Provaboti Barman

Clinical efficacy of gene-modified stem cells in adenosine deaminase–deficient immunodeficiency

BACKGROUND. Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase–deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) &ggr;-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1–2.6) and granulocytes (VCN = 0.01–0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION. ClinicalTrials.gov NCT00794508. FUNDING. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.

Comparison of 2 anal cytology protocols to predict high-grade anal intraepithelial neoplasia.

Objectives Nylon-flocked and Dacron swab anal cytology collection procedures were evaluated for detecting high-grade anal intraepithelial neoplasia. Materials and Methods Cross-sectional data for 42 HIV-infected and 16 uninfected men who have sex with men have been used. Sequentially collected anal cytology specimens, high-resolution anoscopy, and medical biopsy evaluated the sensitivity and specificity of cytology for predicting high-grade anal intraepithelial neoplasia. Men showing atypical squamous cells (ASC) or more severe findings by cytology were compared with those showing negative for intraepithelial lesions. Results The prevalence of high-grade anal intraepithelial neoplasia was 35% (21/58), and findings were approximately 1.5 times higher among HIV-infected compared with uninfected men. Unsatisfactory cytology was twice as common among Dacron compared with nylon-flocked swab protocol specimens (14% [8/58] vs 7% [4/58]). Sensitivity and specificity for the nylon-flocked protocol cytology showing ASC or more severe findings were 81% (58%–95%) and 73% (50%–89%), respectively. Dacron protocol specimens showed 52% (30%–74%) and 58% (34%–80%) sensitivity and specificity, respectively. Men showing ASC or more severe findings using the nylon-flocked protocol cytology showed 3-fold higher odds for high-grade anal intraepithelial neoplasia compared with men with negative results (p < .05), but no statistically significantly higher odds of high-grade anal intraepithelial neoplasia for men showing ASC or more severe findings compared with those with negative results for Dacron protocol cytology (p > .05). Conclusions The nylon-flocked protocol better detects high-grade anal intraepithelial neoplasia than does the Dacron protocol, yields more interpretable results, and classifies men with high-grade anal intraepithelial neoplasia as cytologically abnormal 2.5 times more often, even in this small clinical trial. Clinical trials registration number: NCT00955591.

30. Phase II Clinical Trial of Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA-SCID) Using a γ-Retroviral Vector

We report follow-up of subjects treated in a Phase II study of gene therapy for ADA-SCID. Between 2009 and 2012, ten ADA-deficient SCID patients were treated by γ-retroviral-mediated gene transfer (MND-ADA) to their bone marrow CD34+ cells. The subjects were given non-myeloablative chemotherapy (busulfan @ 90 mg/m2) and were withdrawn from PEG-ADA enzyme replacement therapy (ERT) prior to infusion of autologous gene-modified cells. Subject age at the time of treatment ranged from 3 months to 15 years (median = 11.5 months). Follow-up times range from 2 to 5 years. All but one subject, who was 15-years old at the time of treatment, remain off PEG-ADA ERT with immune reconstitution that reached maximal level between 6 and 12 months after transplant and was maintained thereafter. Vector marking in peripheral blood cells remained consistently detectable (> 0.1 copy/PBMC and ≥ 0.003 copy/granulocyte) at 2 years and later after transplant in subjects who discontinued ERT. These subjects also had PBMC ADA enzymatic activity in the normal range and red blood cell deoxynucleotide levels below 10%. Three subjects have discontinued intravenous immunoglobulin; five subjects have discontinued prophylactic antibiotics. All subjects have polyclonal gene marking with no sign of lymphoproliferative disease. The subjects remain in good health without infections or other complications.

Nylon-flocked swab collection method better predicts high-grade AIN than does dacron swab method

Author(s): Wiley, Dorothy; Bolan, Robert; Voskanian, Alen; Young, Stephen; Elashoff, David; Hsu, Hilary; Masongsong, Emmanuel; Barman, Provaboti; Detels, Roger

Abstract LB-177: HPV16 CpG and de novo-cytosine methylation is differentially associated with low-grade versus high-grade anal intraepithelial neoplasia in HIV-infected men

Objective: To determine if cytosine methylation in CpG and de novo sites is differentially associated with high- or low-grade anal intraepithelial neoplasias (HG-, LG-AIN), HPV16 DNA from182 clinical specimens. Background: Intra-anal cancer (IAC) is an emerging health crisis for gay, bisexual, transgender and other men who have sex with men (MSM) and rates have risen sharply among HIV-infected MSM despite introduction of HAART. High-risk HPVs are a causal risk factor for IAC and are especially common where MSM show HIV-coinfection. Further, cytosine methylation has been posited as an epigenetic transcription regulator that is poorly described in intra-anal dysplasias and cancers. Methods: HPV16 DNA was extracted from anal swab specimens and tested using bisulfite modification, PCR, cloning and sequencing of ∼10 clones/sample. Cervical cancer cell lines, CaSki and SiHa, and paraffin-embedded anal cancer specimens were similarly characterized as controls. Genomic sequences were evaluated using CLUSTAL and BiQ Sequence Alignment analysis software. Descriptive, tabular and multivariate analyses were performed using SAS (Version 9.2). Graphical representations were compiled using SIGMAPLOT (Version 9.0). Findings: Overall, methylation was relatively low in clinical AIN specimens. For 25,085 cytosines, the mean prevalence of methylation was 2.7% (SEM=0.4%) across 3′ L1 and LCR; the prevalence me-CpG, -CpA, -CpT, and -CpC was 3.2% (0.2%), 2.6% (0.1%), 2.5% (0.1%), and 2.7% (0.1%), respectively. However, some variation in HPV16 genomes was observed across specific functional gene sequences for HG- and LG-AIN specimens. Specifically, LG-AINs showed 1.30–2.62 times greater mean prevalence of methylation when compared to HG-AINs, across 24 of the 31 cytosines between nt7428–7564 ( p -values Conclusions: Data suggest the HPV16 5′LCR/enhancer region contains a large concentration of cytosine-containing transcription regulatory elements where binding interference by methylation might alter expression of the p97 promoter. Methylation in the HPV16 5′LCR/enhancer may decrease risk for HG-AIN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-177. doi:10.1158/1538-7445.AM2011-LB-177

Abstract C31: DMBA-induced skin tumors in HPV16 E7 transgenic mice are affected by bovine α-lactalbumin made lethal to tumor cells (BAMLET)

Background and Purpose: Human papillomavirus Type 16 (HPV16) infection is necessary but not alone a sufficient causal risk factor for cervical and other anogenital cancers and nearly 25% of head and neck malignancies (1, 2). HPV16 E7 is one of the important oncoproteins that promotes carcinogenesis. Most treatments for cervical precancers are ablative and few therapeutic drugs are available. Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET), a complex of Human α-Lactalbumin and oleic acid shows promise for treating skin papillomas by differentially inducing apoptosis in transformed cells without adverse effects on normal cells. Bovine α-Lactalbumin Made Lethal to Tumor Cells (BAMLET) has been shown to be biologically equivalent to HAMLET (3) and in vitro studies suggest BAMLET and HAMLET show similar cytotoxic effects in tumor cell lines (4). Further, in vitro studies suggest both a dose- and time-dependent response to BAMLET treatment (3–6). To determine the effect of BAMLET on tumor number, size and histological characteristics in vivo, we compared two groups of BAMLET-treated with normal saline (NS)-treated HPV16 E7 transgenic C57/BL6 mice (TG) and syngeneic mice receiving chemical carcinogen DMBA topically to induce skin tumor. Methods: For 56 TG and syngeneic mice, in six treatment groups, 100nmol/200μl DMBA was applied topically on the dorsal thoracic flank for up to 20 weeks. BAMLET, 0.7 mM, was applied daily for 7d before and either 11d (18d BAMLET) or up to 23 weeks (24w BAMLET) following DMBA initiation. As a comparison, NS was similarly applied to 10 TG and syngeneic mice for a total of 24 weeks. A series of bivariate zero-inflated repeated-measures Poisson (ZIP) models predicted the number of papillomas based on TG/syngeneic groupings, and treatment group. The fully adjusted model evaluated the effect of the TG, treatment group and duration, and time on study for predicting the number of tumors. Least-squares linear regression (LSLR) was used to estimate the effect of treatment on average tumor size at censoring or death, controlling for the effect of gender, among the transgenic mice. Results: On average, papillomas developed after 9 weeks of DMBA treatment. Analyses showed TG-mice developed more tumors than did syngeneic mice (p Discussion and Conclusion: These preliminary data suggest the effect of the transgene is strong on the number of tumors the result from DMBA treatment and on survival. This strong effect suggests we lack the power to detect an effect of BAMLET in transgenic mice. However, some data herein suggest BAMLET may increase average tumor size among HPV16-E7 transgenic mice. In light of positive findings in human clinical studies using the human analogue, HAMLET, understanding whether the effects of α-Lactalbumins and oleic acid complexes are modulated by HPV E6, E7, or E6/E7 oncogenes warrants further exploration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C31.

Cytosine methylation in the HPV16 3' L1/ 5'LCR region characterized from anal epithelia of HPV-HIV coinfected men

Anal specimens derived from 83 HIV-HPV16 co-infected men were analyzed for cytosine methylation using bisulfite modification, PCR amplification, cloning, and sequencing. Data were processed using the ClustalW alignment algorithm within BiQ Analyzer Software and input into SAS 9.2 to generate heat maps and histograms(Figure ​histograms(Figure1).1). Approximately 10 clones were characterized for each specimen across 81 CpA, CpT, CpC (denovo methylation) and 10 CpG sites within the 3’-L1 and 5’LCR region of HPV16 genomes. Analyses were confined to 64 denovo cytosine residues and 10 CpGs outside the primer annealing regions. Clinical outcome evaluation by a single examiner showed seven men with no anal intraepithelial neoplasias (AIN), 26 with AIN-1, and 50 with AIN-2. Data showed the average prevalence (standard deviation) of methylcytosines was 11% (0.3) across 7,553 bases/residues and did not vary across maintained or denovo cytosine groups, i.e., 6,723 denovo sites and 830 CpGs. Evaluation by a single examiner using cytology and histology showed 7 with no anal intraepithelial neoplasias (AIN), 26 showed AIN-1, and 50 showed AIN-2. Mean and median prevalence of denovo and CpG methylation were closely approximated across AIN groups. Maxima were observed in two regions: 7111 to 7119, and 7505 to 7514 that showed a methylcytocine prevalence of 24% (0.4), and 18% (0.4), respectively. Logistic regression suggests that with each year of age, risk for moderate-severe methylation ≥30% at each site decreases; however, among smokers risk for ≥30% methylation increases. Specifically, moderate cytosine-methylation decreased by 5% (OR=0.95, 95% CI, (0.92, 0.99) for each year of age, centered around the mean of the sample. Smokers were nearly twice as likely to show moderate cytosine methylation overall (OR=1.9, (1.1, 3.3) and these estimates did not vary across CpG-denovo sites. Although not statistically significant, moderate cytosine methylation was inversely associated with grade of AIN in these data (when compared to high-grade, low-grade and no AIN showed OR=1.5 (0.8, 2.7) and 2.0 (0.9, 4.5), respectively, p=0.2). Figure 1 Heatmap of Methylcytosines in the 3’L1 and 5’LCR Region of HPV16.

Abstract 2807: The relationship between age and efficiency of cervical cancer screening using population-based surveillance

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Data for in situ cervical carcinomas are not routinely collected by U.S. tumor registries. Some experts suggest that comparing preinvasive and invasive cervical cancers (ICC) rates may represent our best population-based appraisal of screening strategies. From 1991-1997, a total of 5,921 women diagnosed with in situ or ICC were reported to the Markey Cancer Registry in Lexington, Kentucky, a population-based registry. Maximum-likelihood Poisson regression analyses were used to estimate the incidence rate of in situ and ICC (per 100,000), summarized over age-and race-specific groups for each of seven years, 1991 to 1997. U.S. Census age and race data for 1990 were used to estimate denominators for two race groups: African American (AA) and all others (white). Incidence rates and rate ratios are reported. A total of 4,290 and 1,631 were diagnosed with in situ and ICC in the study period. While more than 80% of cases among women 65 years, only 42% (+4%) of tumors were diagnosed as in situ lesions. Specifically, among 20-39 year olds, AA were less likely than white women to be diagnosed with in situ carcinoma (p 55 were less likely to be diagnosed with in situ disease than ICC. While year of diagnosis was significant in the analyses, relationships between age and diagnostic stage persisted across each of 7 years examined. These data suggest that Pap test most efficiently identifies treatable, high-grade precancerous lesions among women younger than 50 years. HPV infection prevalence varies by age, but not race, while ICC disproportionately affects women of color, especially as they age. Several factors may explain these findings. Screening may be performed less often overall or more inconsistently among older women and among AA. Also, if screening in the population decreases with advancing age, symptomatic older women with higher likelihood of malignancy will be over-represented in the data. Directly tailoring screening programs to recruit and retain non- and under-screened women and women with medical record evidence of poorly triaged Pap test abnormalities may improve detection of cervical cancers at a preinvasive stage, where there is a very high likelihood of survival. We believe that these findings are consistent with those of others, and together they suggest our best public health strategy may be to actively recruit middle-age and older women into cervical cancer screening programs who have undergone Pap test screening fewer than three times in a ten year period. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2807.