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Abstract Chimeric Antigen Receptor (CAR) immunotherapy utilizes genetically-engineered immune cells that express a unique cell surface receptor that combines tumor antigen specificity with immune cell activation. In recent clinical trials, the adoptive transfer of CAR-modified immune cells (including CAR-T and CAR-NK cells) into patients has been remarkably successful in treating multiple refractory blood cancers. To improve safety and efficacy, and expand potential applicability to other cancer types, CARs with different target specificities and sequence modifications are being developed and tested by many laboratories. Despite the overall progress in CAR immunotherapy, conventional tools to design and evaluate the efficacy and safety of CAR immunotherapies can be inaccurate, time-consuming, costly, and labor-intensive. Furthermore, existing tools cannot always determine how responsive individual patients will be to a particular CAR immunotherapy. Recent work in our laboratory suggests that the quality of the immunological synapse (IS) can accurately predict CAR-modified cell efficacy (and toxicity) that can correlate with clinical outcomes. Here we review current efforts to develop a Synapse Predicts Efficacy (SPE) system for easy, rapid and cost-effective evaluation of CAR-modified immune cell immunotherapy. Ultimately, we hypothesize the conceptual basis and clinical application of SPE will serve as an important parameter in evaluating CAR immunotherapy and significantly advance precision cancer immunotherapy. Graphical abstract Graphic abstract for manuscript CCAS-D-20-00136 by Liu, D., et al., ‘The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy”. The various branches of evaluating cancer immunotherapy metaphorically represented as a Rubik’s cube. The development of a novel approach to predict the effectiveness of Chimeric Antigen Receptor (CAR)-modified cells by quantifying the quality of CAR IS will introduce a new parameter to the rapidly expanding field of cancer immunotherapy. Currently, no single parameter can predict the clinical outcome or efficacy of a specific type of CAR-modified cell. IS quality will serve as a quantifiable measure to evaluate CAR products and can be used in conjunction with other conventional parameters to form a composite clinical predictor. Much like a Rubik’s cube has countless configurations, several methods and combinations of clinical metrics have arisen for evaluating the ability of a given immunotherapeutic strategy to treat cancer. The quality of IS depicting cancer immunotherapy is metaphorically expressed as a Rubik’s cube. Each face/color represents one aspect of cancer therapy. Each grid in one face indicates one factor within that aspect of cancer therapy. For example, the green color represents the tumor microenvironment, and one out of the nine grids in the green color indicates suppressor cells (suppressors in green). Changes in one factor may completely alter the entire strategy of cancer therapy. However, the quality of IS (illuminated center red grid) makes the effectiveness of CAR immunotherapy predictable.

BackgroundThe rapid emergence of new vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires an equally rapid deployment of diagnostic tests to specifically identify each VOC as soon as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) Alpha/Beta/Gamma RT-PCR melting temperature (Tm) signature-based assay, which now includes modifications that allow specific detection of Delta (B.1.617.2) and Omicron (B.1.529) VOCs.MethodsWe developed a dual SMB assay (SMB-452), which targeted the T22917G (L452R) mutation in the SARS-CoV-2 spike protein to specifically detect the Delta VOC. We also identified a Tm profile in our existing SMB-501 and SMB-484 assays (which detect mutations in codons 501 and 484 of the SARS-CoV-2 spike protein, respectively) that differentiate the Omicron-specific N501Y (A23063T) and E484A (A23013C) mutations from both wild type (WT) and other VOCs. The entire six SMB three-codon assay was tested using reference SARS-CoV-2 RNAs. The assay was then validated using clinical samples from COVID-19 patients tested with a LightCycler 480 (LC480) (74 samples), Bio-Rad CFX96 (34 samples), Rotor-Gene Q (Qiagen) (34 samples) and an ABI-7500 (34 samples) RT-PCR instruments. Six SMB Tm results were then inputted into an Excel Analysis tool to generate specific VOC identifications.ResultsThe limit of detection (LOD) for the new SMB-452 assay, which specifically identified the Delta variant was 1 genomic equivalent (GE) per reaction. The LODs of the SMB-501 and SMB-484 assays, which detect Omicron were 100 and 103 GE respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% of the samples as WT or VOCs in clinical samples and 6% of the tests producing indeterminate results. None of the samples were incorrectly identified as WT or as a different VOC. Thus, excluding samples with indeterminant results, the assay was 100% sensitive and 100% specific compared to sequencing. There was also 100% concordance between the LC480, BioRad, ABI and Qiagen results, excluding negative or indeterminate results; however, the Qiagen assay had significantly more indeterminates than the other assays.ConclusionThis new assay can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

The increased transmission of SARS-CoV-2 variants of concern (VOC), which originated in the United Kingdom (B.1.1.7/alpha), South Africa (B1.351/beta), Brazil (P.1/gamma), the United States (B.1.427/429 or epsilon), and India (B.1.617.2/delta), requires a vigorous public health response, including real-time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time-consuming and expensive. Here, we describe a simple, rapid, and high-throughput reverse transcriptase PCR (RT-PCR) melting-temperature (Tm) screening assay that identifies the first three major VOCs. RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild-type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (wild type [WT]), B.1.1.7, and B.1.351 variant strains. The assay was then validated using clinical samples. The limit of detection for both the WT and variants was 4 and 10 genomic copies/reaction for the 501- and 484-codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples, as confirmed by Sanger sequencing. We have developed an RT-PCR melt screening test for the major VOCs that can be used to rapidly screen large numbers of patient samples, providing an early warning for the emergence of these variants and a simple way to track their spread.

Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion condition. The WHS core phenotype includes developmental delays, intellectual disabilities, seizures, and distinctive facial features. Various other comorbidities have also been reported, such as hearing loss, heart defects, as well as eye problems and kidney problems. In this report, we present a case of WHS accompanied by hyperparathyroidism and hypercalcemia, which has not been previously reported. A girl was born at 37 weeks of gestation by vaginal delivery. She was small for the gestational age (2,045 g) and admitted to neonatal intensive care unit. She had typical WHS facial features and was found to have bilateral small kidneys associated with transient metabolic acidosis and renal insufficiency. She had right-sided sensorineural hearing loss, a small atrial septal defect, and colpocephaly and hypoplasia of corpus callosum. She had a single seizure which was well controlled with an oral antiepileptic medication. Cytogenetic studies demonstrated a large terminal chromosome 4p deletion (21.4 Mb) and 4p duplication (2.1 Mb) adjacent to the deletion. A unique finding in this patient is her consistently elevated levels of parathyroid hormone and serum calcium, suggesting hyperparathyroidism. We present this rare case along with a review of the literature and hope to draw an attention to a potential relationship between WHS and hyperparathyroidism.

Background. The emergence of more transmissible SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B1.351) and Brazil (P.1) requires a vigorous public health response, including real time strain surveillance on a global scale. Although new SARS-CoV-2 variants can be most accurately identified by genomic sequencing, this approach is time consuming and expensive. A simple and more rapid screen for the key SARS-CoV-2 mutations that define variant strains is needed. We developed a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature assay that identifies the SARS-CoV-2 N501Y mutation, a key mutation which is present in all three known variant strains of concern. Methods. RT-PCR primers and two sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) mutation. One SMB was designed with a probe region that was complementary to the wild type sequence (WT) and a second SMB was designed with a probe region that was complementary to the mutant (MT) sequence. Each SMB was labeled with a different fluorophore, and the assay was performed in a single test well. After RT-PCR, WT versus MT SARS-CoV-2 was identified by a characteristic shift in the melting temperature (Tm) of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT) and SARS-CoV-2 hCoV-19/England/204820464/2020 (a B.1.17 variant). The assay was then validated using clinical samples. Results. The limit of detection (LOD) of the assay for both the WT and the B1.1.7 variant was 4 genomic copies/reaction. The two SMBs produced Tm shifts that were 100% sensitive and 100% specific for identifying the N501Y mutation in cultured virus and in clinical samples as confirmed by Sanger sequencing. Conclusion. We have developed a rapid screening test for the SARS-CoV-2 N501Y mutation, which is a characteristic of all three SARS-CoV-2 stains of global concern. This assay can be used to rapidly screen large numbers of patient samples for these variants, providing an early warning for the emergence and spread of these strains of concern.

Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis.

Background MicroRNAs (miRNAs) play a major role in regulating immune responses at post-transcriptional levels. Previously, we have reported fluctuating interlukine-1ß (IL-1ß)/IL-10 ratios produced by peripheral blood monocytes (PBMo) in some patients with autism spectrum disorders (ASD). This study examined whether changes in miRNA expression by PBMo are associated with changes in IL-1ß/IL-10 ratios and how such changes are associated with ASD clinical features. Methods miRNA expression by purified PBMo from ASD subjects (N = 69) and non-ASD controls (N = 27) were determined by high-throughput sequencing. Cytokine production by PBMo in responses to stimuli of innate immunity, and behavioral symptoms [assessed by aberrant behavioral checklist (ABC)] were also evaluated at the same time of sample obtainment. Results As a whole, there was no difference in miRNA expression between ASD and control non-ASD PBMo. However, when ASD cells were subdivided into 3 groups with high, normal, or low IL-1ß/IL-10 ratios as defined in the “Results” section, in comparison with the data obtained from non-ASD controls, we observed marked changes in miRNA expression. Namely, over 3-fold changes in expression of miR-181a, miR-93, miR-223, miR-342, and miR-1248 were observed in ASD PBMo with high or low IL-1ß/IL-10 ratios, but not in ASD PBMo with normal ratios. These miRNAs that had altered in expression are those closely associated with the regulation of key signaling pathways. With changes in IL-1ß/IL-10 ratios, we also observed changes in the production of cytokines (IL-6, TNF-α, and TGF-ß) other than IL-1ß/IL-10 by ASD PBMo. The association between behavioral symptoms and cytokine levels was different when ASD cells exhibit high/low IL-1ß/IL-10 ratios vs. when ASD cells exhibited normal ratios. Non-IgE-mediated food allergy was also observed at higher frequency in ASD subjects with high/low IL-1ß/IL-10 ratios than with normal ratios. Conclusions Changes in cytokine profiles and miRNA expression by PBMo appear to be associated with changes in ASD behavioral symptoms. miRNAs that are altered in expression in ASD PBMo with high/low IL-1ß/IL-10 ratios are those associated with inflammatory responses. Changes in IL-1ß/IL-10 ratios along with changes in miRNA expression may serve as biomarkers for immune-mediated inflammation in ASD. Electronic supplementary material The online version of this article (10.1186/s12974-017-1003-6) contains supplementary material, which is available to authorized users.

The etiology of trauma-hemorrhagic shock (T/HS)-induced acute lung injury has been difficult to elucidate because of, at least in part, the inability of in vivo studies to separate the noninjurious pulmonary effects of trauma-hemorrhage from the tissue-injurious ones. To circumvent this in vivo limitation, we used a model of T/HS in which T/HS lung injury was abrogated by dividing the mesenteric lymph duct. In this way, it was possible to separate the pulmonary injurious response from the noninjurious systemic response to T/HS by comparing the pulmonary molecular responses of rats subjected to T/HS, which did and did not develop lung injury, with those of nonshocked rats. Using high-density oligonucleotide arrays and treatment group comparisons of whole lung tissue collected at 3 h after the end of the shock or sham-shock period, 139 of 8,799 assessed genes were identified by significant analysis of microarrays. Hemorrhage without the secondary effects of lung injury modulated the expression of 21 genes such as interleukin 1beta, metallothionein-2, and myeloctomatosis oncogene (c-myc). In response to injury, 42 genes were identified to be differentially expressed. Upregulated genes included the L1 retroposon and guanine deaminase, whereas downregulated genes included catalase and superoxide dismutase 1. Real-time polymerase chain reaction confirmed the differential expression for selected genes. PathwayAssist analysis identified interleukin 1beta as a central regulator of two subpathways of stress response-related genes (c-myc and superoxide dismutase 1/catalase) as well as several unrelated genes such as lipoprotein lipase. Our model system provided a unique opportunity to distinguish the molecular changes associated with T/HS-induced acute lung injury from the systemic molecular response to T/HS.

Background: We developed and evaluated a genotyping assay for detection of 50 cystic fibrosis (CF) mutations. The assay is based on small (500 μm) electronic chips, radio frequency (RF) microtransponders (MTPs). The chips are analyzed on a unique fluorescence and RF readout instrument. Methods: We divided the CF assay into 4 panels: core, Hispanic, African-American, and Caucasian. We amplified 18 CF transmembrane regulator (CFTR) DNA fragments covering 50 mutations by use of multiplex PCR using 18 CFTR gene-specific primer pairs. PCR was followed by multiplex allele-specific primer extension (ASPE) reactions and hybridization to capture probes synthesized on MTPs. We used 100 ASPE primers and 100 capture probes. We performed fluorescence measurements of hybridized MTP kits and assay analysis using a custom automated bench-top flow instrument. Results: We validated the system by performing the assay on 23 commercial DNA samples in an internal study and 32 DNA samples in an external study. For internal and external studies, correct calls were 98.8% and 95.7%, false-positive calls 1.1% and 3.9%, and false-negative calls 0.12% and 0.36%, respectively. Conclusions: The MTP-based multiplex assay and analysis platform can be used for CF genotyping.

Developmental delay (DD) and mental retardation (MR) are important child heath issues with a one percent prevalence. Karyotyping with or without subtelomeric FISH (fluorescent in situ hybridization), unless the phenotype of the patient suggests a specific aberration for a specific FISH assay, is the most common procedure in cytogenetic evaluation of MR/ DD. In addition, there are several platforms utilizing microarray based comparative genomic hybridization technology (array-CGH) for genetic testing. Array-CGH can detect deletions or duplications in very small segments of chromosomes and the use of this technology is expected to increase the diagnostic yield. The major limitation of the current BAC based array technologies is the low resolution (� 1 Mb) of the chip and suboptimal coverage particularly in the subtelomeric regions. Our aim was to design a novel array-CGH chip with high-density of probes in the subtelomeric regions as well as to maintain sufficient density in other regions of the genome to provide comprehensive coverage for DD/MR. For this purpose, we used Human Genome CGH Microarray 44B chip (Agilent) as the template for the novel design. Using e-array 4.0 (Agilent), one third of the probes were randomly removed from the array and replaced by 14,000 subtelomeric probes. The average density of the probe coverage is 125 kb and 250–400 probes interrogate subtelomeric regions. To evaluate the array, we tested 15 samples (including subtelomeric aberrations and other microdeletion syndromes), which were previously analyzed by karyotyping and/or FISH. The concordance rate between array results and previous results is 100%. In addition we detected two novel aberrations that were not detected by karyotyping. These results demonstrate the utility of this format of array-CGH in detecting genome wide submicroscopic copy number changes as well as providing comprehensive coverage of all subteleomeric regions. 2007 Wiley-Liss, Inc.

The gut mucosal immune system has to maintain an intricate immune homeostasis by maintaining tolerance to macronutrients, commensal flora, and other harmless molecules in the gut lumen while exerting effective immune defense against pathogenic microbes. It takes the first few years of life to establish this intricate immune homeostasis and until then the gut mucosal immune system is rather error-prone. This is reflected in the fact that young infants and children often suffer from temporary intolerance to common food proteins (FP), e.g. food allergy (FA). During this period, most FA manifests as delayed type FA, mediated by cellular immune responses – often called FP induced enterocolitis syndrome (FPIES) (Jyonouchi, 2008; Nowak-Wegrzyn and Muraro, 2009; Sicherer et al., 1998). Although FPIES seldom cause fatal anaphylaxis, as opposed to IgE-mediated FA, this condition is often under-diagnosed and under-treated. This is, in part, due to the delayed onset of symptoms that renders clinical connection more difficult, as well as, the lack of commercially available diagnostic measures. This is in contrast to IgE mediated FA in which a causal relationship is apparent in many cases and reactivity to food allergens is easily detected with prick skin test (PST) and/or presence of FA specific IgE antibody. The delay in the onset of symptoms of FPIES makes it especially difficult to diagnose FPIES clinically in infants and young children, as well as those with limited expressive language including children with autism spectrum disorders (ASD). In general, FPIES has an excellent prognosis provided there is timely implementation of avoidance measures against offending food (Jyonouchi, 2008; Nowak-Wegrzyn and Muraro, 2009). However, delayed diagnosis/treatment of FPIES can lead to failure to thrive (FTT), protein losing enteropathy, and possibly other irreversible complications.

Innate/adaptive immune responses and transcript profiles of peripheral blood monocytes were studied in ASD children who exhibit fluctuating behavioral symptoms following infection and other immune insults (ASD/Inf, N=30). The ASD/Inf children with persistent gastrointestinal symptoms (ASD/Inf+GI, N=19), revealed less production of proinflammatory and counter-regulatory cytokines with stimuli of innate immunity and marked changes in transcript profiles of monocytes as compared to ASD/no-Inf (N=28) and normal (N=26) controls. This included a 4-5 fold up-regulation of chemokines (CCL2 and CCL7), consistent with the production of more CCL2 by ASD/Inf+GI cells. These results indicate dysregulated innate immune defense in the ASD/Inf+GI children, rendering them more vulnerable to common microbial infection/dysbiosis and possibly subsequent behavioral changes.

It is uncertain whether uterine leiomyosarcoma arises de novo or in preexisting leiomyoma. Leiomyoma-like areas can be seen associated with uterine leiomyosarcoma, raising the possibility of precursor lesions for uterine leiomyosarcoma. In this study, we examined cases of uterine leiomyosarcoma associated with leiomyoma-like areas at the histological, immunohistochemical and DNA level to further evaluate if benign-looking leiomyoma-like and uterine leiomyosarcoma areas are related. Cases of uterine leiomyosarcoma observed at the New York University Medical Center from 1994 to 2007 were reviewed for the presence of leiomyoma-like areas. Of the 26 cases of uterine leiomyosarcoma observed during this period, 18 cases had an associated leiomyoma-like area (five cellular leiomyoma, four symplastic leiomyoma, four cellular and symplastic leiomyoma and five usual type leiomyoma). Sixteen of the 18 cases were examined immunohistochemically for Ki-67, for estrogen receptor, progesterone receptor and for p53. Immunohistochemical profiles were as expected for leiomyoma-like (the mean expression of p53, ER, PR and Ki-67 at 0.3, 63, 75 and 0.6%, respectively), symplastic leiomyoma-like areas (the mean expression of p53, ER, PR and Ki-67 at 0.6, 85, 89 and 5.5%, respectively) and uterine leiomyosarcoma areas (the mean expression of p53, ER, PR and Ki-67 at 52, 38, 39 and 61%, respectively). In six cases, the leiomyoma-like and uterine leiomyosarcoma areas from each case were examined using high-density oligonucleotide array-CGH to determine genetic aberrations in the two areas. Nearly all the genetic aberrations found in leiomyoma-like areas were also found in the corresponding uterine leiomyosarcoma areas. In addition, uterine leiomyosarcoma areas had additional genetic aberrations. The immunohistochemical profiles and genetic aberrations of the examined cases suggest that uterine leiomyosarcoma could arise from the preexisting leiomyoma-like areas that often have a symplastic or cellular morphology.

Chondrosarcomas of the bone are malignant hyaline cartilage-forming tumors with an annual incidence rate of 3.6% of all primary bone malignancies in the United States. Specimens of 25 chondrosarcomas (10 grade I, 9 grade II, 1 grade III, and 5 dedifferentiated) from 23 patients were collected from the Department of Pathology at the University Hospital at UMDNJ-New Jersey Medical School from 1996 to 2007. Array-based comparative genomic hybridization (array-CGH) studies were performed on frozen tumor specimens. Recurrent deletions observed in at least in six tumors were 5q13.2, 5q14.2 approximately q21.3, 6q12 approximately q13, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There was a statistically significant association between high-grade tumor (grade III and dedifferentiated) and the recurrent genetic deletions at 5q14.2 approximately q21.3, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There is consistency between increased levels of aneuploidy and the progression of chondrosarcoma from lower to higher grades.

Karyotyping is a major component of the genetic work-up of patients with dysmorphism. Cytogenetic aberrations close to a known tumor suppressor gene raise important clinical issues because deletion of that tumor suppressor gene can cause genetic predisposition to cancer. We present two cancer-free dysmorphic patients with karyotypes of 46,XX,del(5)(q15q22.3) and 46,XX,del(3)(p25.2∼pter). These deletions are close to the APC and VHL genes that confer susceptibility to familial Adenomatous polyposis (OMIM #17510) and von-Hippel-Lindau syndrome (OMIM #193300), respectively. The array-based comparative genomic hybridization (array-CGH) analysis using a custom Agilent 44K oligonucleotide array demonstrated an interstitial 20.7-megabase (Mb) deletion on 5q (chr5: 89,725,638-110,491,345) and a terminal 9.45-Mb deletion on 3p (chr3:pter-9,450,984). According to the March 2006 human reference sequence, the APC gene is located at chr5: 112,101,483-112,209,835 and the VHL gene is located at chr3: 10,158,319-10,168,746. These results indicate that the APC gene is 2,300 kilobases (kb) and the VHL gene is 700 kb away from deleted regions. Southern blot analysis for APC and VHL genes were negative, consistent with array-CGH findings. These results demonstrate the power of array-CCH to assess potential tumor suppressor gene involvement and cancer risk in patients with microscopically visible deletions in areas near tumor suppressors.

Mutations in the 5,10-methylenetetrahydrofolate reductase (MTHFR) and coagulation factors II and V genes have been found at high frequencies in European and American Caucasian populations and are associated with increased risk for thrombophilia, premature coronary artery disease, and a variety of adverse pregnancy outcomes. Hispanic populations in the United States exhibit high levels of some of these conditions, so we initiated a population-based study to determine the frequency of these mutations (MTHFR C677T and A1298C, Factor II G20210A and Factor V G1691A) in this group. We find comparable frequencies of the Factors II and V mutations, but a high incidence of the two MTHFR mutations in a diverse sample of American Hispanics compared to those reported in Caucasians. Prospective studies of Hispanic women with these mutations and pregnancy outcomes will establish if there is a causal relationship.

Mutations in several genes have recently been identified which predispose to thrombosis, specifically Factor V G1691A (Factor V Leiden), Prothrombin G20210A, and Methylene tetrahydrofolate redactase (MTHFR) C677T. The prevalence of these genes in European populations has been studied, but there is little data on their prevalence in minorities. Samples from a predominantly African-American population were analyzed for these mutations. While the G20210A mutation in the prothrombin gene and homozygosity for the C677T mutation of the MTHFR were not found in African-Americans, it appears that the carrier rate for the MTHFR C677T among Hispanics may be higher than in other reported groups.

Treatment for FPIES is the avoidance of offending food, i.e., a restricted diet (RD). While most FPIES children are good responders to the RD (FPIES-GR), some are not (FPIES-poor responders: FPIES-PR) with persistent non-IgE mediated reactivity to multiple foods. This study assessed production of counter-regulatory cytokines crucial for oral tolerance by peripheral blood mononuclear cells (PBMCs) in FPIES-PR (N=18, 0.6 -4.8 yr), FPIES-GR (N=22, 1.1-5.9 yr), and normal control (N=19, 1.0-5.7 yr) children after FPIES children were on the RD. PBMCs were stimulated with agonists of toll like receptors (TLRs) and representative luminal antigens (Ags) (soy, cow’s milk, and wheat proteins, and candida Ag). Microarray analysis of PB moncytes was also performed on samples from some of these children. FPIES-PR children revealed more severe clinical features including failure to thrive, intolerance to probiotics, and anaphylaxis like reaction to rice than FPIES-GR children (p

Introduction There exists a small subset of children with autism spectrum disorders (ASD) characterized by fluctuating behavioral symptoms and cognitive skills following immune insults. Some of these children also exhibit specific polysaccharide antibody deficiency (SPAD), resulting in frequent infection caused by encapsulated organisms, and they often require supplemental intravenous immunoglobulin (IVIG) (ASD/SPAD). This study assessed whether these ASD/SPAD children have distinct immunological findings in comparison with ASD/non-SPAD or non-ASD/SPAD children. Case description We describe 8 ASD/SPAD children with worsening behavioral symptoms/cognitive skills that are triggered by immune insults. These ASD/SPAD children exhibited delayed type food allergy (5/8), treatment-resistant seizure disorders (4/8), and chronic gastrointestinal (GI) symptoms (5/8) at high frequencies. Control subjects included ASD children without SPAD (N = 39), normal controls (N = 37), and non-ASD children with SPAD (N = 12). Discussion and Evaluation We assessed their innate and adaptive immune responses, by measuring the production of pro-inflammatory and counter-regulatory cytokines by peripheral blood mononuclear cells (PBMCs) in responses to agonists of toll like receptors (TLR), stimuli of innate immunity, and T cell stimulants. Transcription profiling of PB monocytes was also assessed. ASD/SPAD PBMCs produced less proinflammatory cytokines with agonists of TLR7/8 (IL-6, IL-23), TLR2/6 (IL-6), TLR4 (IL-12p40), and without stimuli (IL-1ß, IL-6, and TNF-α) than normal controls. In addition, cytokine production of ASD/SPAD PBMCs in response to T cell mitogens (IFN-γ, IL-17, and IL-12p40) and candida antigen (Ag) (IL-10, IL-12p40) were less than normal controls. ASD/non-SPAD PBMDs revealed similar results as normal controls, while non-ASD/SPAD PBMCs revealed lower production of IL-6, IL-10 and IL-23 with a TLR4 agonist. Only common features observed between ASD/SPAD and non-ASD/SPAD children is lower IL-10 production in the absence of stimuli. Transcription profiling of PB monocytes revealed over a 2-fold up (830 and 1250) and down (653 and 1235) regulation of genes in ASD/SPAD children, as compared to normal (N = 26) and ASD/non-SPAD (N = 29) controls, respectively. Enriched gene expression of TGFBR (p < 0.005), Notch (p < 0.01), and EGFR1 (p < 0.02) pathways was found in the ASD/SPAD monocytes as compared to ASD/non-SPAD controls. Conclusions The Immunological findings in the ASD/SPAD children who exhibit fluctuating behavioral symptoms and cognitive skills cannot be solely attributed to SPAD. Instead, these findings may be more specific for ASD/SPAD children with the above-described clinical characteristics, indicating a possible role of these immune abnormalities in their neuropsychiatric symptoms.

s 475 Array-CGH analysis of solid tumors with apparent normal karyotypes Gokce Toruner , Zhen-Ya Tang , Deanna Streck , Lisa Osborne , Rohini Kurvathi a a Institute of Genomic Medicine, UMDNJ-NJ Medical School, Newark, NJ, USA Some solid tumors with apparent normal karyotypes are not normal. These results are false negatives and detected normal result is due to overtake of the culture by normal cells. Discard fresh tissues from 10 cases of solid tumors with apparent normal karyotype (liposarcoma, renal cell carcinoma, lymphoma, breast cancer) were analyzed by array-CGH using Agilent 44K arrays. Four cases (40%) had abnormalities detected with array CGH, which was not previously detected after conventional tissue culture and cytogenetic analysis chromosome analysis. The pilot experiments indicate that incorporation of array-CGH in routine cancer cytogenetics may be beneficial in decreasing false negative results due to cell culture issues. Divergent genomic and epigenomic landscapes of lung cancer subtypes underscore the selection of different oncogenic pathways during tumor development Ian M. Wilson , William W. Lockwood , Bradley P. Coe , Raj Chari , Larissa A. Pikor , Kelsie L. Thu , John Yee , John English , Nevin Murray , Ming-Sound Tsao , John D. Minna , Adi F. Gazdar , Calum E. MacAulay , Stephen Lam , Wan L. Lam b Cancer Genetics Laboratory, Department of Pathology, British Columbia Cancer Agency, Vancouver, BC, Canada; Department of Integrative Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada; Cancer Biology and Genetics Section, Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA; Department of Surgery, Vancouver General Hospital, West, Vancouver, BC, Canada; Department of Pathology, Vancouver General Hospital, Vancouver, BC, Canada; Department of Medical Oncology, British Columbia Cancer Agency, Vancouver, BC, Canada; Department of Pathology, Division of Applied Molecular Oncology, University Health Network e Princess Margaret Hospital and Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA Traditionally non-small cell lung cancer (NSCLC) has been regarded as a single disease in terms of both diagnosis and of therapy; however, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and newly developed chemotherapies. Therefore, identifying the molecular differences between these tumor types will impact diagnostic and therapeutic decisions. Here, we performed a large-scale multidimensional microarray analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating DNA copy number, DNA methylation and gene expression profiles to identify subtype-specific alterations. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtypespecific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for differential behaviors in tumor biology and clinical outcomes. The possibility of using these differing oncogenic pathways to inform therapy design was then explored through an in silico screening of candidate therapeutic compounds. The robust and reproducible differences in both the genomic and epigenomic landscapes of AC and SqCC tumor highlights the future role of microarray technologies in the diagnosis and clinical management of lung and other solid tumors.

ASD is considered a behavioral syndrome associated with multiple genetic and environmental factors. This study focuses on a subset of ASD children characterized by fluctuating behavioral symptoms and cognitive skills, triggered by immune insults, in addition to chronic GI symptoms (ASD-immune subtype; ASD-IS). The study included ASD-IS (N=19), control ASD (N=29), and normal control (N=26) children. We evaluated production of pro-inflammatory and counter-regulatory cytokines by peripheral blood mononuclear cells (PBMCs) in response to agonists of toll like receptors (TLRs), polyclonal T cell stimulants, and recall antigens (Ags), and transcriptional profiles of PB monocytes. ASD-IS PBMCs produced less pro-inflammatory (IL-1β, IL-6, IL-12, and IL-23) and counter-regulatory (IL-10 and sTNFRII) cytokines with TLR agonists and less Th1 cytokines (IFN-γ and TNF-α) with recall Ags than control cells. ASD-IS PBMC also produced higher amounts of CCL2 than controls. Transcript profiles of ASD-IS monocytes were distinguished by altered mRNA expression (>2x) in >900 genes compared with either ASD or normal controls. Among these, 392 genes overlapped in the two control groups, including genes regulating TLR signaling, inflammatory responses, cell migration/adhesion, chemokines (CCL2 and CCL7), and genes associated with risks for autism, sychizophrenia, and depression. Our findings indicate a role of innate immunity in the puzzling clinical features observed in ASD-IS children.

A subset of ASD children (ASD Test Group) characterized by repeated loss of cognitive skills triggered by infection and frequent persistent GI symptoms were studied. We evaluated mRNA expression by peripheral blood (PB) monocytes in ASD test group (N=20, median 8 y) , ASD controls(N=22, median 7.6 y) , and normal control children (N=10, median 5.5 y) as well as the mothers of the ASD test (N=19) and ASD control (N=22) groups. We found that 568 genes were up-regulated and 72 genes were down-regulated (>2 fold) in the ASD test group versus 469 up-regulated and 57 down-regulated in the control groups. Only the ASD control group displayed up-regulation of CCL7 and CCL2 and down-regulation of IL-6 and CD86. The ASD test group mothers revealed up-regulation of 175 genes including CCL7 and down-regulation of 57 genes as compared to the ASD control mothers. The ASD test group also revealed decreased spontaneous production of IL-6 and IL-1β by PB mononuclear cells compared to controls. The 16/20 ASD test group children with persistent GI symptoms revealed up-regulation of CCL7 (>5 fold) and CCL2 and down-regulation of inflammatory mediators (IL-6, IL-23, IL-1α/IL-1β, CD86, and other chemokines) as compared to ASD and normal controls. Our results indicate a distinct pattern of mRNA expression in PB monocytes in the ASD subset with characteristic clinical features. Interestingly, CCL7 and CCL2 are up-regulated in the brain in various neurodegenerative disorders.

We have reported presence of an ASD subset distinguished by recurrent loss of previously acquired cognitive skills with worsening behavioral symptoms triggered by infection (typically viral), which may be associated with altered TLR responses important for neuro-immune interactions involving IL-1β. This study assessed gene expression of peripheral blood (PB) monocytes in this ASD subset (ASD-test group, N=14, median 7.8 yr) in comparison with control ASD (N=11, median 6.8 yr) and normal children (N=5, median 8yr). Both the ASD groups regressed at the onset of ASD and had high frequency of non-IgE mediated food allergy. The ASD test group also had lower IL-10 production with recall antigens than 24 normal controls. Microarray analysis revealed difference of mRNA expression (two fold or more) of 1593 genes in the ASD-test group and 2704 genes ASD-controls as compared to normal controls. IL-1 pathway related genes were over-represented in the ASD-test group (p=0.002) while IL-2, IL-3, IL-9, α-integrin (all p