Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Widespread, human-associated redondoviruses infect the commensal protozoan Entamoeba gingivalis.

Redondoviruses are circular Rep-encoding single-stranded DNA (CRESS) viruses of high prevalence in healthy humans. Redondovirus abundance is increased in oro-respiratory samples from individuals with periodontitis, acute illness, and severe COVID-19. We investigated potential host cells supporting redondovirus replication in oro-respiratory samples and uncovered the oral amoeba Entamoeba gingivalis as a likely host. Redondoviruses are closely related to viruses of Entamoeba and contain reduced GC nucleotide content, consistent with Entamoeba hosts. Redondovirus and E. gingivalis co-occur in metagenomic data from oral disease and healthy human cohorts. When grown in xenic cultures with feeder bacteria, E. gingivalis was robustly positive for redondovirus RNA and DNA. A DNA proximity-ligation assay (Hi-C) on xenic culture cells showed enriched cross-linking of redondovirus and Entamoeba DNA, supporting E. gingivalis as the redondovirus host. While bacteria are established hosts for bacteriophages within the human virome, this work shows that eukaryotic commensals also contribute an abundant human-associated virus.

Clinical neuro-ophthalmic findings in familial dysautonomia.

BACKGROUND: To define the clinical neuro-ophthalmic abnormalities of patients with familial dysautonomia (FD). METHODS: Sixteen patients (32 eyes) with the clinical and molecular diagnoses of FD underwent thorough neuro-ophthalmic clinical evaluation. RESULTS: Visual acuity ranged from 0.05 to 1.0 decimal units and was reduced in 15 of 16 patients. Mild to moderate corneal opacities were found in most patients but were visually significant in only 2 eyes. Red-green color vision was impaired in almost all cases. Depression of the central visual fields was present on automated visual fields in all patients, even in those with normal visual acuity. Temporal optic nerve pallor was present in all cases and was associated with retinal nerve fiber layer loss in the papillomacular region. Various ocular motility abnormalities also were observed. CONCLUSION: Patients with FD have a specific type of optic neuropathy with predominant loss of papillomacular nerve fibers, a pattern similar to other hereditary optic neuropathies caused by mutations either in nuclear or in mitochondrial DNA, affecting mitochondrial protein function. Defects of eye movements, particularly saccades, also appear to be a feature of patients with FD.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Rapid manufacturing of non-activated potent CAR T cells.

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 Isolates.

We report the genome of a B.1.1.7+E484K severe acute respiratory syndrome coronavirus 2 from Southeastern Pennsylvania and compare it with all high-coverage B.1.1.7+E484K genomes (n = 235) available. Analyses showed the existence of at least 4 distinct clades of this variant circulating in the United States and the possibility of at least 59 independent acquisitions of the E484K mutation.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 isolates from Pennsylvania.

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote "escape" from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.

Signatures of COVID-19 severity and immune response in the respiratory tract microbiome.

Rationale: Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Objective: To define the respiratory tract microbiome in COVID-19 and relationship disease severity, systemic immunologic features, and outcomes. Methods and Measurements: We examined 507 oropharyngeal, nasopharyngeal and endotracheal samples from 83 hospitalized COVID-19 patients, along with non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR, and immune features were characterized by lymphocyte/neutrophil (L/N) ratios and deep immune profiling of peripheral blood mononuclear cells (PBMC). Main Results: COVID-19 patients had upper respiratory microbiome dysbiosis, and greater change over time than critically ill patients without COVID-19. Diversity at the first time point correlated inversely with disease severity during hospitalization, and microbiome composition was associated with L/N ratios and PBMC profiles in blood. Intubated patients showed patient-specific and dynamic lung microbiome communities, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Conclusions: The respiratory tract microbiome and commensal virome are disturbed in COVID-19, correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, possible use as biomarkers, and role of bacterial and viral taxa identified here in COVID-19 pathogenesis.

Signatures of COVID-19 Severity and Immune Response in the Respiratory Tract Microbiome.

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

Novel KRIT1/CCM1 mutation in a patient with retinal cavernous hemangioma and cerebral cavernous malformation.

BACKGROUND: Retinal cavernous hemangiomas are rare vascular anomalies, and can be associated with cerebral cavernous malformations (CCM). Distinct mutations have been reported in patients who have both CCMs and retinal cavernous hemangiomas. METHODS: Fluorescein angiography, spectral domain optical coherence tomography, and genetic testing were performed on a patient with a retinal cavernous hemangioma and a CCM. RESULTS: Our patient was heterozygous in the KRIT1/CCM1 gene for a frameshift mutation, c.1088delC. This would be predicted to result in premature protein termination. DISCUSSION: We have identified a novel mutation in the KRIT1/CCM1 gene in a patient with both CCM and retinal cavernous hemangioma. We hypothesize that the occurrence of retinal cavernous hemangiomas and CCMs is underlaid by a common mechanism present in the KRIT1/CCM1 gene.

Association of positive dual-modality positron emission tomography/computed tomography imaging of primary choroidal melanoma with chromosome 3 loss and tumor size.

PURPOSE: To evaluate the positive dual-modality positron emission tomography/computed tomography (PET/CT) of choroidal melanoma with chromosome 3 loss and tumor size. METHODS: Thirty-seven consecutive patients with choroidal melanoma with known chromosome 3 status who underwent whole-body PET/CT imaging were retrospectively reviewed. Cytology and chromosome 3 loss were identified by fine-needle aspiration biopsy. Fluorescent in situ hybridization and whole genome microarray by single-nucleotide polymorphism were used to evaluate the chromosome 3 status. Metabolic activity of primary choroidal melanoma by PET/CT imaging was evaluated. RESULTS: Thirteen of 37 (35%) primary choroidal melanomas had loss of chromosome 3; 7 of the 13 (54%) melanomas were positive for metabolic activity identified by PET/CT imaging. All 24 of 37 melanomas without chromosome 3 loss were inactive for metabolic activity. There was a statistically significant association between positive metabolic activity and chromosome 3 loss (P = 0.00017 Fisher exact test); positive PET/CT imaging was 54% sensitive and 100% specific for loss of chromosome 3. Seven of 37 (19%) choroidal melanomas with positive metabolic activity by PET/CT were statistically significantly larger in size than the 30 metabolically inactive melanomas (P < 0.001, Kruskal-Wallis test). CONCLUSION: Positive metabolic activity of choroidal melanoma identified by PET/CT imaging was statistically significantly associated with chromosome 3 loss and larger tumor size.

A new grading system for retinal pigment epithelial tears.

PURPOSE: The purpose of this study was to assess the prognostic value of a new grading system for retinal pigment epithelium (RPE) tears that developed after antivascular endo-thelial growth factor (VEGF) therapy for exudative age-related macular degeneration. METHODS: The authors performed a retrospective review of consecutive eyes that developed an RPE tear after intravitreal injection of an anti-VEGF agent (pegaptanib, bevacizumab, or ranibizumab) within a 4.5-year period (January 2005 to January 2009) at a single center. Fundus photography, fluorescein angiography, and optical coherence topography were studied for each case of RPE tear, and using fluorescein angiography analysis, a measurement of greatest linear diameter (millimeter) was obtained and a grading scale devised. The grade of RPE tear was correlated with visual and anatomical outcomes and response to continued anti-VEGF therapy. RESULTS: Twenty-one eyes from 20 patients were evaluated in this study. Retinal pigment epithelium tears were graded from one to four based on the greatest length in the vector direction of the tear and involvement of the fovea. Nineteen percent (n = 4) of eyes had Grade 1 tears (diameter smaller than 200 microm), 14% (n = 3) had Grade 2 tears (diameter between 200 microm and 1-disk diameter), 19% (n = 4) had Grade 3 tears (diameter greater than 1-disk diameter), and 48% (n = 10) had Grade 4 tears (Grade 3 tears that involved the foveal center). Lower grade tears were more likely to have better visual acuity and better response to continued anti-VEGF therapy and less likely to develop a disciform scar but were at risk of progressing to a higher grade tear over time. CONCLUSION: The grading of RPE tears according to greatest linear diameter may have prognostic value in predicting visual acuity and anatomical outcome with or without continued anti-VEGF therapy. Lower grade tears have better visual acuity and response to anti-VEGF therapy. Grade 4 tears have a very poor prognosis with or without anti-VEGF therapy.

CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.

Retinal vessel abnormalities are associated with elevated fasting insulin levels and cerebral atrophy in nondiabetic individuals.

OBJECTIVE: To determine the impact of insulin resistance short of diabetes on the arteriolar-to-venular ratio (AVR) and whether AVR is related to cerebral atrophy. DESIGN: Cross-sectional study. PARTICIPANTS: Forty-six nondiabetic subjects with normal glucose tolerance and varying degrees of insulin resistance ranging in age from 43 to 77 years. METHODS: Insulin resistance was assessed by fasting insulin and the homeostasis model assessment. Arteriolar-to-venular ratio was determined using digital retinal photography with a nonmydriatic camera, and retinal data were analyzed using a reliable semiautomated method. Cerebral atrophy was derived by means of manual tracing and thresholding procedures on structural magnetic resonance images. MAIN OUTCOME MEASURES: Arteriolar-to-venular ratio and cerebral atrophy. RESULTS: Hyperinsulinemia negatively impacted AVR. Furthermore, AVR was associated with cerebral atrophy. Both of these findings were independent of the effects of age and hypertension. CONCLUSIONS: These novel findings indicate that insulin resistance short of diabetes and independent of age and hypertension has a negative impact on retinal vessel health. Moreover, impaired retinal vessel health related to brain atrophy also was independent of hypertension and white matter hyperintensities. Given the connections between retinal and cerebral vasculature, this may offer a partial explanation for the presence of cognitive and brain abnormalities among individuals with insulin resistance. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.