Neutrophilic dermatosis in a patient with an IKZF1 variant and a review of monogenic autoinflammatory disorders presenting with neutrophilic dermatoses.

Monogenic diseases of immune dysregulation should be considered in the evaluation of children presenting with recurrent neutrophilic dermatoses in association with systemic signs of inflammation, autoimmune disease, hematologic abnormalities, and opportunistic or recurrent infections. We report the case of a 2-year-old boy presenting with a neutrophilic dermatosis, found to have a novel likely pathogenic germline variant of the IKAROS Family Zinc Finger 1 (IKZF1) gene; the mutation likely results in a loss of function dimerization defective protein based on reports and studies of similar variants. IKZF1 variants could potentially lead to aberrant neutrophil chemotaxis and development of neutrophilic dermatoses. Long-term surveillance is required to monitor the development of hematologic malignancy, autoimmunity, immunodeficiency, and infection in patients with pathogenic IKZF1 germline variants.

Analytical Principles of Cancer Next Generation Sequencing.

This article covers analytical principles of cancer next generation sequencing (NGS). Cancer samples require special considerations due to the cancer-specific applications of testing, as well as cancer sample specific issues, including low input, low tumor purity, or fixation-related artifacts. Laboratories typically use a combination of approaches around specimen processing, assay design, and bioinformatics analysis to allow for successful detection of actionable biomarkers. Examples of these approaches for cancer NGS testing are discussed and reviewed here.

Analytical performance of lateral flow immunoassay for SARS-CoV-2 exposure screening on venous and capillary blood samples.

OBJECTIVES: We validate the use of a lateral flow immunoassay (LFI) intended for rapid screening and qualitative detection of anti-SARS-CoV-2 IgM and IgG in serum, plasma, and whole blood, and compare results with ELISA. We also seek to establish the value of LFI testing on blood obtained from a capillary blood sample. METHODS: Samples collected by venous blood draw and finger stick were obtained from patients with SARS-CoV-2 detected by RT-qPCR and control patients. Samples were tested with Biolidics 2019-nCoV IgG/IgM Detection Kit lateral flow immunoassay, and antibody calls were compared with ELISA. RESULTS: Biolidics LFI showed clinical sensitivity of 92% with venous blood at 7 days after PCR diagnosis of SARS-CoV-2. Test specificity was 92% for IgM and 100% for IgG. There was no significant difference in detecting IgM and IgG with Biolidics LFI and ELISA at D0 and D7 (p = 1.00), except for detection of IgM at D7 (p = 0.04). Capillary blood of SARS-CoV-2 patients showed 93% sensitivity for antibody detection. CONCLUSIONS: Clinical performance of Biolidics 2019-nCoV IgG/IgM Detection Kit is comparable to ELISA and was consistent across sample types. This provides an opportunity for decentralized rapid testing and may allow point-of-care and longitudinal self-testing for the presence of anti-SARS-CoV-2 antibodies.

Sequencing identifies multiple early introductions of SARS-CoV-2 to the New York City region.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 864 SARS-CoV-2 sequences from cases in the New York City metropolitan area during the COVID-19 outbreak in spring 2020. The majority of cases had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that early transmission was most linked to cases from Europe. Our data are consistent with numerous seeds from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of genomic surveillance in addition to traditional epidemiological indicators.

Sequencing identifies multiple early introductions of SARS-CoV-2 to the New York City Region.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 864 SARS-CoV-2 sequences from cases in the New York City metropolitan area during the COVID-19 outbreak in Spring 2020. The majority of cases had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that early transmission was most linked to cases from Europe. Our data are consistent with numerous seeds from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of genomic surveillance in addition to traditional epidemiological indicators.

Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types.

AIM: Microsatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may have Lynch syndrome and predicting prognosis in patients with colorectal tumours. Next-generation sequencing (NGS) provides the opportunity to interrogate large numbers of microsatellite loci concurrently with genomic variants. We sought to develop a method to detect MSI that would not require paired normal tissue and would leverage the sequence data obtained from a broad range of tumours tested using our 467-gene NGS Columbia Combined Cancer Panel (CCCP). METHODS: Altered mononucleotide and dinucleotide microsatellite loci across the CCCP region of interest were evaluated in clinical samples encompassing a diverse range of tumour types. The number of altered loci was used to develop a decision tree classifier model trained on the retrospectively collected cohort of 107 clinical cases sequenced by the CCCP assay. RESULTS: The classifier was able to correctly classify all cases and was then used to analyse a test set of clinical cases (n=112) and was able to correctly predict their MSI status with 100% sensitivity and specificity. Analysis of recurrently altered loci identified alterations in genes involved in DNA repair, signalling and transcriptional regulation pathways, many of which have been implicated in MSI tumours. CONCLUSION: This study highlights the utility of this approach, which should be applicable to laboratories performing similar testing.

Clonal T cell receptor gene rearrangements in coeliac disease: implications for diagnosing refractory coeliac disease.

AIMS: Refractory coeliac disease type II (RCDII), a rare complication of coeliac disease (CD) associated with high morbidity, requires identification of a clonal population of phenotypically aberrant intraepithelial lymphocytes (IELs) for diagnosis. However, data regarding the frequency and significance of clonal T cell receptor (TCR) gene rearrangements (TCR-GRs) in small bowel (SB) biopsies of patients without RCDII are limited. METHODS: We analysed results of TCR-GR analyses performed on SB biopsies at our institution over a 3-year period, which were obtained from eight active CD, 172 CD on gluten-free diet (GFD), 33 RCDI, and three RCDII patients and 14 patients without CD. TCR-GR patterns were divided into clonal, polyclonal and prominent clonal peaks (PCPs) and these patterns were correlated with clinical and pathological features. RESULTS: Clonal TCR-GR products were detected in biopsies from 67% of patients with RCDII, 17% of patients with RCDI and 6% of patients with GFD. PCPs were observed in all disease phases (range 12%-33%). There was no significant difference in the TCR-GR patterns between the non-RCDII disease categories (p=0.39). A higher frequency of surface CD3(-) IELs was noted in cases with clonal TCR-GR, but the PCP pattern did not show associations with any clinical or pathological feature. Persistence of clonal or PCP pattern on repeat biopsy was seen for up to 2 years without evidence of RCDII. CONCLUSIONS: Clonal TCR-GRs are not infrequent in cases lacking features of RCDII, while PCPs are frequent in all disease phases. TCR-GR results should be assessed in conjunction with immunophenotypic, histological and clinical findings for appropriate diagnosis and classification of RCD.

Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody.

Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ∼10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.

Peptidomimetic blockade of MYB in acute myeloid leukemia.

Aberrant gene expression is a hallmark of acute leukemias. MYB-driven transcriptional coactivation with CREB-binding protein (CBP)/P300 is required for acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP/P300 complex and rapidly accumulates in the nuclei of AML cells. Treatment of AML cells with MYBMIM led to the dissociation of the MYB:CBP/P300 complex in cells, its displacement from oncogenic enhancers enriched for MYB binding sites, and downregulation of MYB-dependent gene expression, including of MYC and BCL2 oncogenes. AML cells underwent mitochondrial apoptosis in response to MYBMIM, which was partially rescued by ectopic expression of BCL2. MYBMIM impeded leukemia growth and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells. These findings elucidate the dependence of human AML on aberrant transcriptional coactivation, and establish a pharmacologic approach for its therapeutic blockade.

Antibodyomics: bioinformatics technologies for understanding B-cell immunity to HIV-1.

Numerous antibodies have been identified from HIV-1-infected donors that neutralize diverse strains of HIV-1. These antibodies may provide the basis for a B cell-mediated HIV-1 vaccine. However, it has been unclear how to elicit similar antibodies by vaccination. To address this issue, we have undertaken an informatics-based approach to understand the genetic and immunologic processes controlling the development of HIV-1-neutralizing antibodies. As DNA sequencing comprises the fastest growing database of biological information, we focused on incorporating next-generation sequencing of B-cell transcripts to determine the origin, maturation pathway, and prevalence of broadly neutralizing antibody lineages (Antibodyomics1, 2, 4, and 6). We also incorporated large-scale robotic analyses of serum neutralization to identify and quantify neutralizing antibodies in donor cohorts (Antibodyomics3). Statistical analyses furnish another layer of insight (Antibodyomics5), with physical characteristics of antibodies and their targets through molecular dynamics simulations (Antibodyomics7) and free energy perturbation analyses (Antibodyomics8) providing information-rich output. Functional interrogation of individual antibodies (Antibodyomics9) and synthetic antibody libraries (Antibodyomics10) also yields multi-dimensional data by which to understand and improve antibodies. Antibodyomics, described here, thus comprise resolution-enhancing tools, which collectively embody an information-driven discovery engine aimed toward the development of effective B cell-based vaccines.

Clinical Genomic Profiling of a Diverse Array of Oncology Specimens at a Large Academic Cancer Center: Identification of Targetable Variants and Experience with Reimbursement.

Large cancer panels are being increasingly used in the practice of precision medicine to generate genomic profiles of tumors with the goal of identifying targetable variants and guiding eligibility for clinical trials. To facilitate identification of mutations in a broad range of solid and hematological malignancies, a 467-gene oncology panel (Columbia Combined Cancer Panel) was developed in collaboration with pathologists and oncologists and is currently available and in use for clinical diagnostics. Herein, we share our experience with this testing in an academic medical center. Of 255 submitted specimens, which encompassed a diverse range of tumor types, we were able to successfully sequence 92%. The Columbia Combined Cancer Panel assay led to the detection of a targetable variant in 48.7% of cases. However, although we show good clinical performance and diagnostic yield, third-party reimbursement has been poor. Reimbursement from government and third-party payers using the 81455 Current Procedural Terminology code was at 19.4% of billed costs, and 55% of cases were rejected on first submission. Likely contributing factors to this low level of reimbursement are the delays in valuation of the 81455 Current Procedural Terminology code and in establishing national or local coverage determinations. In the absence of additional demonstrations of clinical utility and improved patient outcomes, we expect the reimbursement environment will continue to limit the availability of this testing more broadly.

Free Energy Perturbation Calculation of Relative Binding Free Energy between Broadly Neutralizing Antibodies and the gp120 Glycoprotein of HIV-1.

Direct calculation of relative binding affinities between antibodies and antigens is a long-sought goal. However, despite substantial efforts, no generally applicable computational method has been described. Here, we describe a systematic free energy perturbation (FEP) protocol and calculate the binding affinities between the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class. The protocol has been adapted from successful studies of small molecules to address the challenges associated with modeling protein-protein interactions. Specifically, we built homology models of the three antibody-gp120 complexes, extended the sampling times for large bulky residues, incorporated the modeling of glycans on the surface of gp120, and utilized continuum solvent-based loop prediction protocols to improve sampling. We present three experimental surface plasmon resonance data sets, in which antibody residues in the antibody/gp120 interface were systematically mutated to alanine. The RMS error in the large set (55 total cases) of FEP tests as compared to these experiments, 0.68kcal/mol, is near experimental accuracy, and it compares favorably with the results obtained from a simpler, empirical methodology. The correlation coefficient for the combined data set including residues with glycan contacts, R2=0.49, should be sufficient to guide the choice of residues for antibody optimization projects, assuming that this level of accuracy can be realized in prospective prediction. More generally, these results are encouraging with regard to the possibility of using an FEP approach to calculate the magnitude of protein-protein binding affinities.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

MLL/KMT2A translocations in diffuse large B-cell lymphomas.

Translocations of the histone-lysine N-methyltransferase 2A (KMT2A) gene, formerly known as myeloid lymphoid leukemia/mixed-lineage leukemia gene, are commonly associated with high-risk de novo or therapy-associated B-cell and T-cell lymphoblastic leukemias and myeloid neoplasms. Rare B-cell non-Hodgkin lymphomas harboring KMT2A translocations have been reported, but information regarding the clinical behavior of such cases is limited. Here, we describe two extranodal diffuse large B-cell lymphomas (DLBCLs): a primary thyroid DLBCL and a large cell transformation of a splenic marginal zone lymphoma, which displayed complex karyotypes and translocations involving chromosome 11q23 targeting the KMT2A gene. The pathological and clinical characteristics of these cases are discussed in the context of previously reported lymphomas associated with different types of KMT2A genetic aberrations. In contrast to the poor clinical outcomes of patients with acute leukemias and myeloid neoplasms associated with KMT2A translocations, patients with B-cell non-Hodgkin lymphomas, exhibiting similar translocations, appear to respond well to immunochemotherapy. Our findings add to the growing list of histone methyltransferase genes deregulated in DLBCL and highlight the diversity of mechanisms, altering the function of epigenetic modifier genes in lymphomas.

Brentuximab vedotin (SGN-35) in a 3-year-old child with relapsed systemic anaplastic large cell lymphoma.

The treatment of anaplastic large cell lymphoma (ALCL) has largely relied on anthracycline-based chemotherapy. Targeted lymphoma therapy, using an anti-CD30 antibody, provides an innovative treatment modality for specific lymphomas, particularly ALCL. Brentuximab vedotin (BV; SGN-35) uses a CD30 monoclonal antibody to generate apoptosis of targeted lymphoma cells. We present a pediatric patient with ALCL who experienced second relapse after standard chemotherapy and autologous and allogeneic hematopoietic stem cell transplantation. This patient has been treated with BV as a single agent and has sustained remission for several months. BV offers a targeted treatment for relapsed ALCL, even after multiple relapses.

Downbeat nystagmus secondary to familial hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis is a rare autosomal recessive disorder characterized by severe inflammation induced by defective natural killer cell function, which triggers a state of highly stimulated but ineffective immune response. This disorder can affect multiple organ systems, and neurologic manifestations include irritability, seizures, impaired consciousness, meningismus, and cranial nerve palsies. We describe a unique case of hemophagocytic lymphohistiocytosis in which downbeat nystagmus developed due to cerebellar swelling with compression of the cervicomedullary junction.

T-cell acute lymphoblastic leukaemia after liver transplantation: post-transplant lymphoproliferative disorder or coincidental de novo leukaemia?

Post-transplant lymphoproliferative disorders of T-cell origin are quite uncommon, and the vast majority represent neoplasms of mature, post-thymic T- or natural killer cells. Here, we report a rare case of T-cell acute lymphoblastic leukaemia (T-ALL), which occurred in an 18-year-old man who had undergone three liver transplants, initially for biliary atresia and subsequently for graft failure due to chronic rejection. He had received immunosuppression with cyclosporine and tacrolimus, as well as short-term treatment with OKT3. The T-ALL occurred 16 years after the first liver transplant. This case highlights the challenge for classifying rare neoplasms occurring in recipients of solid organ transplants that are currently not recognized to lie within the spectrum of post-transplant lymphoproliferative disorders. Given the long interval between the liver transplants and the development of T-ALL, a coincidental occurrence of the leukaemia cannot be ruled out. However, the potential roles of immunosuppressive therapy and other co-morbid conditions of the individual as possible risk factors for the pathogenesis of T-ALL are discussed.

Calculation of the free energy and cooperativity of protein folding.

Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.

Unfolded state of polyalanine is a segmented polyproline II helix.

Definition of the unfolded state of proteins is essential for understanding their stability and folding on biological timescales. Here, we find that under near physiological conditions the configurational ensemble of the unfolded state of the simplest protein structure, polyalanine alpha-helix, cannot be described by the commonly used Flory random coil model, in which configurational probabilities are derived from conformational preferences of individual residues. We utilize novel effectively ergodic sampling algorithms in the presence of explicit aqueous solvation, and observe water-mediated formation of polyproline II helical (P(II)) structure in the natively unfolded state of polyalanine, and its facilitation of alpha-helix formation in longer peptides. The segmented P(II) helical coil preorganizes the unfolded state ensemble for folding pathway entry by reducing the conformational space available to the diffusive search. Thus, as much as half of the folding search in polyalanine is accomplished by preorganization of the unfolded state.