Deanna Streck

Molecular and immunohistochemical evidence for the origin of uterine leiomyosarcomas from associated leiomyoma and symplastic leiomyoma-like areas

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.

An oligonucleotide based array-CGH system for detection of genome wide copy number changes including subtelomeric regions for genetic evaluation of mental retardation†

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.

Children with autism spectrum disorders (ASD) who exhibit chronic gastrointestinal (GI) symptoms and marked fluctuation of behavioral symptoms exhibit distinct innate immune abnormalities and transcriptional profiles of peripheral blood (PB) monocytes

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.

Immunological characterization and transcription profiling of peripheral blood (PB) monocytes in children with autism spectrum disorders (ASD) and specific polysaccharide antibody deficiency (SPAD): case study

IntroductionThere 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 descriptionWe 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 EvaluationWe 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.ConclusionsThe 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.

Genome profiling of chondrosarcoma using oligonucleotide array-based comparative genomic hybridization.

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.

A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays

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.

High Incidence of Two Methylenetetrahydrofolate Reductase Mutations (C677T and A1298C) in Hispanics

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.

Impaired Oral Tolerance in ASD Children with Food Protein Induced Enterocolitis Syndrome (FPIES) – Altered Innate and Adaptive Immune Responses in ASD Children Recovered from FPIES in Comparison with non-ASD/FPIES and ASD/non-FPIES Children

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.

MicroRNA expression changes in association with changes in interleukin-1ß/interleukin10 ratios produced by monocytes in autism spectrum disorders: their association with neuropsychiatric symptoms and comorbid conditions (observational study)

BackgroundMicroRNAs (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.MethodsmiRNA 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.ResultsAs 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.ConclusionsChanges 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.