Verification of prognostic expression biomarkers is improved by examining enriched leukemic blasts rather than mononuclear cells from acute myeloid leukemia patients.

BACKGROUND: Studies have not systematically compared the ability to verify performance of prognostic transcripts in paired bulk mononuclear cells versus viable CD34-expressing leukemic blasts from patients with acute myeloid leukemia. We hypothesized that examining the homogenous leukemic blasts will yield different biological information and may improve prognostic performance of expression biomarkers. METHODS: To assess the impact of cellular heterogeneity on expression biomarkers in acute myeloid leukemia, we systematically examined paired mononuclear cells and viable CD34-expressing leukemic blasts from SWOG diagnostic specimens. After enrichment, patients were assigned into discovery and validation cohorts based on availability of extracted RNA. Analyses of RNA sequencing data examined how enrichment impacted differentially expressed genes associated with pre-analytic variables, patient characteristics, and clinical outcomes. RESULTS: Blast enrichment yielded significantly different expression profiles and biological pathways associated with clinical characteristics (e.g., cytogenetics). Although numerous differentially expressed genes were associated with clinical outcomes, most lost their prognostic significance in the mononuclear cells and blasts after adjusting for age and ELN risk, with only 11 genes remaining significant for overall survival in both cell populations (CEP70, COMMD7, DNMT3B, ECE1, LNX2, NEGR1, PIK3C2B, SEMA4D, SMAD2, TAF8, ZNF444). To examine the impact of enrichment on biomarker verification, these 11 candidate biomarkers were examined by quantitative RT/PCR in the validation cohort. After adjusting for ELN risk and age, expression of 4 genes (CEP70, DNMT3B, ECE1, and PIK3CB) remained significantly associated with overall survival in the blasts, while none met statistical significance in mononuclear cells. CONCLUSIONS: This study provides insights into biological information gained/lost by examining viable CD34-expressing leukemic blasts versus mononuclear cells from the same patient and shows an improved verification rate for expression biomarkers in blasts.

Coordinated missplicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome.

SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.

AML risk stratification models utilizing ELN-2017 guidelines and additional prognostic factors: a SWOG report.

BACKGROUND: The recently updated European LeukemiaNet risk stratification guidelines combine cytogenetic abnormalities and genetic mutations to provide the means to triage patients with acute myeloid leukemia for optimal therapies. Despite the identification of many prognostic factors, relatively few have made their way into clinical practice. METHODS: In order to assess and improve the performance of the European LeukemiaNet guidelines, we developed novel prognostic models using the biomarkers from the guidelines, age, performance status and select transcript biomarkers. The models were developed separately for mononuclear cells and viable leukemic blasts from previously untreated acute myeloid leukemia patients (discovery cohort, N = 185) who received intensive chemotherapy. Models were validated in an independent set of similarly treated patients (validation cohort, N = 166). RESULTS: Models using European LeukemiaNet guidelines were significantly associated with clinical outcomes and, therefore, utilized as a baseline for comparisons. Models incorporating age and expression of select transcripts with biomarkers from European LeukemiaNet guidelines demonstrated higher area under the curve and C-statistics but did not show a substantial improvement in performance in the validation cohort. Subset analyses demonstrated that models using only the European LeukemiaNet guidelines were a better fit for younger patients (age < 55) than for older patients. Models integrating age and European LeukemiaNet guidelines visually showed more separation between risk groups in older patients. Models excluding results for ASXL1, CEBPA, RUNX1 and TP53, demonstrated that these mutations provide a limited overall contribution to risk stratification across the entire population, given the low frequency of mutations and confounding risk factors. CONCLUSIONS: While European LeukemiaNet guidelines remain a critical tool for triaging patients with acute myeloid leukemia, the findings illustrate the need for additional prognostic factors, including age, to improve risk stratification.

Identification of differentially expressed proteins in retinoblastoma tumors using mass spectrometry-based comparative proteomic approach.

In India, retinoblastoma is among the top five childhood cancers. Children mostly present with extraocular extension and high risk features that results in unsatisfactory treatment and low survival rate. In addition, lack of potential therapeutic and prognostic targets is another challenge in the management of retinoblastoma. We studied comparative proteome of retinoblastoma patients (HPV positive and negative (n=4 each) and controls (n=4), in order to identify potential retinoblastoma-specific protein targets. 2D-DIGE coupled MALDI-TOF/TOF mass spectrometry identified 39 unique proteins. Highly deregulated proteins were GFAP,RBP3,APOA1,CRYAA,CRABP1,SAG and TF. Gene ontology (Panther 7.0) revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). 8 proteins were significantly upregulated in HPV positive vis-a-vis HPV negative cases. Patient group exhibited 12 upregulated and 18 downregulated proteins compared to controls. Pathway and network analysis (IPA software) revealed CTNNB1 as most significantly regulated signalling pathway in HPV positive than HPV negative retinoblastoma. The trends in transcriptional change of 9 genes were consistent with those at proteomic level. The Western blot analysis confirmed the expression pattern of RBP3,GFAP and CRABP1. We suggest GFAP,RBP3,CRABP1,CRYAAA,APOA1 and SAG as prospective targets that could further be explored as potential candidates in therapy and may further assist in studying the disease mechanism. SIGNIFICANCE: In this study we evaluated tumor tissue specimens from retinoblastoma patients and identified 39 differentially regulated proteins compared to healthy retina. From these, we propose RBP3, CRABP1, GFAP, CRYAA, APOA1 and SAG as promising proteomic signatures that could further be explored as efficient prognostic and therapeutic targets in retinoblastoma. The present study is not only a contribution to the ongoing endeavour for the discovery of proteomic signatures in retinoblastoma, but, may also act as a starting point for future studies aimed at uncovering novel targets for further therapeutic interventions and improving patient outcomes.

Prevalence of human papillomavirus, Epstein-Barr virus, and cytomegalovirus in fine needle aspirates from lung carcinoma: A case-control study with review of literature.

BACKGROUND: Oncogenic viruses have recently been allied with lung carcinoma, however, the causal association has not been established till date. The study was conducted to determine the prevalence of high-risk Human papillomavirus (HPV; subtypes 16, 18, 31, 33 and 45), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) in lung carcinoma using polymerase chain reaction (PCR) on fine needle aspirates. METHODS: Fine needle aspirates of patients with lung carcinoma were included as cases. The control samples included normal lung tissue, collected at the time of medico legal autopsies. DNA was extracted from samples of both cases and controls and analysed by PCR for the presence of HPV, EBV and CMV. RESULTS: A total of 5/73 (6.8%) cases demonstrated the presence of HPV. Of these, 3 were positive for HPV-16 and one each for HPV-18 and HPV-45. A significant association of HPV with squamous cell carcinoma (SCC) (P = 0.01) was observed. Two cases were positive for EBV; however, the difference was not statistically significant for EBV (P = 0.5) as well as CMV. None of the controls were positive for HPV, EBV or CMV. CONCLUSION: We conclude that fine needle aspirates can serve as reliable sample for PCR based detection of viruses. A significantly higher prevalence of HPV in lung cancer and a significant association with SCC was observed, thereby, indicating a positive link between HPV and etiopathogenesis of lung carcinoma. Diagn. Cytopathol. 2016;44:987-993. © 2016 Wiley Periodicals, Inc.

Proteomic analysis of differentially expressed proteins in vitreous humor of patients with retinoblastoma using iTRAQ-coupled ESI-MS/MS approach.

There is close proximity of vitreous humor with the tumor bulk in eyes with retinoblastoma. This renders vitreous humor a promising source to evaluate disease-specific protein targets in retinoblastoma. We studied the differential proteome of vitreous fluid in retinoblastoma tumors (n = 4) as compared to controls (n = 4). The vitreous humor was depleted off the high abundant fraction using MARS-6 affinity column. Subsequently, the tryptic peptides were derivatised with iTRAQ labels. The labelled peptides were pooled and subjected to fractionation using bRPLC. This was followed by protein identification and quantification using electrospray ionisation mass spectrometry (ESI-MS/MS) approach. The identified proteins were subjected to bioinformatics analysis utilizing PANTHER 7.0 and IPA software. Four hundred and thirty-one non-redundant (362 upregulated and 69 downregulated) proteins (≥2 unique peptides, ± 1.5 folds, p < 0.05) were identified. The majority of the proteins were cytoplasmic (40 %), majorly involved in catalytic (32.7 %) and binding activities (26.3 %). Highly deregulated proteins included MMP2, TNC, CD44, SUZ12 and CRABP1. The protein expression of GFAP, CRABP1, MMP2 and TNC was validated by western blotting. Pathway and network analyses revealed p38MAPK and Akt signalling to be the most significantly regulated pathways in retinoblastoma. This is the first report of differential vitreous proteome of retinoblastoma and highlights novel protein targets, such as MMP2, TNC and CRABP1. Further investigations into unravelling the biological role of the proteins and their prospects of being utilised as potential candidates in therapeutics are warranted.

Identification of potential protein biomarkers for early detection of pregnancy in cow urine using 2D DIGE and label free quantitation.

BACKGROUND: An early, reliable and noninvasive method of early pregnancy diagnosis is prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also help in to reduce the calving interval and rebreeding time which is beneficial for industries as well as farmers. The aim of this work is to identify potential biomarker for pregnancy detection at earlier stages (16-25 days). To achieve this goal we performed DIGE and LFQ for identification of protein which has significant differential expression during pregnancy. RESULTS: DIGE experiment revealed a total of eleven differentially expressed proteins out of which nine were up regulated having fold change ≥1.5 in all time points. The LFQ data analysis revealed 195 differentially expressed proteins (DEPs) out of 28 proteins were up-regulated and 40 down regulated having significant fold change ≥1.5 and ≤0.6 respectively. Bioinformatics analysis of DEPs showed that a majority of proteins were involved in regulation of leukocyte immunity, endopeptidase inhibitor activity, regulation of peptidase activity and polysaccharide binding. CONCLUSION: This is first report on differentially expressed protein during various time points of pregnancy in cow to our best knowledge. In our work, we identified few proteins such MBP, SERPIN, IGF which were differentially expressed and actively involved in various activities related to pregnancy such as embryo implantation, establishment and maintenance of pregnancy. Due to their involvement in these events, these can be considered as biomarker for pregnancy but further validation of is required.

HLA-DRB1 in Henoch-Schönlein purpura: A susceptibility study from North India.

Etiology of Henoch-Schönlein purpura (HSP) a small vessel vasculitis remains elusive. Susceptibility may be conferred by major histocompatibility complex. There are limited reports on the association of human leucocyte antigens (HLA) and HSP. The aim was to investigate the association of HLA-DRB1 (HLA class II antigen) with HSP. Forty three patients, <14years, who fulfilled the diagnostic criteria of HSP, laid by 'European League Against Rheumatism' were enrolled. Fifty four age-matched, healthy controls were included. PCR with 24 sequence specific primers for HLA-DRB1 allotypes was performed. Commercially available HLA-DR tissue typing kit (Inno-train, Kronberg im Taunus, Hesse, Germany) was utilized. The mean age of patients and controls was 8.5±3.2 and 7.6±3.6years, respectively (p=0.18). Gastrointestinal and renal involvement was observed in 25 (58.1%) and 14 (32.6%) patients, respectively. A greater frequency of HLA-DRB1*11 was observed in patients (11.6%) as compared to controls (1.9%), however, the results were not significant following Bonferroni correction. No significantly protective HLA genotype was observed. None of the HLA-DRB1 antigen was noted to increase the susceptibility to gastrointestinal or renal involvement. In conclusion, in the first study from India, none of the HLA-DRB*1 genotypes were observed to increase the susceptibility of North Indian children to HSP.

Comparative Proteomic Analysis of Advanced Ovarian Cancer Tissue to Identify Potential Biomarkers of Responders and Nonresponders to First-Line Chemotherapy of Carboplatin and Paclitaxel.

Conventional treatment for advanced ovarian cancer is an initial debulking surgery followed by chemotherapy combination of carboplatin and paclitaxel. Despite initial high response, three-fourths of these women experience disease recurrence with a dismal prognosis. Patients with advanced-stage ovarian cancer who underwent cytoreductive surgery were enrolled and tissue samples were collected. Post surgery, these patients were started on chemotherapy and followed up till the end of the cycle. Fluorescence-based differential in-gel expression coupled with mass spectrometric analysis was used for discovery phase of experiments, and real-time polymerase chain reaction, Western blotting, and pathway analysis were performed for expression and functional validation of differentially expressed proteins. While aldehyde reductase, hnRNP, cyclophilin A, heat shock protein-27, and actin are upregulated in responders, prohibitin, enoyl-coA hydratase, peroxiredoxin, and fibrin-ß are upregulated in the nonresponders. The expressions of some of these proteins correlated with increased apoptotic activity in responders and decreased apoptotic activity in nonresponders. Therefore, the proteins qualify as potential biomarkers to predict chemotherapy response.

HPV-16 Detected in One-Fourth Eyes With Retinoblastoma: A Prospective Case-control Study From North India.

The incidence of nonfamilial retinoblastoma (RB) is believed to be higher in developing countries. The reports on association of human papillomavirus (HPV) with RB are limited and contradictory. The aim was to investigate the prevalence of HPV in RB tumor tissue. In the prospective study, consecutive eyes enucleated for RB from patients lacking a family history of RB were enrolled as cases over a 3-year period. Controls included donor eyes obtained from the eye bank. Normal retinal tissue from the donor eyes and tumor tissue from eyes with RB was subjected to DNA isolation. Polymerase chain reaction followed by dot-blot hybridization was performed to detect 21 HPV genotypes. The study cohort included 39 RB and 42 normal retinal tissues. A positive result for HPV-polymerase chain reaction was obtained in 10 (25.6%) tumor tissues and none of the control eyes. HPV-16 was the only subtype detected. Socioeconomic status (P=0.58) or maternal age (P=0.58) was not associated with presence of HPV. All HPV-positive patients had undergone a vaginal delivery (P=0.60). HPV-16 was detected in one-fourth cases of nonfamilial RB. None of the control cases (donor eyes) tested positive. Implication of the presence of HPV in RB tissue and role in carcinogenesis needs further elucidation.

Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins.

Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors. This article is part of a Special Issue entitled: Proteomics in India.

DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): lactating vis-a-vis heifer.

Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland. BIOLOGICAL SIGNIFICANCE: This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

Prevalence and genotypes of HPV in female renal transplant recipients in North India.

Estimation of the prevalence of high-risk human papillomavirus (HPV) genotypes in female renal transplant recipients is important for formulating strategies for prevention and screening of cervical cancer in the susceptible group. Data from developing countries are very limited. The study was prospective, cross-sectional, and hospital-based. Female renal transplant recipients, who had received the graft at least 6 mo earlier, were enrolled. Women who visited the outpatient unit for varied complaints and who underwent a normal cervical examination were recruited as controls. A pap smear was obtained in all women. HPV genotyping array kit was utilized for identifying 21 HPV genotypes. Forty renal transplant recipient women and 80 controls were enrolled. The median age of cases and controls was 40 yr (range, 24-69 yr) and 38 yr (range, 23-72 yr), respectively. The mean duration since transplant was 53±42.6 mo (range, 6-168 mo). There was no evidence of cervical dysplasia in any pap smear. High-risk HPV was detected in 32.5% (13/40) and 17.5% (14/80) of cases and controls, respectively (P=0.18). Of the 21 genotypes screened, 7 subtypes were detected. HPV 16 and 31 were the most common (5/13; 38.5%) subtypes observed in the cases, followed by HPV 18 (30.7%). HPV 16 was the most common subtype in controls (10/14; 71.4%). Five (38.5%) renal transplant recipients harbored multiple HPV genotypes, as compared with 4 (28.6%) controls (P=1.0). The prevalence of high-risk HPV in female renal transplant recipients was 1.9 times that observed among controls, although there was no evidence of cervical dysplasia.

HSV-1 as well as HSV-2 is frequent in oral mucosal lesions of children on chemotherapy.

PURPOSE: Prevalence data of herpes simplex virus (HSV) in oral mucositis in children on treatment for cancer is limited. Quantitative polymerase chain reaction (PCR) has been seldom utilized for detection of HSV-1/2 in oral mucosa. METHODS: Children on treatment for cancer with oral mucositis were enrolled as cases and healthy children as controls. An oral swab from the lesion in cases and mucosal scraping in controls were obtained. Both qualitative and real-time quantitative PCR for HSV-1/2 were performed. Serum ELISA-IgG/IgM for HSV-1/2 antibodies (NovaLisa™-Dietzenbach-Germany) were measured. RESULTS: Thirty-two cases (Age, 6.3±3.4 years) and 30 controls were enrolled. Majority (69%) of cases had ALL. All patients had febrile neutropenia, except two. ELISA-IgM-HSV-1/2 was not positive in any case or control. ELISA-IgG-HSV-1/2 was positive in 11 (34%) cases and nine (30%) controls (p=1.0). Qualitative PCR for HSV-1 detected the virus in eight (25%) cases and nil controls (p=0.009). HSV-2 was not detected in any case/control by qualitative PCR. Quantitative PCR detected HSV-1 in 21 (66%) and HSV-2 in 22 (69%) cases. In controls, quantitative PCR detected HSV-1 in three (10%) and HSV-2 in none. In patients, the mean viral load of HSV-1 (5,500±15,987×10(4) copies/nanogram DNA) was more than HSV-2 (4.03±8.5×10(4)) (p=0.11). There was no correlation of HSV-1/2 with grading of mucositis. CONCLUSIONS: Both HSV-1/2 are commonly shed from oral mucosal lesions in children receiving chemotherapy. In a novel finding, real-time PCR detected copies of HSV-2 in 69% cases, all missed by conventional PCR. Implication for morbidity, if any, or treatment needs to be determined.

HPV genotyping in north Indian women infected with HIV.

Investigating the prevalence of high-risk human papilloma virus (HPV) genotypes in human immunodeficiency virus (HIV)-infected women is vital to generate data for formulating guidelines for prevention/screening of cervical cancer in this vulnerable group. The study was aimed to analyze the HPV genotypes in HIV-infected women. It was a prospective, hospital-based, and cross-sectional study. HIV-infected women were enrolled from the antiretroviral clinic and controls from the gynecology outpatient. The HPV genotyping array kit was used for identifying 21 HPV genotypes. Detection of HPV was confirmed by performing an HPV type-specific polymerase chain reaction. A Pap smear was collected in all women. One hundred thirty HIV-infected women and 64 controls were enrolled. All women with low CD4 counts (n=97) were receiving antiretroviral therapy. Twenty-six (20%) HIV-infected women and 12 (18.7%) women in the control group tested positive for high-risk HPV (P=1.0). HPV 16 was the most common type, detected in 42% of HPV-positive women in the HIV-infected cohort, followed by HPV 45 (15%), HPV 18/52/31/58 (11.5% each), and HPV 33 (7.6%). The corresponding figures in the control group were as follows: HPV 16 (66.6%), HPV 45/18/31 (16.6% each), and HPV 33/58/68 (8.3% each). Cervical intraepithelial neoplasia was detected in 2.3% of HIV-infected women. The prevalence of high-risk HPV in HIV-infected women (20%) was similar to the prevalence in controls (18.7%). This and the incidence of cervical intraepithelial neoplasia are lower than those in previous reports. It is plausible that administration of antiretroviral therapy contributed to the reduced prevalence. The currently available vaccine would likely be beneficial to the local HIV-infected population, as nearly half the HPV-infected women harbored genotypes 16 or 18.