Longitudinal study of traumatic-stress related cellular and cognitive aging.

Traumatic stress is associated with both accelerated epigenetic age and increased risk for dementia. Accelerated epigenetic age might link symptoms of traumatic stress to dementia-associated biomarkers, such as amyloid-beta (Aß) proteins, neurofilament light (NFL), and inflammatory molecules. We tested this hypothesis using longitudinal data obtained from 214 trauma-exposed military veterans (85 % male, mean age at baseline: 53 years, 75 % White) who were assessed twice over the course of an average of 5.6 years. Cross-lagged panel mediation models evaluated measures of lifetime posttraumatic stress disorder and internalizing and externalizing comorbidity (assessed at Time 1; T1) in association with T1 epigenetic age (per the GrimAge algorithm) and T1 plasma markers of neuropathology along with bidirectional temporal paths between T1 and T2 epigenetic age and the plasma markers. Results revealed that a measure of externalizing comorbidity was associated with accelerated epigenetic age (ß = 0.30, p <.01), which in turn, was associated with subsequent increases in Aß-40 (ß = 0.20, p <.001), Aß-42 (ß = 0.18, p <.001), and interleukin-6 (ß = 0.18, p <.01). T1 advanced epigenetic age and the T1 neuropathology biomarkers NFL and glial fibrillary acidic protein predicted worse performance on T2 neurocognitive tasks assessing working memory, executive/attentional control, and/or verbal memory (ps = 0.03 to 0.009). Results suggest that advanced GrimAge is predictive of subsequent increases in neuropathology and inflammatory biomarkers as well as worse cognitive function, highlighting the clinical significance of this biomarker with respect to cognitive aging and brain health over time. The finding that advanced GrimAge mediated the association between psychiatric comorbidity and future neuropathology is important for understanding potential pathways to neurodegeneration and early identification of those at greatest risk.

B-lymphoblastic leukemia/lymphoma with MYC and BCL2 gene rearrangements shows evidence for clonal evolution and mitotic recombination.

BACKGROUND: B-lymphoblastic leukemia/lymphomas (B-ALL/LBL) are uncommon neoplasms that may be associated with a variety of cytogenetic and molecular changes. The mechanisms by which these changes arise have not been fully described. AIMS/PURPOSE: This report describes an unusual case of B-ALL/LBL with complex clonal evolution that includes BCL2 and MYC gene rearrangements. METHODS: Immunophenotyping was performed by immunohistochemistry and flow cytometry. Traditional G-band karyotyping was accompanied by fluorescence in-situ hybridization (FISH) using break-apart and dual fusion probes. Single nucleotide polymorphisms were assessed using a high-density DNA microarray. RESULTS: The karyotype of the blasts showed reciprocal translocation of chromosomes 4 and 18, reciprocal translocation of chromosomes 8 and 14 with two copies of the oncogenic translocation derivative(14)t(8;14), and no normal chromosome 14. FISH studies showed complex IGH-BCL2 and IGH-MYC fusion signals. CONCLUSIONS: A clonal evolution model involving multiple chromosomal translocations and mitotic recombination is postulated to account for the karyotype, FISH, and microarray results but leaves unresolved the exact order of the evolutionary changes.

An epigenome-wide association study of posttraumatic stress disorder in US veterans implicates several new DNA methylation loci.

BACKGROUND: Previous studies using candidate gene and genome-wide approaches have identified epigenetic changes in DNA methylation (DNAm) associated with posttraumatic stress disorder (PTSD). METHODS: In this study, we performed an EWAS of PTSD in a cohort of Veterans (n = 378 lifetime PTSD cases and 135 controls) from the Translational Research Center for TBI and Stress Disorders (TRACTS) cohort assessed using the Illumina EPIC Methylation BeadChip which assesses DNAm at more than 850,000 sites throughout the genome. Our model included covariates for ancestry, cell heterogeneity, sex, age, and a smoking score based on DNAm at 39 smoking-associated CpGs. We also examined in EPIC-based DNAm data generated from pre-frontal cortex (PFC) tissue from the National PTSD Brain Bank (n = 72). RESULTS: The analysis of blood samples yielded one genome-wide significant association with PTSD at cg19534438 in the gene G0S2 (p = 1.19 × 10-7, padj = 0.048). This association was replicated in an independent PGC-PTSD-EWAS consortium meta-analysis of military cohorts (p = 0.0024). We also observed association with the smoking-related locus cg05575921 in AHRR despite inclusion of a methylation-based smoking score covariate (p = 9.16 × 10-6), which replicates a previously observed PGC-PTSD-EWAS association (Smith et al. 2019), and yields evidence consistent with a smoking-independent effect. The top 100 EWAS loci were then examined in the PFC data. One of the blood-based PTSD loci, cg04130728 in CHST11, which was in the top 10 loci in blood, but which was not genome-wide significant, was significantly associated with PTSD in brain tissue (in blood p = 1.19 × 10-5, padj = 0.60, in brain, p = 0.00032 with the same direction of effect). Gene set enrichment analysis of the top 500 EWAS loci yielded several significant overlapping GO terms involved in pathogen response, including "Response to lipopolysaccharide" (p = 6.97 × 10-6, padj = 0.042). CONCLUSIONS: The cross replication observed in independent cohorts is evidence that DNA methylation in peripheral tissue can yield consistent and replicable PTSD associations, and our results also suggest that that some PTSD associations observed in peripheral tissue may mirror associations in the brain.

Acquired Amegakaryocytic Thrombocytopenia Misdiagnosed as Immune Thrombocytopenia: A Case Report.

INTRODUCTION: Acquired amegakaryocytic thrombocytopenia (AATP) is a rare bleeding disorder that causes severe thrombocytopenia with preserved hematopoiesis of other cell lineages. Many cases are misdiagnosed and treated as immune thrombocytopenia. CASE PRESENTATION: We report a case of AATP, in a 50-year-old man, that was treated as immune thrombocytopenia for years with no clinical response. The disorder later was diagnosed as AATP after bone marrow biopsy and was successfully treated with cyclosporine. DISCUSSION: The exact mechanism of AATP remains unclear; it is suspected to be an immune-mediated process. Patients with AATP present with severe bleeding and thrombocytopenia, which is usually unresponsive to high-dose corticosteroids. There are no standard treatment guidelines for AATP. Cyclosporine and antithymocyte globulin are found to be effective in some cases. The prompt diagnosis of AATP is vital because it carries high mortality because of excessive bleeding, and it can progress into aplastic anemia or myelodysplastic syndrome.

The goddess who spins the thread of life: Klotho, psychiatric stress, and accelerated aging.

BACKGROUND: Longevity gene klotho (KL) is associated with age-related phenotypes but has not been evaluated against a direct human biomarker of cellular aging. We examined KL and psychiatric stress, including posttraumatic stress disorder (PTSD), which is thought to potentiate accelerated aging, in association with biomarkers of cellular aging. METHODS: The sample comprised 309 white, non-Hispanic genotyped veterans with measures of epigenetic age (DNA methylation age), telomere length (n = 252), inflammation (C-reactive protein), psychiatric symptoms, metabolic function, and white matter neural integrity (diffusion tensor imaging; n = 185). Genotyping and DNA methylation were obtained on epi/genome-wide beadchips. RESULTS: In gene by environment analyses, two KL variants (rs9315202 and rs9563121) interacted with PTSD severity (peak corrected p = 0.044) and sleep disturbance (peak corrected p = 0.034) to predict advanced epigenetic age. KL variant, rs398655, interacted with self-reported pain in association with slowed epigenetic age (corrected p = 0.048). A well-studied protective variant, rs9527025, was associated with slowed epigenetic age (p = 0.046). The peak PTSD interaction term (with rs9315202) also predicted C-reactive protein (p = 0.049), and white matter microstructural integrity in two tracts (corrected ps = 0.005 - 0.035). This SNP evidenced a main effect with an index of metabolic syndrome severity (p = 0.015). Effects were generally accentuated in older subjects. CONCLUSIONS: Rs9315202 predicted multiple biomarkers of cellular aging such that psychiatric stress was more strongly associated with cellular aging in those with the minor allele. KL genotype may contribute to a synchronized pathological aging response to stress and could be a therapeutic target to alter the pace of cellular aging.

MYC amplification in multiple marker chromosomes and EZH2 microdeletion in a man with acute myeloid leukemia.

The role of MYC and EZH2 in acute myeloid leukemia (AML) pathogenesis is poorly understood. Herein we present a case of AML with MYC amplification in marker chromosomes and a microdeletion of chromosome 7 below cytogenetic resolution. The karyotype of the patient's bone marrow aspirate showed three to five marker chromosomes in all dividing cells without other structural or numerical chromosomal abnormalities. Analysis by fluorescence in situ hybridization (FISH) with a probe specific for the human MYC gene revealed amplification of the oncogene localized to the marker chromosomes. Using whole genome single nucleotide polymorphism (SNP) microarray analysis, an approximately 4.4 Mb amplicon containing the MYC gene was identified with an estimated amplification of about 30 copies per leukemic cell and, thus, an average of about 8 copies per marker chromosome. A 6.4 Mb hemizygous microdeletion of chromosome 7 within band q36.1 was also found by SNP microarray analysis in a cellular-equivalent dosage of 50%. The microdeletion spans multiple genes, including EZH2, a gene with well-known cancer association. No mutation was found in the remaining EZH2 allele by next generation gene sequencing. The combination of MYC amplification and EZH2 deletion, which has not been described previously in AML, may suggest a synergistic role of the two oncogenes in the pathogenesis of the patient's acute leukemia.

Natural history of chronic myelomonocytic leukemia: gene sequencing identifies multiple clonal molecular abnormalities associated with rapid progression to acute myeloid leukemia.

KEY CLINICAL MESSAGE: Gene panel sequencing in a CMML patient without any detectable genetic abnormality by conventional genetic studies identified four concurrent somatic mutations in three genes. Gene panel mutation analysis is a rapidly emerging clinical tool to demonstrate the clonality in hematologic malignancies, and to identify the potential targets for therapy.

Increased microsatellite instability and epigenetic inactivation of the hMLH1 gene in head and neck squamous cell carcinoma.

OBJECTIVE: The study is designed to elucidate the relationship between epigenetic silencing of the hMLH1 (human MutL homologue 1) gene and microsatellite instability (MSI) and the prognostic values of hMLH1 promoter methylation and MSI in head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Cross-sectional study. SETTING: Tertiary referral center. SUBJECTS AND METHODS: A total of 120 cases of HNSCC were analyzed for hMLH1 promoter hypermethylation, protein expression, and MSI by using methylation-specific polymerase chain reaction, immunohistochemical staining, and polymerase chain reaction amplification with the use of 16 fluorescent-labeled microsatellite markers, followed by fragment analysis. RESULTS: Of 120 HNSCCs, hMLH1 promoter hypermethylation and decreased hMLH1 protein expression were shown in 39 (32.5%) and 22 (18.3%), respectively. hMLH1 promoter hypermethylation was detected in 13 of 52 (25%) normal-appearing squamous mucosa adjacent to invasive carcinoma. MSI was detected in 21 (17.5%) tumors at two or more markers and in 99 (82.5%) tumors with no evidence of MSI or at only one marker. Hypermethylation of the hMLH1 gene is significantly associated with decreased hMLH1 protein expression (P < 0.001). High-frequency MSI was significantly associated with promoter hypermethylation (P = 0.01) but not with decreased protein expression (P = 0.069) of hMLH1 gene. hMLH1 promoter hypermethylation is significantly associated with decreased cause-specific survival for HNSCC patients (P = 0.03). CONCLUSIONS: Promoter hypermethylation of the hMLH1 gene could be detected early in head and neck squamous carcinogenesis and may be associated with increased MSI and poor survival in HNSCC.

Concordant loss of heterozygosity of DNA repair gene, hOGG1, in melanoma in situ and atypical melanocytic hyperplasia.

BACKGROUND: One major risk factor for cutaneous melanoma is chronic sun-exposure and oxidative stress. Among various oxidative DNA damages, 8-oxoquanine is the most abundant and is potentially mutagenic if not sufficiently repaired. The human 8-oxoquanine DNA glycosylase 1 (hOGG1) gene specifically repairs 8-oxoguanine, and this gene shows frequent loss of heterozygosity (LOH) in human tumors. In this study, we investigate whether hOGG1 LOH occurs in melanoma in situ (MIS) and adjacent atypical melanocytic hyperplasia (AMH). METHODS: Twelve skin biopsies with MIS and adjacent AMH were included. DNA samples derived from manual microdissection of tissues were subjected to polymerase chain reaction amplification using three fluorescent-labeled microsatellite makers, followed by fragment analysis. RESULTS: Five of 12 cases were informative for both telomeric (3S1297) and centromeric (3S1289 or 3S1300) markers, bordering the hOGG1 locus. Among them, four (80%) MIS and three (60%) AMH showed hOGG1 LOH at both markers. CONCLUSIONS: These results shows that LOH at hOGG1 gene is associated with MIS and AMH and suggest that the hOGG1 gene may play a role in melanocytic tumor progression.

Epstein-Barr viral load as a marker of lymphoma in AIDS patients.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) lymphoma, and viral DNA is present within the malignant cells in about half of affected patients. We examined the extent to which EBV viral load is elevated in the plasma of AIDS lymphoma patients compared to AIDS patients with opportunistic infections. Sixty-one AIDS patients were studied including 35 with lymphoma (24 non-Hodgkin, six Hodgkin, and five brain lymphoma) and 26 with various opportunistic infections. In situ hybridization revealed EBV encoded RNA (EBER) expression in the malignant cells of 17/28 AIDS lymphomas (61%). In 232 serial plasma samples from 35 lymphoma patients and in 128 samples from AIDS controls, EBV viral load was assayed by quantitative-polymerase chain reaction (Q-PCR) using a TaqMan probe targeting the BamH1W sequence. EBV was detected in plasma from all 17 EBER-positive AIDS lymphoma patients, with viral loads ranging from 34 to 1,500,000 copies per ml (median 3,210). Viral load usually fell rapidly upon initiation of lymphoma therapy and remained undetectable except in two patients with persistent tumor. In 11 AIDS patients, whose lymphoma lacked EBER expression, and in 26 control patients without lymphoma, levels of EBV in plasma were usually low or undetectable (range 0-1,995 and 0-2,409, median 0 and 0, respectively). There was no association between EBV viral load and human immunodeficiency virus (HIV) load or CD4 count. In conclusion, EBV viral load shows promise as a tool to assist in diagnosis and management of EBV-related lymphoma patients.

HIV and plasmablastic lymphoma manifesting in sinus, testicles, and bones: a further expansion of the disease spectrum.

Patients with HIV infection are at increased risk for B-cell neoplasms and plasma cell dyscrasias. Both B cell and plasma cell tumors tend to be intermediate or high grade and are frequently associated with Epstein-Barr virus. Patients with HIV infection are also at higher risk of acquiring plasmablastic lymphoma. Until this time, only sinus, oral gastrointestinal, and lung manifestation have been noted. In this report we describe a 41-year-old male with HIV infection who developed multiple pleomorphic, extramedullary plasmablastic lymphomas associated with Epstein-Barr virus. We review the clinical and immunological features of his malignancy and thereby expand the spectrum of disease to include additional sites (bones, testicles) not previously reported.

Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments: a novel approach to screen for the virus in paraffin-embedded tissue and plasma.

Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.

Veterans with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) have a markedly increased rate of second malignancy, which is the most common cause of death.

Patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) have an increased incidence of high-grade lymphoid malignancy. The risk of non-lymphoid second malignancy in this population is not well-defined to date. To test the hypothesis that patients with CLL/SLL have an increased risk of second malignancy, we studied the rate of second malignancy in 132 CLL/SLL patients and compared it to the rate of malignancy (excluding non-melanomatous skin cancer) in the Central Arkansas Veterans Healthcare System population of approximately 38,000 veterans over a period of 11.5 years. The rate of second malignancy, diagnosed concomitantly or after CLL/SLL, and the age-adjusted rate of malignancy calculated from tumor registry reports and demographic data, were used to calculate a Standardized Morbidity Ratio (SMR) with 95% confidence interval (CI). Twenty-one (16%) of the CLL/SLL patients had second malignancies (19 non-lymphoid, 1 Richter's transformation and 1 Hodgkin's disease), which were fatal in 15 (71%) patients. The SMR for the CLL/SLL population was 2.97 (95% CI 1.84-4.55) for second malignancy and 2.69 (95% CI 1.62-4.21) for non-lymphoid second malignancy. This study of a well-defined CLL/SLL population shows a significantly increased risk of second malignancy, which was the primary cause of death for 9% of all CLL/SLL patients (34% of all patient deaths).

Epstein-Barr Virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies.

Epstein-Barr Virus (EBV), a ubiquitous gamma herpes virus, infects more than 95% of the human population before adulthood. Life-long persistence, usually without adverse health consequences, relies on a balance between viral latency, viral replication, and host immune response. Patients with EBV-related disease often have high levels of EBV DNA in their plasma. This study addresses whether this circulating, cell-free EBV DNA is encapsidated in virions or exists as naked genomes. First, an assay was developed, combining DNase I and quantitative real-time PCR, to discriminate encapsidated from naked EBV DNA. EBV DNA was almost always naked in the plasma of AIDS-related lymphoma patients (n = 11) and immunosuppressed/posttransplantation patients (n = 8). In contrast, infectious mononucleosis patients (n = 30) often had a mixture of encapsidated and naked EBV DNA. These findings may be important in understanding how viral load relates to disease status and in predicting response to nucleoside analogs and other antiviral therapies.

Alemtuzumab (CAMPATH 1H) does not kill chronic lymphocytic leukemia cells in serum free medium.

The mechanism of action of alemtuzumab (CAMPATH 1H) in chronic lymphocytic leukemia (CLL) is uncertain. We tested the hypothesis that alemtuzumab alone can induce apoptosis in cultured CLL cells. Purified peripheral blood B-lymphocytes from CLL patients were treated in serum free medium (AIM-V). There was minimal spontaneous apoptosis in untreated cells. Alemtuzumab ligation did not alter the membrane distribution of CD52 in single cells but many cells formed transient, small, tightly adherent clusters. Alemtuzumab alone did not induce apoptosis. In contrast, alemtuzumab plus complement was rapidly cytotoxic. We conclude that alemtuzumab does not cause apoptosis in purified CLL B cells cultured in serum free medium.

Autoimmune cytopenia does not predict poor prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is characterized by an acquired immune defect that can cause autoimmune complications, including anemia and thrombocytopenia. We conducted an observational study of the epidemiology, clinical presentation and significance of autoimmune complications of CLL/SLL in 132 patients from a large population (>45,000 veterans), in which at least 90% of patients with CLL/SLL have been previously identified. Over a period of 12.5 years, 12 patients (9.1%) had autoimmune complications; of these, 6 (4.5%) had autoimmune hemolytic anemia (AIHA), 5 (3.8%) had immune thrombocytopenia (ITP), and 1 (0.8%) had pure red blood cell aplasia (PRBA). All 6 cases of AIHA had a positive direct immunoglobulin test for IgG and C3d. In 6 patients, CLL/SLL was an incidental finding at the time of presentation with autoimmune cytopenia. Nine out of 10 patients responded to immunosuppressive therapy, which was complicated by serious infection in 7 cases, one of which was fatal. The major cause of mortality in patients with autoimmune complications of CLL/SLL was secondary malignancy. Survival of patients with immune cytopenia was not significantly different from CLL/SLL patients without immune cytopenia. Among patients with anemia or thrombocytopenia, mortality was significantly higher in those with bone marrow failure compared to an autoimmune etiology. We show that in a non-referred population with a high incidence of CLL/SLL, autoimmune cytopenia can occur early in the natural history of the disease. These data suggest that the Rai and Binet classifications for CLL need to be modified for patients with autoimmune cytopenia. .

The distinct gene expression profiles of chronic lymphocytic leukemia and multiple myeloma suggest different anti-apoptotic mechanisms but predict only some differences in phenotype.

We compared gene expression in purified tumor cells from untreated patients with chronic lymphocytic (CLL) (n=24) and newly diagnosed multiple myeloma (MM) (n=29) using the Affymetrix HuGeneFL microarray with probes for approximately 6800 genes. Hierarchical clustering analysis showed that CLL and MM have distinct expression profiles (class prediction). Gene and protein expression (measured by flow cytometry) correlated well for CD19, CD20, CD23, and CD138 in CLL and MM, but not for immunoglobulin light chain, CD38 and CD79b in CLL, or CD45 and CD52 in MM. CLL and MM differentially expressed 18% of 130 apoptosis related genes, suggesting differences in mechanisms of cell survival.

Bone marrow transplant engraftment analysis with loss of an informative allele.

Short tandem repeats (STRs) are highly polymorphic DNA sequences in the human genome. STR genotype analysis is used for human identity testing and to monitor bone marrow engraftment after allogeneic transplantation. Engraftment analysis requires one or more informative STR loci that distinguish recipient from donor. The following case illustrates that chromosome loss in tumor cells during the course of disease may cause corresponding loss of an STR locus. This circumstance is a potential source of error in the interpretation of engraftment analysis, especially if only one informative allele is used to monitor engraftment.