Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization

Therapeutic strategies directed at the tumor surfaceome (TS), including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with preserved tissue architecture. Moreover, TS-MAP specifically identifies proteins capable of endocytosis as tractable targets for ADCs and other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome (e.g., integrins, proteoglycans, semaphorins, and cancer stem cell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. The TS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected gliomas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data provide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming at a better understanding of how to harness the TS for personalized immunotherapy.

Cell-surface proteins have a key role in drug development, and approximately two-thirds of approved human drugs target a cell-surface protein (1). Recently, tumor cell–surface proteins integrated with the plasma membrane (tumor surfaceome [TS]) have attracted considerable attention as targets for immunotherapies in cancer. Immune checkpoint-blocking antibodies (e.g., ipilimumab and nivolumab), antibody drug conjugates (ADCs, e.g., trastuzumab emtansin), radioimmunotherapy (RIT, e.g., ⁹⁰Y ibritumomab tiuxetan), and chimeric antigen receptor T (CAR-T) cells are all directed at the TS and currently revolutionize cancer treatment (2–6). With the impressive development of creative methods for antibody and T cell engineering, a remaining challenge is the lack of strategies to comprehensively map potential TS target antigens for the design of more rational, individualized treatments (7). Although advancements in DNA and RNA sequencing provide high throughput data for prediction algorithms, e.g., personalized peptide vaccine trials (8, 9), the predicted proteome derived from these platforms is not necessarily expressed and available for targeting. Moreover, proteomics-based strategies involve analysis of the bulk from disintegrated tumor tissue, resulting in loss of spatial information and limited coverage of the less abundant and hydrophobic TS proteins (10, 11). Of particular relevance, ADC, RIT, and other intracellular drug delivery strategies rely on TS targets that functionally engage in endocytic internalization (12). Clearly, despite its great targeting potential in cancer immunotherapy, the TS remains an elusive treasure for further discovery.Procedures for unbiased mapping of the TS and target identification should include specific labeling of the TS in freshly resected patient tumors with preserved tissue architecture. Enrichment of TS proteins and reduction of noise from intracellular proteins as well as abundant extracellular matrix collagens and glycoproteins would greatly improve downstream mass spectrometry analysis. Moreover, the approach should allow functional and dynamic profiling of TS internalization in an intact tissue environment. With the aim to address these challenges and to provide insight into the complexity of the TS, we have developed a versatile technology for TS mapping (TS-MAP). As proof of concept, we focused on primary brain tumors that remain among the most aggressive forms of cancer and for which attempts to conquer the most common variant, glioblastoma (GBM) (World Health Organization [WHO] grade IV) have failed so far (13). TS-MAP is compatible with spheroids from primary human stem cell–like GBM cultures, as well as mouse and patient brain tumors, and separately profiles surface resident and internalized TS proteins. Moreover, a TS classifier (SURFME) was curated for filtering and categorization of bona fide membrane proteins exposed to the extracellular space. We find significant differences in the TS between the 2D and 3D spheroid format, which underlines the importance of cellular spatial organization. In strong support of the need of individualized approaches, our findings suggest substantial intertumoral heterogeneity in the relative abundance of TS proteins in a cohort of freshly resected patient gliomas.     

Signet-ring cell lymphoma of bone marrow.

A case of signet-ring cell lymphoma affecting bone marrow is reported. The tumour presented as multiple lytic lesions in the lumbosacral spine. A bone biopsy specimen showed the typical appearances of signet-ring cell lymphoma, and the cells stained positively with antiserum to CD20, though neither immunoglobulin light or heavy chains could be shown within the vacuoles. The patient subsequently responded to chemotherapy.     

Evaluation of the NINCDS-ADRDA criteria in the differentiation of Alzheimer's disease and frontotemporal dementia

OBJECTIVES: The diagnosis of Alzheimer's disease (AD) is now reliant on the use of NINCDS-ADRDA criteria. Other diseases causing dementia are being increasingly recognised--for example, frontotemporal dementia (FTD). Historically, these disorders have not been clearly demarcated from AD. This study assesses the capability of the NINCDS-ADRDA criteria to accurately distinguish AD from FTD in a series of pathologically proved cases. METHODS: The case records of 56 patients (30 with AD, 26 with FTD) who had undergone neuropsychological evaluation, brain imaging, and ultimately postmortem, were assessed in terms of whether at initial diagnosis the NINCDS-ADRDA criteria were successful in diagnosing those patients who had AD and excluding those who did not. RESULTS: (1) The overall sensitivity of the NINCDS-ADRDA criteria in diagnosing "probable" AD from 56 patients with cortical dementia (AD and FTD) was 0.93. However, the specificity was only 0.23; most patients with FTD also fulfilled NINCDS-ADRDA criteria for AD. (2) Cognitive deficits in the realms of orientation and praxis significantly increased the odds of a patient having AD compared with FTD, whereas deficits in problem solving significantly decreased the odds. Neuropsychological impairments in the domains of attention, language, perception, and memory as defined in the NINCDS-ADRDA statement did not contribute to the clinical differentiation of AD and FTD. CONCLUSION: NINCDS-ADRDA criteria fail accurately to differentiate AD from FTD. Suggestions to improve the diagnostic specificity of the current criteria are made.     

Dysbindin-1 is a synaptic and microtubular protein that binds brain snapin

Variations in the gene encoding the novel protein dysbindin-1 (DTNBP1) are among the most commonly reported genetic variations associated with schizophrenia. Recent studies show that those variations are also associated with cognitive functioning in carriers with and without psychiatric diagnoses, suggesting a general role for dysbindin-1 in cognition. Such a role could stem from the protein's known ability to affect neuronal glutamate release. How dysbindin-1 might affect glutamate release nevertheless remains unknown without the discovery of the protein's neuronal binding partners and its subcellular locus of action. We demonstrate here that snapin is a binding partner of dysbindin-1 in vitro and in the brain. Tissue fractionation of whole mouse brains and human hippocampal formations revealed that both dysbindin-1 and snapin are concentrated in tissue enriched in synaptic vesicle membranes and less commonly in postsynaptic densities. It is not detected in presynaptic tissue fractions lacking synaptic vesicles. Consistent with that finding, immunoelectron microscopy showed that dysbindin-1 is located in (i) synaptic vesicles of axospinous terminals in the dentate gyrus inner molecular layer and CA1 stratum radiatum and in (ii) postsynaptic densities and microtubules of dentate hilus neurons and CA1 pyramidal cells. The labeled synapses are often asymmetric with thick postsynaptic densities suggestive of glutamatergic synapses, which are likely to be derived from dentate mossy cells and CA3 pyramidal cells. The function of dysbindin-1 in presynaptic, postsynaptic and microtubule locations may all be related to known functions of snapin.     

Strain variation among Bordetella pertussis isolates from Québec and Alberta provinces of Canada from 1985 to 1994

Pulsed-field gel electrophoresis and gene typing were able to differentiate among 3,597 Bordetella pertussis isolates circulating in Alberta and Québec Provinces, Canada, from 1985 to 1994 and distinguish them from the strains used in vaccine production. This study provides a baseline for continued surveillance of prevalent and emerging strains of B. pertussis in Canada.     

Multicentre age-related reference intervals for cerebrospinal fluid serine concentrations: Implications for the diagnosis and follow-up of serine biosynthesis disorders

The disorders of serine biosynthesis are a group of inborn errors of metabolism characterised by congenital microcephaly, seizures and severe psychomotor retardation. Although these disorders are rare the prompt recognition of serine deficiency is important as these disorders are treatable. The diagnosis is based on decreased concentrations of serine in cerebrospinal fluid (CSF). It has previously been reported that CSF serine concentrations are inversely associated with age. However, accurate age-related reference intervals have not been generated which has contributed to cases not being identified. In a multicentre study involving 9 different laboratories a total of 424 CSF serine results were obtained. Regression based analyses were performed to calculate age-specific reference intervals. Lower reference intervals for subjects aged 1 week, 1 month, 6 months, 1 year, 3 years and 15 years were 35.0, 31.0, 26.0, 24.0, 21.0 and 17.0 μmol/L respectively. Assessment of CSF serine concentrations in 11 patients (aged 1 day to 13 years) previously diagnosed with disorders of serine biosynthesis (serine concentrations ranging from 5 to 18 μmol/L) were clearly decreased compared to our age-related reference intervals and would have correctly identified all cases, thus enabling prompt treatment. However, if age had not been taken into consideration a reference interval of 12.6–69.4 μmol/L would be obtained for the combined data set and would have resulted in 2 cases being missed. In conclusion, appropriate age-related reference intervals for CSF serine should be used to diagnose patients with inborn errors of serine biosynthesis.     

The standard error of measurement is a more appropriate measure of quality for postgraduate medical assessments than is reliability: an analysis of MRCP(UK) examinations

Background  

Cronbach's alpha is widely used as the preferred index of reliability for medical postgraduate examinations. A value of 0.8-0.9 is seen by providers and regulators alike as an adequate demonstration of acceptable reliability for any assessment. Of the other statistical parameters, Standard Error of Measurement (SEM) is mainly seen as useful only in determining the accuracy of a pass mark. However the alpha coefficient depends both on SEM and on the ability range (standard deviation, SD) of candidates taking an exam. This study investigated the extent to which the necessarily narrower ability range in candidates taking the second of the three part MRCP(UK) diploma examinations, biases assessment of reliability and SEM.