Alba Lucía Cómbita

Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding.

Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus‐host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20‐mer peptides, covering the entire protein, HPLC‐purified, 125I‐radiolabeled and tested in VERO and HeLa cell‐binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54–77) and 18294 (274–308) from HPV16 L1, as well as 18312 (59–78) and 18322 (259–278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface‐exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase‐treated cells. Cross‐linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV‐16 VLP binding to HeLa cells. According to the L1‐ and VLP‐reported structure, both peptides are close on the VLP‐surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell‐binding regions.

Age-specific seroprevalence of human papillomavirus 16, 18, 31, and 58 in women of a rural town of Colombia.

Objective The study’s objective was to estimate human papillomavirus (HPV) genotype–specific seroprevalence to determine population HPV exposure and inform vaccine policy. Methods This study is a cross-sectional prevalence survey of 878 women of Pueblorrico, a rural town of Colombia. A standardized questionnaire was used to obtain information on demographic characteristics, sexual and reproductive history, and smoking habits. Seropositivity to HPV-16, -18, -31, and -58 was determined by virus-like particles in an enzyme-linked immunosorbent assay. Results Overall seropositivity to any HPV genotype was 27.9%. The combined seroprevalence of women 15 to 19 and 20 to 24 years old was 35.4% (95% confidence interval [CI], 25.9–46.2) and 36.0% (95% CI, 27.7–45.3), respectively. Seroprevalence for HPV-16 was 17% (95% CI, 14.6–19.6); for HPV-18, 9.8% (95% CI, 8.0–11.9); for HPV-31, 11.4% (95% CI, 9.5–13.7); and for HPV 58, 12.5% (95% CI, 10.5–14.9). Higher HPV seropositivity was associated with the lifetime number of occasional sexual partners (odds ratio, 3.05; 95% CI, 1.26–7.37) and having more than 2 regular sexual partners (odds ratio, 3.00; 95% CI, 1.21–7.45) in women younger than 44 and older than 45 years old, respectively. Use of oral contraceptives and tobacco/cigarettes was significantly associated with reduced HPV seropositivity in women older than 45 but not in women younger than 44 years old. Conclusions Human papillomavirus seropositivity is associated with measures of sexual behavior, particularly a greater lifetime number of sexual partners. Hormonal and tobacco/cigarette use may be factors influencing the HPV seropositivity in women older than 45 years old.

High expression of ID family and IGJ genes signature as predictor of low induction treatment response and worst survival in adult Hispanic patients with B-acute lymphoblastic leukemia

BackgroundB-Acute lymphoblastic leukemia (B-ALL) represents a hematologic malignancy with poor clinical outcome and low survival rates in adult patients. Remission rates in Hispanic population are almost 30 % lower and Overall Survival (OS) nearly two years inferior than those reported in other ethnic groups. Only 61 % of Colombian adult patients with ALL achieve complete remission (CR), median overall survival is 11.3 months and event-free survival (EFS) is 7.34 months. Identification of prognostic factors is crucial for the application of proper treatment strategies and subsequently for successful outcome. Our goal was to identify a gene expression signature that might correlate with response to therapy and evaluate the utility of these as prognostic tool in hispanic patients.MethodsWe included 43 adult patients newly diagnosed with B-ALL. We used microarray analysis in order to identify genes that distinguish poor from good response to treatment using differential gene expression analysis. The expression profile was validated by real-time PCR (RT-PCT).ResultsWe identified 442 differentially expressed genes between responders and non-responders to induction treatment. Hierarchical analysis according to the expression of a 7-gene signature revealed 2 subsets of patients that differed in their clinical characteristics and outcome.ConclusionsOur study suggests that response to induction treatment and clinical outcome of Hispanic patients can be predicted from the onset of the disease and that gene expression profiles can be used to stratify patient risk adequately and accurately. The present study represents the first that shows the gene expression profiling of B-ALL Colombian adults and its relevance for stratification in the early course of disease.

Prognostic stratification improvement by integrating ID1/ID3/IGJ gene expression signature and immunophenotypic profile in adult patients with B-ALL

BackgroundSurvival of adults with B-Acute Lymphoblastic Leukemia requires accurate risk stratification of patients in order to provide the appropriate therapy. Contemporary techniques, using clinical and cytogenetic variables are incomplete for prognosis prediction.MethodsTo improve the classification of adult patients diagnosed with B-ALL into prognosis groups, two strategies were examined and combined: the expression of the ID1/ID3/IGJ gene signature by RT-PCR and the immunophenotypic profile of 19 markers proposed in the EuroFlow protocol by Flow Cytometry in bone marrow samples.ResultsBoth techniques were correlated to stratify patients into prognostic groups. An inverse relationship between survival and expression of the three-genes signature was observed and an immunophenotypic profile associated with clinical outcome was identified. Markers CD10 and CD20 were correlated with simultaneous overexpression of ID1, ID3 and IGJ. Patients with simultaneous expression of the poor prognosis gene signature and overexpression of CD10 or CD20, had worse Event Free Survival and Overall Survival than patients who had either the poor prognosis gene expression signature or only CD20 or CD10 overexpressed.ConclusionBy utilizing the combined evaluation of these two immunophenotypic markers along with the poor prognosis gene expression signature, the risk stratification can be significantly strengthened. Further studies including a large number of patients are needed to confirm these findings.

Evaluation of the immune response to human papillomavirus types 16, 18, 31, 45 and 58 in a group of Colombian women vaccinated with the quadrivalent vaccine

Objective: To analyze whether the immune response to HPV-16, -18, -31, -45 and -58 capsids in women vaccinated with the quadrivalent vaccine induces cross-reactivity against other HPV virus-like particles (VLPs). Methods: A total of 88 women aged between 18 and 27 years attending the HPV clinic at the Instituto Nacional de Cancerologia were enrolled and vaccinated against HPV. Follow-up visits were scheduled at months 7, 12, and 24. Samples were collected for cytology, HPV-DNA typing, and detection of HPV antibodies. IgG antibodies were measured by ELISA using HPV-16, -18, -31, -45, and -58 VLPs. HPV-DNA detection was done by GP5+/GP6+PCR-ELISA and HPV typing was performed by Reverse Line-Blot assay. Results: Pre-vaccination, the seroprevalence of HPV-16, -18, -31, -45, and -58 was 39%, 31.7%, 15.9%, 31.7%, and 23.2%, respectively. One month post-vaccination, the seroprevalence increased close to 100% for all types. At month 24, this response was maintained only for HPV-16 and -18. For HPV-31, -45 and -58, the seroprevalence decreased to below 50%. The prevalence of HPV DNA types 16, 18 and 58 before vaccination was little changed 1 month after vaccination. No new infections were observed at 24 months. For HPV-16 and -18 related types, no differences were observed before vaccination and at month 24. For other high-risk HPV types, the prevalence increased 18 months post-vaccination (15.5%) compared with pre-vaccination (9.8%). Conclusion: Immune response to all HPV types increased after vaccination, but this increase was maintained only for HPV-16 and -18. These results suggest a possible cross-reactivity against HPV types 31, 45 and 58, but this cross-reactivity wanes with time.

Abstract B56: RNA-seq analysis of prostate cancer samples from Hispanic patients reveal progressive characteristics in Gleason patterns

Background. One of the prognostic markers widely used in clinic to predict survival in prostate cancer (PC) patients is the Gleason score. Differences have been observed in biochemical progression-free and overall survival in patients with different Gleason scores. Because each Gleason represents a different state of tumor progression, we decided to characterize expression profiles and pathways in Gleason 3, 4 and 5 in order to explore the tumor biology context in each pattern, and thus determine possible prognosis biomarkers in PC. Methods. We extracted RNA from normal and malignant formalin-fixed paraffin-embedded tissues from radical prostatectomy samples of Colombian patients and analyzed expression using data generated with next generation sequencing methods. Pathway analysis was done with MetaCore. Results. Eleven gene networks (7 genes) were common between all Gleason scores and represented cellular processes possibly involved with damage of the prostatic tissue including: cell adhesion, cytoskeleton remodeling and metabolism of phosphatidyl compounds. According to the analysis in Gleason pattern 3, the principal pathways were associated with cytoskeleton remodeling, gap junctions signaling and regulation of cell adhesion. These pathways could contribute to de-differentiation process experienced by cells towards tumor transformation in early stages of progression. Also, signaling of epithelial-to-mesenchymal transition (EMT) were observed, this findings could suggests not only cytoskeleton remodeling during de-differentiation, but also appearance of metastatic cells in this Gleason pattern. On the other hand, the action of the Androgen Receptor (AR) is suggested by protein-protein interactions characterized by rapid activation of signaling cascades of proliferation, cell growth and survival, via MAPK and PI3K/AKT. The latter in turn induces AR overexpression through control of regulatory protein MDM-2 and optimizes binding of AR to promoter/enhancer regions over its target genes. Also, AR inhibits WNT signaling via interaction with B-catenin. However, AR is a LEF-1/TCF transcriptional target, which is activated by WNT/B-catenin signaling. The pathways identified in Gleason pattern 4 were similar to those of Gleason 3, with a more significant participation of pathways leading to cytoskeleton remodeling, regulation of cell adhesion molecules and of EMT. In addition, there is ligand-independent activation of AR, wherein activating mutations, splicing variants and activation by other signaling cascades, may lead to directed transactivation of target genes in a deregulated manner. Further, NGF/TrkA/PI3K-mediated signaling may lead to inhibition of apoptosis and survival of these cells. Finally, pathways analysis in Gleason pattern 5 showed that the most significant was cell adhesion and ECM remodeling, which favors the metastatic process and regulation of cell motility and adhesion, processes aided by migration and angiogenesis signaling pathways. Likewise, AR activation and downstream signaling has an important role, transactivating transcription factors, which together lead to cell proliferation, inhibition of apoptosis, expression of tumor growth factors, regulation of cell adhesion molecules and from inflammatory response. The latter is reflected in another pathway identified, as is the antigen presentation by MHC class II. Conclusion. The pathways identified by different Gleason patterns exhibit progression of localized prostate tumors into metastatic ones, where there is cell growth and dysregulation in signaling pathways at the beginning, and then, as they progress, they acquire the ability to migrate and to regulate AR signaling through different mechanisms so that allows AR to be a central regulator in PC. Citation Format: Natalia L. Acosta, Melody C. Baddoo, Alba L. Combita, Rodolfo Varela, Jorge Mesa, Maria C. Sanabria-Salas, Jovanny Zabaleta. RNA-seq analysis of prostate cancer samples from Hispanic patients reveal progressive characteristics in Gleason patterns. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B56.

Abstract B36: Biological and clinical significance of ID1/ID3/IGJ expression signature modulation in B-Acute lymphoblastic leukemia

Background. Acute lymphoblastic leukemia (ALL) in Hispanic populations represents a public health problem that requires priority attention because its incidence and mortality increase annually. Intensive chemotherapeutic schemes in the last decade in the United States and other countries and have led to higher rates of durable remissions. However, these schemes have shown disappointing results in Hispanic populations. In Colombia, for example, the median overall survival (OS) is less than 11.3 months, the event-free survival (EFS) is 7.34 months, and only 61% of patients achieve complete remission (CR). Therefore, it is interesting to explore the underlying molecular characteristics of the disease in Hispanic patients. Previously we identified a gene expression signature associated with response to induction treatment and outcome of adult Hispanic patients with B-ALL. These ID1, ID3 and IGJ seem to be involved in many tumorigenic processes. The goal of our study is identify the role of these genes in B-ALL and its relevance in the prognosis and progression of disease in an in vitro model. Methods. CRISPR-Cas9 Gene Engineering and expression-ready ORF cDNA clones were used to either down-regulate or over-express, respectively, the expression of ID1, ID3 and IGJ genes in the adult B-ALL cell line NALM6. We used TaqMan to quantify the levels of mRNA expression of ID1/ID3/IGJ genes after over-expression. Protein expression by Flow Cytometry was used to verify silencing and over-expression of our 3-genes signature. Cell viability and proliferation were determined by anexin V-PI and MTT assay, respectively at 0, 24, 48, 72 and 96 hours after modulation. Cell cycle analysis was performed using BrdU and 7-AAD. Separately, increasing doses of cyclophosphamide, doxorubicin and cytarabine were used to determine the role of ID1, ID3, and IGJ genes in chemo resistance at 24, 48, 72, 96, and 144 hours. Total RNA from leukemic cells was isolated using Trizol method. We used microarray analysis to identify genes differentially expressed after silencing/overexpression. We performed Immunophenotype analysis of 43 B-ALL adult Hispanic patient Bone Marrow samples using the panel of antibodies recommended and standardized by the European consortium Euroflow. Correlation analysis were used to establish the association between our 3-Gene expression signature and the expression of surface markers currently used for diagnostic and follow-up of B-ALL patients. Results. We observed that both overexpression and silencing of ID1, ID3 and IGJ modulated B-ALL cells cell cycle. ID1/ID3/IGJ overexpression resulted in a loss of S phase and arrest of the cells in the G0/G1 phase of the cell cycle. On the contrary, ID1/ID3/IGJ silencing induced a loss of G0/G1 phase and gain in the number of cells in G2/M and S phases. Gain-of-expression and loss-of-expression analyses using incremental doses of chemotherapy agents currently used in the B-ALL treatment, demonstrate that ID1/ID3/IGJ signature promotes drug resistance of the NALM6 B-ALL cell line. Clinically, patients with higher ID1/ID3/IGJ expression have high expression of CD20 and CD10 markers (aberrantly expressed in immature cells) and shorter OS and EFS. In addition, more than 2,000 genes that are differentially expressed in the cells with modulation of the expression of ID1/ID3/IGJ suggesting a complex network of biological processes implicated in the drug resistance and poor prognosis present in the patients with this 3-gene signature. Conclusions. Our data identify the ID1/ID3/IGJ signature as possible modulator of molecular events during B-ALL differentiation, proliferation and drug resistance. These results highlight the potential role of this gene signature both as a risk stratification tool and as a candidate therapeutic target in Hispanic population. Citation Format: Nataly Cruz-Rodriguez, Alba Lucia Combita, Jone Garai, Leonardo Jose Enciso, Sandra Milena Quijano, Silvia Serrano-Gomez, Li Li, Jovanny Zabaleta. Biological and clinical significance of ID1/ID3/IGJ expression signature modulation in B-Acute lymphoblastic leukemia. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B36.

Abstract 3121: Gene expression signature predicts induction treatment response and clinical outcome in adult Colombian patients with acute lymphoblastic leukemia

Background. In Colombia ALL in adults represents a public health problem because its incidence and mortality increase annually. Only 61% of Colombian adult patients with ALL achieve complete remission. The median overall survival to the disease is less than 11.3 months and the event-free survival is 7.34 months. Identification of prognostic factors in patients with ALL is crucial for the proper planning of treatment strategies and the optimal results of therapy. Our goal was to determine gene expression signatures correlated with response to therapy and to evaluate the utility of these expression patterns as predictors of risk prior to therapy of adult Colombian patients with B-ALL. Methods. This study included 43 adult patients newly diagnosed with B-cell precursor or common B-ALL. Patients were recruited at the Colombian National Cancer Institute and Hospital Universitario San Ignacio, both in Bogota, Colombia. The leukemic blast population from diagnostic samples was separated with magnetic microbeads coated with either anti-CD19 or anti-CD34 antibodies followed by column enrichment using standard procedures and MACS (Miltenyi, Bergisch Gladbach,Germany). Total RNA from purified leukemic cells was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer9s protocols. We used microarray analysis to identify genes that distinguish poor from good response to induction treatment using differential gene expression analysis and the response group as reference and the Illumina Custom algorithm embedded in the GenomeStudio software (Illumina). The expression profile was validated by real-time PCR (RT-PCT) using TaqMan probes. The 2-ΔΔCT method was used to estimate the fold induction of each gene using GAPDH and an internal calibrator as controls. Assays were done in triplicate. Results. We identified 442 genes differentially expressed between 22 leukemia patients who responded and 5 who did not respond to induction chemotherapeutic treatment. Hierarchical analysis with the 99 most differentially expressed genes between the two groups revealed 3 sets of patients that differed in their clinical characteristics giving these genes high prognostic clinical outcome impact capacity. We validated the expression of 7 genes by RT PCR in 43 patients and, in addition to finding a correlation with gene expression profiles, we established correlations with good and poor prognosis from the time of diagnosis. Conclusions. Our study suggests that the response to induction treatment and clinical outcome of patients can be predicted from the onset of the disease and that gene expression profiles can be used to stratify patient risk adequately and accurately. The present study represents the first showing that gene expression profiling could become a clinically relevant tool for stratification in the early course of disease of Colombian adults B-ALL. Citation Format: Nataly Cruz-Rodriguez, Sandra M. Quijano, Leonardo J. Enciso, Alba L. Combita, Jovanny Zabaleta. Gene expression signature predicts induction treatment response and clinical outcome in adult Colombian patients with acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3121.

Abstract C88: HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer

Background: There is limited information of the role of hTERT methylation, hTERT expression and their association with type specific HPV infections in patients with invasive cervical cancer Objective: To analyze the possible association between methylation status of the hTERT promoter gene, hTERT expression and HPV infection in patients with invasive cervical cancer. Methodology: Eighty seven frozen samples of women with invasive cervical cancer were analyzed for type specific HPV infection using a GP5+/GP6+ mediated PCR- RLB. hTERT DNA methylation analysis was performed on bisulfite modified DNA using a new PCR-RLB-hTERT methylation assay targeting two regions flanking the hTERT [region 1 (nt -240 to -1) and region 2 (nt +1 to +120) relative to first ATG]. Expression of hTERT was detected by immunohistochemistry using an Anti-hTERT (348-358) rabbit pAb). Results: All samples with HPV types belonging to the α7 species (HPV 18, 45 and 59) showed no hTERT methylation in region 1 (core promoter) and an increase in% of partial methylation in region 2. Samples with HPV types belonging to the α9 specie (16, 52, 35, 31 and 58) showed similar% of methylation in region 1 and region 2 according viral type but the percentages of methylation were different between them (ranging from 0% for HPV58 samples to 66.7% for HPV31). Strong expression of hTERT was observed in 77% of the samples independent of the HPV genotype. Conclusion: hTERT methylation seems to be associated with specific genotype HPV infection; however expression of the hTERT protein seems to depend of additional molecular events and worth further investigation. Citation Format: Pablo Moreno-Acosta, Nicolas Morales, Marcela Burgos, Oscar Gamboa, Juan Carlos Mejia, Alfredo Romero-Rojas, Alba Lucia Combita, Monica Molano. HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C88.