUND: Expert knowledge is often shared among multidisciplinary academic teams at tumor boards (TBs) across the country, but these conversations exist in silos and do not reach the wider oncology community. OBJECTIVE: Using an oncologist-only question and answer (Q&A) website, we sought to document expert insights from TBs at National Cancer Institute-designated Comprehensive Cancer Centers (NCI-CCCs) to provide educational benefits to the oncology community. METHODS: We designed a process with the NCI-CCCs to document and share discussions from the TBs focused on areas of practice variation on theMednet, an interactive Q&A website of over 13,000 US oncologists. The faculty translated the TB discussions into concise, non-case-based Q&As on theMednet. Answers were peer reviewed and disseminated in email newsletters to registered oncologists. Reach and engagement were measured. Following each Q&A, a survey question asked how the TB Q&As impacted the readers' practice. RESULTS: A total of 23 breast, thoracic, gastrointestinal, and genitourinary programs from 16 NCI-CCC sites participated. Between December 2016 and July 2021, the faculty highlighted 368 questions from their TBs. Q&As were viewed 147,661 times by 7381 oncologists at 3515 institutions from all 50 states. A total of 277 (75%) Q&As were viewed every month. Of the 1063 responses to a survey question on how the Q&A affected clinicians' practices, 646 (61%) reported that it confirmed their current practice, 163 (20%) indicated that a Q&A would change their future practice, and 214 (15%) reported learning something new. CONCLUSIONS: Through an online Q&A platform, academics at the NCI-CCCs share knowledge outside the walls of academia with oncologists across the United States. Access to up-to-date expert knowledge can reassure clinicians' practices, significantly impact patient care in community practices, and be a source of new knowledge and education.

Hypoplastic left heart syndrome (HLHS): molecular pathogenesis and emerging drug targets for cardiac repair and regeneration.

INTRODUCTION: Hypoplastic left heart syndrome (HLHS) is a severe developmental defect characterized by the underdevelopment of the left ventricle along with aortic and valvular defects. Multiple palliative surgeries are required for survival. Emerging studies have identified potential mechanisms for the disease onset, including genetic and hemodynamic causes. Genetic variants associated with HLHS include transcription factors, chromatin remodelers, structural proteins, and signaling proteins necessary for normal heart development. Nonetheless, current therapies are being tested clinically and have shown promising results at improving cardiac function in patients who have undergone palliative surgeries. AREAS COVERED: We searched PubMed and clinicaltrials.gov to review most of the mechanistic research and clinical trials involving HLHS. This review discusses the anatomy and pathology of HLHS hearts. We highlight some of the identified genetic variants that underly the molecular pathogenesis of HLHS. Additionally, we discuss some of the emerging therapies and their limitations for HLHS. EXPERT OPINION: While HLHS etiology is largely obscure, palliative therapies remain the most viable option for the patients. It is necessary to generate animal and stem cell models to understand the underlying genetic causes directly leading to HLHS and facilitate the use of gene-based therapies to improve cardiac development and regeneration.

Using a Collaborative, Virtual Discussion Platform to Mobilize Oncologic Expertise for the COVID-19 Pandemic.

PURPOSE: COVID-19 is a rapidly emerging worldwide pandemic that has drastically changed health care across the United States. Oncology patients are especially vulnerable. Novel point-of-care resources may be useful to rapidly disseminate peer-reviewed information from oncology experts nationwide. We describe our initial experience with distributing this information through a private, curated, virtual collaboration question-and-answer (Q&A) platform for oncologists. METHODS: The Q&A database was queried for a 2-month period from March 12 to May 12, 2020. We collected the total number of views and unique viewers for the questions. We classified the questions according to their emphasis (practice management, clinical management, both) and disease type across radiation oncology, medical oncology, gynecologic oncology, and pediatric oncology. RESULTS: Seventy-nine questions were approved, 67 of which were answered and generated 49,494 views with 5,148 unique viewers. Most discussions covered clinical management, with breast cancer being the most active disease site. Ten questions covered pediatric oncology and gynecologic oncology. Forty-seven percent of the 11,010 users of the platform visited the website during the 2-month period. CONCLUSION: Discussions on the Q&A platform reached a substantial number of oncologists throughout the nation and may help oncologists to modify their treatment in real time with the rapidly evolving COVID-19 pandemic.

Systemic Therapy for Primary Liver Tumors: Cholangiocarcinoma and Hepatocellular Carcinoma.

In the past decade, there has been significant progress in the treatment of primary liver cancer. There has been increasing knowledge of the molecular alterations occurring in these tumors, which is now being translated into patient care. Ongoing clinical trials will further advance the therapeutic options available to patients, including the introduction of molecular targeted therapeutics and immunotherapy approaches. Critical to the success of these new drugs, is the appropriate use of them in the clinic to maximize efficacy and limit toxicity.

B-cell acute lymphoblastic leukemia as a secondary malignancy following diffuse large B-cell lymphoma.

Secondary acute lymphoblastic leukemia (ALL) is a rare disease that has not been well characterized compared with secondary myelodysplastic syndrome or secondary acute myeloid leukemia. We present a report of two patients who developed ALL following complete remission of diffuse large B-cell lymphoma (DLBCL). The first case is more consistent with a therapy- related ALL as a PCR analysis of bone marrow aspirate revealed a distinct clone and the mixed-lineage leukemia gene rearrangement, commonly associated with exposure to topoisomerase II inhibitors. The second case is more consistent with clonal evolution given positive MYC and BCL2 fusion signals in the original diagnosis of DLBCL and the secondary ALL.

Histological Assessment of Cre-loxP Genetic Recombination in the Aging Subventricular Zone of Nestin-CreERT2/Rosa26YFP Mice.

The use of inducible transgenic Nestin-CreERT2 mice has proved to be an essential research tool for gene targeting and studying the molecular pathways implicated in adult neurogenesis, namely, inside the adult subgranular zone (SGZ) of the dentate gyrus and the adult subventricular zone (SVZ) lining the lateral ventricles. Several lines of Nestin-CreER-expressing mice were generated and used in adult neurogenesis research in the past two decades; however, their suitability for studying neurogenesis in aged mice remains elusive. Here, we assessed the efficiency of Cre-loxP genetic recombination in the aging SVZ using the Nestin-CreERT2/Rosa26YFP line designed by Lagace et al. (J Neurosci 27(46):12623-12629, 2007). This analysis was performed in 12-month-old (middle-aged) mice and 20-month-old (old) mice compared to 2-month-old (young adult) mice. To evaluate successful recombination, our approach relies on the histological assessment of Cre mRNA level of expression and the YFP reporter gene's expression inside the aging SVZ by combining in situ hybridization and immunohistochemistry. Using co-immunolabeling, this approach also provides the advantage of estimating the percentage of recombined progeny [(GFP+Nestin+)/Nestin+] and the rate of cell proliferation [(GFP+Ki67+)/GFP+] inside the aging SVZ niche.

Hepatic Abscess in a Returning Traveler with Crohn's Disease: Differentiating Amebic from Pyogenic Liver Abscess.

Liver abscess is a rare but serious complication of Crohn's disease. Patients with Crohn's disease are at risk for pyogenic liver abscesses due to immunosuppressive therapy, fistulous disease, and intraabdominal abscesses. Inflammatory bowel disease patients are also known to have a greater prevalence of amebiasis compared to the rest of the population; however, a higher incidence of amebic liver abscess has not been reported. We describe a case of a liver abscess in a patient with Crohn's disease that was initially presumed pyogenic but later determined to be amebic in origin. Epidemiology, clinical presentation, diagnosis, and treatment of amebic and pyogenic liver abscesses are discussed.