Quantification of cerebrospinal fluid tumor DNA in lung cancer patients with suspected leptomeningeal carcinomatosis.

Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman's ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

Type IV pili facilitated natural competence in Fusobacterium nucleatum.

OBJECTIVES: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. METHODS: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. RESULTS: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. CONCLUSIONS: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Long-term Outcomes of Diffuse or Recurrent Tenosynovial Giant Cell Tumor Treated with Postoperative External Beam Radiation Therapy.

PURPOSE: Tenosynovial giant cell tumor (TGCT) is a rare proliferative disorder of synovial membrane that previously was known as pigmented villonodular synovitis. Primary treatment involves surgical resection; however, complete removal of all disease involvement is difficult to achieve. Radiation may be useful to reduce the risk of recurrence. We report and update our institutional experience treating diffuse and recurrent TGCT with postsurgical external beam radiation therapy. METHODS AND MATERIALS: We performed a retrospective chart review of 30 patients with TGCT from 2003 to 2019 treated with radiation therapy. Each patient was evaluated for demographics, radiation treatment parameters, surgical management, complications, and outcome. RESULTS: With mean follow-up of 82 months (range, 3-211), 24 patients (80%) who underwent surgery followed by radiation therapy did not experience any further relapse, and all 30 patients achieved local control (100%) with additional salvage therapy after radiation therapy. The most common site of disease was the knee (n = 22, 73%), followed by the ankle (n = 5, 16%) and the hand (n = 3, 10%). Seven patients (24%) presented at time of initial diagnosis and 23 (76%) presented with recurrent disease after surgical resection, with an average of 2.6 surgical procedures before radiation therapy. After resection, 18 of 30 patients (67%) demonstrated residual TGCT by imaging. The median radiation therapy dose delivered was 36 Gy (range, 34-36 Gy) in 1.8 to 2.5 Gy/fractions for 4 weeks. In the assessment of posttreatment joint function, 26 sites (86%) exhibited excellent or good function, 2 (7%) fair, and 2 poor (7%) as determined by our scoring system. There were no cases of radiation-associated malignancy. CONCLUSIONS: Among patients with diffuse or recurrent TGCT, postsurgical external beam radiation therapy provided excellent local control and good functional status, with minimal treatment-related complications. Postsurgical radiation therapy is a well-tolerated noninvasive treatment that should be considered after maximal cytoreductive resection to prevent disease progression and recurrence.

Isolated Nodal Recurrence After Definitive Stereotactic Ablative Radiation Therapy for Non-Small Cell Lung Cancer.

PURPOSE: Stereotactic ablative radiation therapy (SABR) results in high rates of primary tumor control for early-stage non-small cell lung cancer (NSCLC). For patients with isolated hilar or mediastinal nodal recurrence (INR) after SABR, the optimal salvage treatment strategy is unclear. The purpose of this study was to determine the rate of INR after SABR for early-stage NSCLC and to describe patterns of care and treatment outcomes after salvage therapy. METHODS AND MATERIALS: This retrospective cohort study included 342 patients with stage T1-3N0M0 NSCLC treated with definitive SABR from 2003 to 2018. We evaluated the incidence of INR and baseline factors between patients who did and did not experience INR. Among patients who experienced INR, we described treatment patterns and outcomes including overall survival (OS) and progression free survival (PFS) from the time of nodal failure using the Kaplan-Meier method. RESULTS: With a median follow-up of 3.3 years, the 3-year INR rate was 10.6% (95% CI, 6.6%-13.4%). Among the 34 patients experiencing INR, the 3-year rates of OS and PFS were 39.3% (24.4%-63.3%) and 26.7% (14.1%-0.3%), respectively. The 34 patients with INR were treated with radiation therapy alone (26.7%), concurrent chemoradiation therapy (43.3%), chemotherapy alone (13.3%), or observation (16.7%). Patients treated with concurrent chemoradiation therapy had the best survival outcomes, with a 3-year OS and PFS of 81.5% (61.1%-100.0%) and 63.9% (40.7%-100.0%), respectively. Of the patients treated with salvage radiation therapy or concurrent chemoradiation therapy, 14.3% experienced grade 3 toxic effects, and no patients had grade ≥4 toxic effects. CONCLUSIONS: In this study, INR occurred in approximately 10% of patients treated with SABR for early-stage NSCLC. The highest rates of OS and PFS among patients with INR were observed in those treated with salvage chemoradiation therapy.

Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation.

F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4+ T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.

Stage I-II diffuse large B-cell lymphoma treated with rituximab and chemotherapy with or without radiotherapy.

We set to identify prognostic factors in a retrospective cohort of consecutive patients with stage I-II diffuse large B-cell lymphoma treated with rituximab-chemotherapy with or without radiotherapy from 2001 through 2017 at our institution. We identified 143 patients with median follow-up of 7.7 years. The majority were male (59.4%), had stage II (53.1%), had stage-modified IPI 0-1 (smIPI, 58.1%), and had non-bulky disease (<7 cm, 68.5%). 99 patients (69.2%) received rituximab-chemotherapy followed by radiotherapy, and 44 patients (30.8%) received rituximab-chemotherapy alone. The 5-year progression-free survival (PFS) and overall survival (OS) were 81.2% and 88.9%, respectively. The 5-year PFS for those with smIPI 0-1 versus 2-4 was 89.5% versus 69.7%, respectively (P = 0.005). Bulky disease (≥7 cm) was associated with worse PFS and OS on univariable and multivariable analyses (P < 0.05). Patients with smIPI 0-1 without bulky disease have excellent outcomes. However, patients with smIPI 2-4 or bulky disease have a high risk of progression.

Low-dose total skin electron beam therapy for refractory cutaneous CD30 positive lymphoproliferative disorders.

We describe a case of a 48-year-old woman with a refractory cutaneous CD30 positive lymphoproliferative disorder treated successfully with total skin electron beam radiotherapy (TSEBT).

Noninvasive Early Identification of Therapeutic Benefit from Immune Checkpoint Inhibition.

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.

KEAP1/NFE2L2 Mutations Predict Lung Cancer Radiation Resistance That Can Be Targeted by Glutaminase Inhibition.

Tumor genotyping is not routinely performed in localized non-small cell lung cancer (NSCLC) due to lack of associations of mutations with outcome. Here, we analyze 232 consecutive patients with localized NSCLC and demonstrate that KEAP1 and NFE2L2 mutations are predictive of high rates of local recurrence (LR) after radiotherapy but not surgery. Half of LRs occurred in tumors with KEAP1/NFE2L2 mutations, indicating that they are major molecular drivers of clinical radioresistance. Next, we functionally evaluate KEAP1/NFE2L2 mutations in our radiotherapy cohort and demonstrate that only pathogenic mutations are associated with radioresistance. Furthermore, expression of NFE2L2 target genes does not predict LR, underscoring the utility of tumor genotyping. Finally, we show that glutaminase inhibition preferentially radiosensitizes KEAP1-mutant cells via depletion of glutathione and increased radiation-induced DNA damage. Our findings suggest that genotyping for KEAP1/NFE2L2 mutations could facilitate treatment personalization and provide a potential strategy for overcoming radioresistance conferred by these mutations. SIGNIFICANCE: This study shows that mutations in KEAP1 and NFE2L2 predict for LR after radiotherapy but not surgery in patients with NSCLC. Approximately half of all LRs are associated with these mutations and glutaminase inhibition may allow personalized radiosensitization of KEAP1/NFE2L2-mutant tumors.This article is highlighted in the In This Issue feature, p. 1775.

Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration.

Fusobacterium nucleatum is implicated in accelerating colorectal cancer (CRC) and is found within metastatic CRC cells in patient biopsies. Here, we found that bacterial invasion of CRC cells and cocultured immune cells induced a differential cytokine secretion that may contribute to CRC metastasis. We used a modified galactose kinase markerless gene deletion approach and found that F. nucleatum invaded cultured HCT116 CRC cells through the bacterial surface adhesin Fap2. In turn, Fap2-dependent invasion induced the secretion of the proinflammatory cytokines IL-8 and CXCL1, which are associated with CRC progression and promoted HCT116 cell migration. Conditioned medium from F. nucleatum-infected HCT116 cells caused naïve cells to migrate, which was blocked by depleting CXCL1 and IL-8 from the conditioned medium. Cytokine secretion from HCT116 cells and cellular migration were attenuated by inhibiting F. nucleatum host-cell binding and entry using galactose sugars, l-arginine, neutralizing membrane protein antibodies, or fap2 deletion. F. nucleatum also induces the mobilization of immune cells in the tumor microenvironment. However, in neutrophils and macrophages, the bacterial-induced secretion of cytokines was Fap2 independent. Thus, our findings show that F. nucleatum both directly and indirectly modulates immune and cancer cell signaling and migration. Because increased IL-8 and CXCL1 production in tumors is associated with increased metastatic potential and cell seeding, poor prognosis, and enhanced recruitment of tumor-associated macrophages and fibroblasts, we propose that inhibition of host-cell binding and invasion, potentially through vaccination or novel galactoside compounds, could be an effective strategy for reducing F. nucleatum-associated CRC metastasis.

Integrating genomic features for non-invasive early lung cancer detection.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.

Circulating Tumor DNA Analysis to Assess Risk of Progression after Long-term Response to PD-(L)1 Blockade in NSCLC.

PURPOSE: Treatment with PD-(L)1 blockade can produce remarkably durable responses in patients with non-small cell lung cancer (NSCLC). However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression. EXPERIMENTAL DESIGN: In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (progression-free survival ≥ 12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing. Tumor tissue was available for 24 patients and was profiled by whole-exome sequencing (n = 18) or by targeted sequencing (n = 6). RESULTS: Thirty-one patients with NSCLC with long-term benefit to PD-(L)1 blockade were identified, and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; in contrast, all 4 patients with detectable ctDNA eventually progressed [Fisher P < 0.0001; positive predictive value = 1, 95% confidence interval (CI), 0.51-1; negative predictive value = 0.93 (95% CI, 0.80-0.99)]. CONCLUSIONS: ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 blockade and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression.

Circulating Tumor DNA Dynamics Predict Benefit from Consolidation Immunotherapy in Locally Advanced Non-Small Cell Lung Cancer.

Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. We applied CAPP-Seq ctDNA analysis to 218 samples from 65 patients receiving chemoradiation therapy (CRT) for locally advanced NSCLC, including 28 patients receiving consolidation immune checkpoint inhibition (CICI). Patients with undetectable ctDNA after CRT had excellent outcomes whether or not they received CICI. Among such patients, one died from CICI-related pneumonitis, highlighting the potential utility of only treating patients with MRD. In contrast, patients with MRD after CRT who received CICI had significantly better outcomes than patients who did not receive CICI. Furthermore, the ctDNA response pattern early during CICI identified patients responding to consolidation therapy. Our results suggest that CICI improves outcomes for NSCLC patients with MRD and that ctDNA analysis may facilitate personalization of consolidation therapy.

Outcomes for pediatric patients with osteosarcoma treated with palliative radiotherapy.

BACKGROUND: Few studies have addressed the efficacy of palliative radiotherapy (RT) for pediatric osteosarcoma (OS), a disease generally considered to be radioresistant. We describe symptom relief, local control, and toxicity associated with palliative RT among children with OS. PROCEDURE: Patients diagnosed with OS at age 18 and under and treated with RT for palliation of symptomatic metastases or local recurrence at the primary site from 1997 to 2017 were included. We retrospectively reviewed details of RT, symptom improvement, local control, survival, and toxicity. RESULTS: Thirty-two courses of palliative RT were given to 20 patients with symptomatic metastatic and/or locally recurrent primary disease. The median equivalent dose in 2 Gy fractions (EQD2) was 40.0 Gy (range, 20.0-60.4). The median number of fractions per course was 15 (range, 5-39). Symptom improvement occurred in 24 (75%) courses of RT at a median time of 15.5 days (range, 3-43). In nine courses (37.5%), symptoms recurred after a median duration of symptom relief of 140 days (range, 1-882). Higher EQD2 correlated with longer duration of response (r = 0.39, P = 0.0003). Imaging revealed local failure in 3 of 14 courses followed with surveillance imaging studies (21.4%). The median time to progression was 12.9 months (range, 4.4-21.8). The median follow-up time following the first course of palliative RT was 17.5 months (range, 1.74-102.24), and median time to overall survival was 19.4 months. Toxicity was mild, with grade 2 toxicity occurring in one course (3.1%). CONCLUSIONS: RT is an effective method of symptom palliation for patients with recurrent or metastatic OS, with higher delivered dose correlating with longer symptom relief and with little associated toxicity.

Central Nervous System Relapse After Stem Cell Transplantation in Adolescents and Young Adults with Acute Lymphoblastic Leukemia: A Single-Institution Experience.

Purpose: To evaluate outcomes and central nervous system (CNS) relapse in adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL), who underwent total body irradiation (TBI) before allogeneic hematopoietic stem cell transplantation (allo-SCT). Methods: A total of 136 AYA patients with ALL who received TBI before allo-SCT between 1998 and 2018 were reviewed. Twenty patients received cranial radiation in their initial treatment before conditioning for transplant and were excluded. Competing risk analysis was used to estimate the cumulative incidence of relapse. Kaplan-Meier and log-rank tests were used to calculate overall survival (OS) and to identify factors predictive of relapse. OS and time to relapse were calculated from date of allo-SCT. Results: One hundred sixteen patients were included in the analysis. Median age was 27 years and median follow-up time was 42 months. Twenty-six patients suffered a disease relapse and 49 died, 26 of posttransplantation complications. The median time to relapse was 7 months and the 5-year OS was 60%. Seven patients had a CNS relapse: 4 of 20 patients (25%) with pre-SCT CNS disease had a post-allo-SCT CNS relapse compared to 3 of 97 (3.1%) without pre-SCT CNS disease. Median time to CNS relapse was 7 months. Patients with post-SCT CNS relapse had median OS of 19 months. Conclusions: AYA patients with CNS disease who undergo an allo-SCT have a high rate of CNS relapse. The addition of additional CNS-directed therapy to transplant protocols warrants further investigation.

Peroxisome proliferator-activated receptor gamma controls prostate cancer cell growth through AR-dependent and independent mechanisms.

BACKGROUND: Prostate cancer (PC) remains a leading cause of cancer mortality and the most successful chemopreventative and treatment strategies for PC come from targeting the androgen receptor (AR). Although AR plays a key role, it is likely that other molecular pathways also contribute to PC, making it essential to identify and develop drugs against novel targets. Recent studies have identified peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor that regulates fatty acid (FA) metabolism, as a novel target in PC, and suggest that inhibitors of PPARγ could be used to treat existing disease. We hypothesized that PPARγ acts through AR-dependent and independent mechanisms to control PC development and growth and that PPARγ inhibition is a viable PC treatment strategy. METHODS: Immunohistochemistry was used to determine expression of PPARÒ¯ in a cohort of patients with PC. Standard molecular techniques were used to investigate the PPARÒ¯ signaling in PC cells as well a xenograft mouse model to test PPARÒ¯ inhibition in vivo. Kaplan-Meier curves were created using cBioportal. RESULTS: We confirmed the expression of PPARÒ¯ in human PC. We then showed that small molecule inhibition of PPARγ decreases the growth of AR-positive and -negative PC cells in vitro and that T0070907, a potent PPARγ antagonist, significantly decreased the growth of human PC xenografts in nude mice. We found that PPARγ antagonists or small interfering RNA (siRNA) do not affect mitochondrial activity nor do they cause apoptosis; instead, they arrest the cell cycle. In AR-positive PC cells, antagonists and siRNAs reduce AR transcript and protein levels, which could contribute to growth inhibition. AR-independent effects on growth appear to be mediated by effects on FA metabolism as the specific FASN inhibitor, Fasnall, inhibited PC cell growth but did not have an additive effect when combined with PPARγ antagonists. Patients with increased PPARÒ¯ target gene expression, but not alterations in PPARÒ¯ itself, were found to have significantly worse overall survival. CONCLUSIONS: Having elucidated the direct cancer cell effects of PPARγ inhibition, our studies have helped to determine the role of PPARγ in PC growth, and support the hypothesis that PPARγ inhibition is an effective strategy for PC treatment.

Comparison of type 5d autotransporter phospholipases demonstrates a correlation between high activity and intracellular pathogenic lifestyle.

Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane ß-barrel domain and an exported passenger. In the poorly studied type 5d subclass, the passenger is a patatin-like lipase. The prototype of this secretion pathway is PlpD of Pseudomonas aeruginosa, an opportunistic human pathogen. The PlpD passenger is a homodimer with phospholipase A1 (PLA1) activity. Based on sequencing data, PlpD-like proteins are present in many bacterial species. We characterized the enzymatic activity, specific lipid binding and oligomeric status of PlpD homologs from Aeromonas hydrophila (a fish pathogen), Burkholderia pseudomallei (a human pathogen) and Ralstonia solanacearum (a plant pathogen) and compared these with PlpD. We demonstrate that recombinant type 5d-secreted patatin domains have lipase activity and form dimers or higher-order oligomers. However, dimerization is not necessary for lipase activity; in fact, by making monomeric variants of PlpD, we show that enzymatic activity slightly increases while protein stability decreases. The lipases from the intracellular pathogens A. hydrophila and B. pseudomallei display PLA2 activity in addition to PLA1 activity. Although the type 5d-secreted lipases from the animal pathogens bound to intracellular lipid targets, phosphatidylserine and phosphatidylinositol phosphates, hydrolysis of these lipids could only be observed for FplA of Fusobacterium nucleatum Yet, we noted a correlation between high lipase activity in type 5d autotransporters and intracellular lifestyle. We hypothesize that type 5d phospholipases are intracellularly active and function in modulation of host cell signaling events.

Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families.

Fusobacterium spp. are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including a link between the oral pathogen Fusobacterium nucleatum and the progression and severity of colorectal cancer. The identification and characterization of virulence factors in the genus Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the identification and corrected annotation of verified and potential virulence factors from the type 5 secreted autotransporter, FadA, and MORN2 protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and type strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we used sequence similarity networks to identify protein subsets and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have identified unique subsets of type 5a autotransporters, which are key proteins associated with virulence in F. nucleatum However, we coupled our bioinformatic data with bacterial binding assays to show that a predicted weakly invasive strain of F. necrophorum that lacks a Fap2 autotransporter adhesin strongly binds human colonocytes. These analyses confirm a gap in our understanding of how autotransporters, MORN2 domain proteins, and FadA adhesins contribute to host interactions and invasion. In summary, we identify candidate virulence genes in Fusobacterium, and caution that experimental validation of host-microbe interactions should complement bioinformatic predictions to increase our understanding of virulence protein contributions in Fusobacterium infections and disease.IMPORTANCEFusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease.

Genetic Predisposition to Symptomatic Lumbar Disk Herniation in Pediatric and Young Adult Patients.

STUDY DESIGN: Case-control whole-genome sequencing analysis of a highly select, young cohort with symptomatic lumbar disk herniation (LDH) compared with the standard variation in a large reference population. OBJECTIVE: To assess genetic influences predisposing pediatric and young adult patients to symptomatic LDH. SUMMARY OF BACKGROUND DATA: LDH has traditionally been attributed to natural weakening or mechanical insult, but recent literature supports a potential genetic influence. METHODS: Young patients with symptomatic, clinically confirmed LDH who underwent surgical treatment were included. Patients were younger than the average age of presentation, limiting the influence of environmental risks. DNA collected from these patients was compared with a reference genome (1000 Genomes Project). A genome-wide association study using whole-genome sequencing was used to characterize genetic mutations potentially associated with LDH. RESULTS: Among the 61 candidate genes flagged, 20 had missense mutations in 2 or more LDH cases. Missense mutations in collagen-encoding genes were observed in 12 of 15 patients (80%). A potential association with clinical presentation was indicated by odds ratios of key single-nucleotide polymorphism (SNP) variants in genes that encode collagen. Relative to the reference population, the LDH cohort demonstrated two statistically significant SNP variants in the gene encoding for aggrecan, a protein that facilitates load-bearing properties in the cartilaginous end plate. Aggrecan genes SNPs rs3817428 and rs11638262 were significantly associated with decreased odds of symptomatic LDH: odds ratio 0.05 (0.02-0.11) and 0.04 (0-0.26), respectively (P < 1 × 10 for both). CONCLUSION: These results suggest that collagen-encoding variants may be a genetic risk factor for LDH. They also shed new light on the role of variants that impact aggrecan, which sustains the cartilaginous end plate. Genetic predisposition to LDH may therefore be related to a multimodal combination of mutations that affect the nucleus pulposus, annulus fibrosus, and the cartilaginous end plates. LEVEL OF EVIDENCE: 4.

Orthotopic Liver Transplantation After Stereotactic Body Radiotherapy for Pediatric Hepatocellular Carcinoma with Central Biliary Obstruction and Nodal Involvement.

Here we describe the case of a 10-year-old boy with a history of chronic hepatitis B who was diagnosed with hepatocellular carcinoma (HCC) with a large central hepatic mass and metastatic disease in a celiac lymph node. His tumor was unresectable, due to location and lack of clear margins, and he could not receive chemotherapy due to elevated bilirubin. He was treated with stereotactic body radiotherapy (SBRT) to the primary site and involved nodal region. After completing radiotherapy, his total bilirubin level fell below 1.0 mg/dL, allowing him to begin systemic therapy with cisplatin and doxorubicin. At three months after SBRT, his bilirubin was 0.1 mg/dL, alpha-fetoprotein (AFP) was 88 ng/mL, and imaging demonstrated a decrease in tumor size (total volume 28.7 cc), with no evidence of local or distant disease progression. He then developed distant disease within the liver, but his disease remained controlled at the primary site and nodes that had been treated with SBRT. He underwent orthotopic liver transplantation (OLT) with an uneventful operative course and remains with no evidence of disease at seven months after OLT. This is one of the first reported cases of successful downstaging of pediatric HCC with nodal involvement to allow for OLT, and it argues for consideration of similar patients for OLT.