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To verify the analytical performance of a new mass spectrometry–based method, termed MASS-FIX, when screening for plasma cell disorders in a routine clinical laboratory.

ObjectivesTo report the expanded neurological presentations and oncological associations of tripartite motif-containing protein 46 (TRIM46)-IgG seropositive patients.MethodsArchived sera/cerebrospinal fluid (CSF) were evaluated by tissue-based immunofluorescence assay to identify patients with identical axon initial segment (AIS)-specific staining pattern. Phage immunoprecipitation sequencing (PhIP-Seq) was used to identify the putative autoantigen.ResultsIgG in serum (17) and/or CSF (16) from 25 patients yielded unique AIS-specific staining on murine central nervous system (CNS) tissue. An autoantibody specific for TRIM46 was identified by PhIP-Seq, and autoantigen specificity was confirmed by transfected COS7 cell-based assay. Clinical information was available for 22 TRIM46-IgG seropositive patients. Fifteen were female (68%). Median age was 67 years (range 25–87). Fifteen (68%) patients presented with subacute cerebellar syndrome (six isolated; nine with CNS accompaniments: encephalopathy (three), brainstem signs (two), myelopathy (two), parkinsonism (one)). Other phenotypes included limbic encephalitis (three), encephalopathy with/without seizures (two), myelopathy (two). Eighteen (82%) had cancer: neuroendocrine carcinomas (9; pancreatic (3), small-cell lung (4), oesophagus (1), endometrium (1)), adenocarcinomas (6; lung (2), ovarian (2), endometrial (1), breast (1)), sarcoma (2) and gastrointestinal tumour (1). Neurological symptoms in three followed immune checkpoint inhibitor (ICI) administration.ConclusionsThis study supports TRIM46-IgG being a biomarker of paraneoplastic CNS disorders and expands the neurological phenotypes, oncological and ICI-related adverse event associations.

IMPORTANCE: Myelin oligodendrocyte glycoprotein-IgG1–associated disorder (MOGAD) is a distinct central nervous system–demyelinating disease. Positive results on MOG-IgG1 testing by live cell-based assays can confirm a MOGAD diagnosis, but false-positive results may occur. OBJECTIVE: To determine the positive predictive value (PPV) of MOG-IgG1 testing in a tertiary referral center. DESIGN, SETTING, AND PARTICIPANTS: This diagnostic study was conducted over 2 years, from January 1, 2018, through December 31, 2019. Patients in the Mayo Clinic who were consecutively tested for MOG-IgG1 by live cell-based flow cytometry during their diagnostic workup were included. Patients without research authorization were excluded. MAIN OUTCOMES AND MEASURES: Medical records of patients who were tested were initially reviewed by 2 investigators blinded to MOG-IgG1 serostatus, and pretest probability was classified as high or low (suggestive of MOGAD or not). Testing of MOG-IgG1 used a live-cell fluorescence-activated cell-sorting assay; an IgG binding index value of 2.5 or more with an end titer of 1:20 or more was considered positive. Cases positive for MOG-IgG1 were independently designated by 2 neurologists as true-positive or false-positive results at last follow-up, based on current international recommendations on diagnosis or identification of alternative diagnoses; consensus was reached for cases in which disagreement existed. RESULTS: A total of 1617 patients were tested, and 357 were excluded. Among 1260 included patients tested over 2 years, the median (range) age at testing was 46 (0-98) years, and 792 patients were female (62.9%). A total of 92 of 1260 (7.3%) were positive for MOG-IgG1. Twenty-six results (28%) were designated as false positive by the 2 raters, with an overall agreement on 91 of 92 cases (99%) for true and false positivity. Alternative diagnoses included multiple sclerosis (n = 11), infarction (n = 3), B12 deficiency (n = 2), neoplasia (n = 2), genetically confirmed adrenomyeloneuropathy (n = 1), and other conditions (n = 7). The overall PPV (number of true-positive results/total positive results) was 72% (95% CI, 62%-80%) and titer dependent (PPVs: 1:1000, 100%; 1:100, 82%; 1:20-40, 51%). The median titer was higher with true-positive results (1:100 [range, 1:20-1:10000]) than false-positive results (1:40 [range, 1:20-1:100]; P < .001). The PPV was higher for children (94% [95% CI, 72%-99%]) vs adults (67% [95% CI, 56%-77%]) and patients with high pretest probability (85% [95% CI, 76%-92%]) vs low pretest probability (12% [95% CI, 3%-34%]). The specificity of MOG-IgG1 testing was 97.8%. CONCLUSIONS AND RELEVANCE: This study confirms MOG-IgG1 as a highly specific biomarker for MOGAD, but when using a cutoff of 1:20, it has a low PPV of 72%. Caution is advised in the interpretation of low titers among patients with atypical phenotypes, because ordering MOG-IgG1 in low pretest probability situations will increase the proportion of false-positive results.

IMPORTANCE: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management. OBJECTIVE: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis. DESIGN, SETTING, AND PARTICIPANTS: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays). MAIN OUTCOMES AND MEASURES: Outcome variables included modified Rankin score and gait aid use. RESULTS: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell–predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis. CONCLUSIONS AND RELEVANCE: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.

Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.

ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2–like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737–shDhx9 synthetic lethal relationship.

ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2–like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737–shDhx9 synthetic lethal relationship.

Elimination of Myeloid Leukemia Cell 1 (Mcl-1) is an early event in the onset of cell death following DNA damage and in many settings plays a critical role in dictating the success of chemotherapeutic agents. Following DNA damage, Mcl-1 is rapidly and efficiently targeted to the 26S proteasome through the action of E3 ubiquitin ligases. Tumors having acquired lesions that lead to stabilization of Mcl-1 are highly aggressive and have a poorer prognosis. Herein, we further characterize an additional mechanism of Mcl-1 proteolysis that is proteasome-independent but mitochondrial-dependent. A mitochondrial targeting signal located in the N-terminus of Mcl-1 is essential for targeting Mcl-1 to this alternative degradative avenue. We demonstrate that the Akt/mTORC1 survival pathway protects Mcl-1 from mitochondrial-dependent proteolysis. Disrupting Mcl-1 mitochondrial targeting improves the pro-survival capacity of Mcl-1 both ex vivo and in vivo in the well characterized mouse Eμ-Myc lymphoma model. Our data uncover an important relationship between the mitochondria and the Mcl-1 N-terminus in dictating cell fate following DNA damage.

Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAiMetternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.

Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAiMetternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.

Background: The fast growing Middle Eastern (ME) population has rarely been studied in the U.S.. The purpose of this study was to compare the epidemiology of primary brain tumors in this ethnic population with the non-Hispanic, non-Middle Eastern White (NHNMW) in California. Methods: ME cases were identified by surname in the California cancer registry and ME population estimates were based on ancestry. Data for 683 cases of primary brain tumors (429 benign, 238 malignant, 16 uncertain) in the ME and 15,589 cases (8352 benign, 6812 malignant, 425 uncertain) in the NHNMW were available for this study. Results: ME patients were significantly (p<0.05) younger and their age-adjusted incidence rates per 100,000 for benign tumors of 10.0 in men and 17.6 in women were higher than similar rates of 7.3 and 10.6 in the NHNMW group (p<0.05). Rates for malignant tumors were similar. Meningioma was the main histology responsible for the observed increase in patients over 40 years of age. Also increased were benign tumors of the pituitary and pineal glands. The overall mortality in patients with benign tumors was significantly lower than malignant tumors. Conclusions: This study presents a significantly high incidence of benign meningioma in the ME population in California. This may be due to higher susceptibility or exposure of this ethnic group to the risk factor(s) for this neoplasm. Considering the reported causal association of benign meningioma with childhood radiation exposure from Israel, exposure to this risk factor in this ethnic group needs to be evaluated in future studies.

Longitudinal studies assessing durability of the anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) humoral immune response have generated conflicting results. This has been proposed to be due to differences in patient populations, the lack of standardized methodologies, and the use of assays that measure distinct aspects of the humoral response.

To determine the frequency, age and sex associations for autoimmune/paraneoplastic encephalitis antibody biomarkers (AE-Abs).

Yersinia enterocoliticaenteritis is a potentially treatable infection. To understand its seasonal incidence and clinical presentation in children, we reviewed case records of children seen in Cardinal Glennon Children's Hospital in St. Louis, MO. We found the incidence of Yersiniaenteritis to be as frequent as enteritis caused by Campylobacter.It occurred more frequently during the winter months (P< 0.002) than during the rest of the year. Fever was common in infants with Yersiniaenteritis. Abdominal pain and distention were infrequent. Seventeen (35) patients were 3 months of age or younger; 4 of 17 (28) developed Yersiniasepsis as a complication of the enteritis. Physicians should perform stool cultures for Y. enterocoliticain young infants who present with high fever and diarrhea in winter months, especially when there is blood in stools or the patient appears septic.

ABSTRACTPTP1B and TC-PTP are highly related protein-tyrosine phosphatases (PTPs) that regulate the JAK/STAT signaling cascade essential for cytokine-receptor activation in immune cells. Here, we describe a novel immunotherapy approach whereby monocyte-derived dendritic cell (moDC) function is enhanced by modulating the enzymatic activities of PTP1B and TC-PTP. To downregulate or delete the activity/expression of these PTPs, we generated mice with PTP-specific deletions in the dendritic cell compartment or used PTP1B and TC-PTP specific inhibitor. While total ablation of PTP1B or TC-PTP expression leads to tolerogenic DCs via STAT3 hyperactivation, downregulation of either phosphatase remarkably shifts the balance toward an immunogenic DC phenotype due to hyperactivation of STAT4, STAT1 and Src kinase. The resulting increase in IL-12 and IFNγ production subsequently amplifies the IL-12/STAT4/IFNγ/STAT1/IL-12 positive autocrine loop and enhances the therapeutic potential of mature moDCs in tumor-bearing mice. Furthermore, pharmacological inhibition of both PTPs improves the maturation of defective moDCs derived from pancreatic cancer (PaC) patients. Our study provides a new advance in the use of DC-based cancer immunotherapy that is complementary to current cancer therapeutics.

Background:Current laboratory methods for detection of monoclonal gammopathies include a panel of serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and serum free light chain quantitation (FLC). Our group has recently described a new assay based on matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) which is capable of detecting, isotyping and quantitating M-proteins in a single assay. The basic principle of the method involves leveraging the unique mass resulting from light chain (LC) Ig gene rearrangement in B-cells. In a similar fashion to SPEP, the mass distribution of LCs after dissociation from their heavy chains can be examined for the presence of an over expressed light chain. In this study we retrospectively compared the MALDI-TOF MS method to SPEP, IFE and Hevylite ratios for monitoring patients with monoclonal gammopathies.

Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed. Mills:Mayo Clinci: Patents & Royalties: Patent Application Filed. Barnidge:Mayo Clinic: Patents & Royalties: Patent Application Filed.

Murray: Mayo Clinic: Patent Application filed Patents & Royalties. Barnidge:Mayo Clinic: Patent filed Patents & Royalties.

Detection of an M-protein (monoclonal immunoglobulin) has been used to diagnose and monitor multiple myeloma (MM). As therapies for MM have improved, more sensitive methods have been used to define response: immunofixation electrophoresis (IFX) of serum and urine, normalization of the serum immunoglobulin free light chain (FLC) ratio, and high sensitivity flow cytometry to detect clonal plasma bone marrow cells. It is hoped that these more sensitive approaches will differentiate those patients with minimal residual disease (MRD) versus no residual disease (NRD), that later which could mean a cure. Flow cytometry of plasma cells requires bone marrow aspiration, which is inconvenient and expensive and is potentially limited by sampling bias. More sensitive methods to differentiate MRD from NRD using serum would be advantageous.