Consensus Primers Research Articles

Low etiologic fraction for human papillomavirus in larynx squamous cell carcinoma

BackgroundHuman Papillomavirus (HPV) is a cause of oropharyngeal squamous cell carcinoma (OPSCC), but its pathogenic role in larynx squamous cell carcinoma (LSCC) remains unclear. Material and methodsA single-institutional, retrospective case-series was performed to estimate the etiological fraction (EF) for HPV in LSCC. Eligible cases included 436 consecutive cases of LSCC diagnosed (2005–2014) at The Ohio State University Medical Center. HPV DNA presence was detected by consensus primer PCR (Inno-LiPa) and HPV type-specific qPCR. HPV E6/E7 mRNA expression was detected by type-specific qRT-PCR. Tumor p16 expression was evaluated by immunohistochemistry (IHC). ResultsHPV DNA was detected by Inno-LiPa in 54 of 404 (13.4%, 95% CI 10.2–17.1) evaluable samples but was confirmed by HPV type-specific qPCR in only 14 (3.5%, 95% CI 1.9–5.7). Only 7 of 404 (1.7%, 95% CI 0.7–3.5) LSCC were positive for HPV E6/E7 mRNA expression, including HPV16 (n=4) and 1 each for 11, 26 and 33. In the HPV11-positive tumor, Sanger sequencing discovered 6 nucleotide mutations in the upstream regulation region, E6 and E7. Of 404 LSCC, 18 had strong and diffuse p16 expression.In comparison to a gold standard of HPV E6/E7 mRNA expression, p16 expression had a sensitivity of 71.4% (95% CI 29.0–96.3), specificity of 96.7% (95% CI 94.5–98.3), positive-predictive-value (PPV) of 27.8% (95% CI 9.7–53.5) and negative-predictive-value of 99.5% (95% CI 98.1–99.9). ConclusionThe EF for HPV in LSCC is low (1.7%) in a geographic region with high EF for OPSCC. Low-risk HPV may rarely cause LSCC. Finally, p16 expression has poor PPV for HPV in LSCC.

Prevalence of High-Risk Human Papillomavirus (HR-HPV) Genotypes and Multiple Infections in Cervical Abnormalities from Northern Xinjiang, China.

Multiple human papillomavirus (HPV) genotypes often coexist within the cervical epithelia and are frequently detected together in various grades of the cervical neoplasia. To date, only a few reports exist on multiple HPV infections of HPV in Xinjiang Uygur Autonomous Region (XUAR). In the present study, we investigated the prevalence of High-Risk HPV (HR-HPV) genotypes and multiple infections. Cervical cytology samples were collected from 428 women who presented cervical abnormalities. Genotyping of HPV was performed by polymerase chain reaction–sequencing based typing (PCR-SBT) using consensus primers and specific primers. Of them, 166 samples were positive for HPV according to PCR results using the consensus primers. These samples contained cervical abnormalities enriched with inflammation (n = 107), cervical intraepithelial neoplasia (CIN) I (n = 19), CINII-III (n = 9) and cervical cancer (n = 31). Of the 166 HPV positive samples as determined by PCR analysis, 151 were further typed by PCR-SBT using 19 pairs of genotype-specific primers. Using this method, 17 different HR-HPV genotypes were identified. The most frequently observed HPV genotypes were HPV16 (44.0%, 73/166), 53 (28.9%, 48/166), 52 (25.3%, 42/166), 58 (22.3%, 37/166) and 35 (17.5%, 29/166). The proportions of single and multiple infections in the HPV-positive specimens were 34.9% and 65.1%, respectively. Multiple HPV types were most prevalent in the inflammatory state (63.0%), followed by cervical cancer (24.1%), CINI (11.1%), and CINII-III (1.9%). The results of our data analyses suggested that i) multiple HPV infection is not necessarily correlated with the severity of cervical abnormalities; and ii) among the multiple HPV infections, double infections combined with HPV16 is the most common. In addition, L1 full-length sequences of the top five high-risk HPV genotypes were amplified and sequenced. According to the L1 sequence of the epidemic genotypes that were amplified, we found that these genotypes contained the sequence point mutation, and that some of these genotypes further showed amino acid modifications. These results provide a basis for the construction of a polyvalent vaccine that is suitable for use in the XUAR, even in economically challenged communities located in China.

Prevalence of HPV 16/18 Infection: A Hospital-Based Study

The objective of the present study was to determine the prevalence of human papillomavirus (HPV) 16/18 infection among the women attending the gynecological outpatient clinic at SMGS Hospital and to find out the correlation between cervical cytology and HPV DNA testing. Women with ≥30 years of age visiting the gynecology outpatient with varied complaints were subjected to Pap smear. In total, 300 samples were subjected to PCR using consensus primer for HPV. The samples that were positive for HPV DNA were subsequently assessed for HPV types 16 and 18. Out of 300 patients, 113 (37.6 %) women tested positive for HPV DNA and out of 113 HPV-positive samples 23 (7.66 %) tested positive for high-risk HPV, i.e., HPV 16 (5 samples) and HPV 18 (18 samples). A statistically higher prevalence of HPV was observed in rural, low socioeconomic and poorly educated group. No association of HPV prevalence was noted with parity, age at marriage and age at first child birth. Increase in HPV prevalence was observed with the increase in abnormality in cervical cytology. HPV infection was present in 18.91 % of women with normal cytology, 31.11 % in inflammatory smear, 32.14 % in ASCUS, 54.16 % in LSIL, 77.77 % in HSIL and 100 % in cancer cervix patients. HPV 16/18 infection was present in 1.35 % of women with normal cytology and 20.63 % of women with abnormal cytology report. The study generates epidemiological data of prevalence of subclinical HPV in the women visiting a tertiary care institute. The data generated will be useful for laying guidelines for mass screening of HPV, treatment and prophylaxis in the local population.

High-Risk Human Papillomavirus Infection in Latvian Male Kidney Transplant Recipients / Augsta Riska Cilvēka Papilomas Vīrusa Infekcija Vīriešu Dzimuma Nieru Transplantāta Saņēmējiem Latvijā

Abstract Kidney transplant recipients have higher incidence of human papillomavirus (HPV)-related malignancies, but studies on the natural history of HPV infection are insufficient, especially regarding in male recipients. The aim of this study was to evaluate the course of high-risk HPV (HR-HPV) infection after kidney allograft transplantation in male recipients: to estimate frequency and activity of HR-HPV infection under immune system suppression. Twenty male renal recipients (age 20 - 68) were enrolled in this investigation and examined in dynamics. Peripheral EDTA-blood samples and urine samples were collected from each patient 2 weeks, 6 months and 12 months after transplantation. Polymerase chain reaction (PCR) with consensus primers was used for initial detection of high range HPV types, a commercial qPCR kit for detection of HR-HPV load in urine samples and ELISA for detection of serum IgG class antibodies to HR-HPV L1-capsid protein. Overall, combining molecular (HR-HPV genomic sequences detected by real-time PCR) and serological studies (IgG class antibodies to HR-HPV L1-capsids’ protein), high frequency of HRHPV infection among male kidney transplant recipients (9/20; 45%) was showed. However, the majority of HR-HPV positive recipients (7/9; 78%) showed signs of infection clearance. It means that, despite the applied immune suppressive therapy, the host’s immune system is capable of dealing with HR-HPV infection up to the 12th month after transplantation. However, the sample size should be increased to enable through statistical analysis before final conclusions are made.

Abstract 846: Improving T-cell receptor clonotyping of T-cell lymphomas using hybrid-Capture and next-generation sequencing

Abstract Background T-cell clonality assays (TCAs) support a variety of clinical and research interests including T-cell malignancy clone identification, minimal-residual disease (MRD) testing and T-cell receptor (TCR) repertoire characterization for the purposes of immunotherapy. Unfortunately, many TCAs are unable to interrogate alpha, beta, gamma, and delta TCR loci simultaneously. Aiming to overcome these limitations, we designed a T-cell clonality assay (the “NTRA”), capable of identifying T-cell gene rearrangements from all four TCR loci, using hybrid-capture followed by deep next-generation sequencing (NGS). A novel informatics package exploiting the Burrows Wheeler Alignment algorithm and finding of CDR3 seed sequences was also deployed. We then validated the assay using orthogonal methods and in a series of clinical T-cell malignancy specimens. Methods We used DNA probes for hybrid-capture of all V and J segments in the TCR loci, followed by NGS on the Illumina NextSeq 500 platform. Analytical validation used a series of 10 specimens, including 6 flow-cytometry characterized T-cell specimens of variable degrees of immunophenotypic uniformity and 4 cell-lines with known TCR rearrangements. PCR/gel electrophoresis and Sanger sequencing were used for orthogonal validation, using primer sets designed to test each specimen for all 90th-centile V-J configurations. Subsequently, 61 clinical T-cell lymphoma specimens were tested, each previously assessed for T-cell clonality using the clinical gold standard (i.e. PCR/ electrophoresis using the BIOMED-2 consensus primers for the TCR-beta (TRB) and TCR-gamma (TRG) loci). Results PCR confirmed the NTRA-identified V-J configurations with an area-under-the-curve (AUC) by receiver-operator characteristic (ROC) analysis of 0.91. Relative to single-strand Sanger sequencing, the NTRA CDR3 sequence results showed a ROC AUC of 0.83. In a DNA dilution series employing the results of a “clonal” specimen (a Jurkat cell line) spiked into a “polyclonal” specimen (a mononuclear peripheral blood specimen), we could identify “clonal” specimen-specific V-J combinations and error-corrected CDR3 sequences down to less than 10⁁-5. In the final clinical validation, the NTRA performed with a ROC AUC of 0.82 relative to the current TRB & TRG BIOMED-2 clinical assay. Conclusions Our novel assay can overcome the current TCR locus data-yield limitations resulting from primer-based TCAs in a single-tube. The NTRA shows comparable sensitivity and specificity relative to current standard assays and excellent performance relative to current MRD techniques when applied to clinical T-cell lymphoma specimens. Citation Format: Etienne Mahe, David Mulder, Mark Dowar, Mahadeo Sukhai, Linh Nguyen, Pamela Ohashi, Jan Delabie, Tracy L. Stockley, Trevor Pugh, Suzanne Kamel-Reid. Improving T-cell receptor clonotyping of T-cell lymphomas using hybrid-Capture and next-generation sequencing. [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 846.

Prevalence of human papillomavirus in oral squamous cell carcinomas in northern Spain.

The aim of this study was to analyze the prevalence of high-risk HPV in oral squamous cell carcinoma (OSCC) in a northern Spanish population, as well as to ascertain the prognostic role of p16INK4a expression. The examination samples were collected from paraffin tissue blocks, from 125 patients surgically treated between 1996 and 2007. All cases were histologically evaluated, and the presence of HPV was assessed by p16 and p53immunohistochemistry followed by DNA detection by in situ hybridization (ISH) and polymerase chain reaction (PCR) amplification using the combination of consensus primers MY11/GP6+. Fourteen cases (11%) were p16-immunopositive, and p53 was scored positive in 73 cases (58%). Five cases (4%) showed a simultaneous p16-positive and p53-negative immunostaining. ISH was negative in all the cases. Among the p16INK4a-immunopositive cases, PCR amplification failed to reveal HPV DNA in any tumor samples. There were no statistically significant differences in any clinical or pathological characteristics of the patients regarding p16INK4a expression. T classification, neck-node metastasis, and clinical stage showed outcome relevance. However, no significant differences in cause-specific survival based on p16INK4a were observed. We did not find any high-risk HPV types in our patients, thus, are unlikely that HPV has an important role in the etiology of OSCC. p16INK4a protein was neither an accurate marker of HPV infection nor a prognosis marker in OSCC.

Metabarcoding of marine zooplankton: prospects, progress and pitfalls

Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few orthologous DNA regions, called barcodes) is revolutionizing analysis of biodiversity of marine zooplankton assemblages. Metabarcoding relies on high-throughput DNA sequencing (HTS) technologies, which yield millions of DNA sequences in parallel and allow large-scale analysis of environmental samples. Metabarcoding studies of marine zooplankton have used various regions of nuclear small- (18S) and large-subunit (28S) rRNA, which allow accurate classification of novel sequences and reliable amplification with consensus primers, but- due to their relatively conserved nature- may underestimate species diversity in a community. To discriminate species, more variable genes are needed. A limited number of metabarcoding studies have used mitochondrial cytochrome oxidase I (COI), which ensures detection of species-level diversity, but may require group-specific primers and thus result in inconsistent amplification success rates. Reference databases with sequences for accurately-identified species are critically needed to allow taxonomic designation of molecular operational taxonomic units (MOTU) and comparison with previous studies of zooplankton diversity. Potential and promising applications of metabarcoding include rapid detection of impacts of climate change, monitoring and assessment of ecosystem health, calculation of biotic indices, characterization of food webs and detection of introduced, non-indigenous species.

MP36-03 PRESENCE OF URINARY UREAPLASMA UREALYTICUM OR UREAPLASMA PARVUM IS NOT ASSOCIATED WITH THE OCCURRENCE OF CHRONIC PROSTATITIS/CHRONIC PELVIC PAIN SYNDROME

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Prostate & Genitalia II1 Apr 2016MP36-03 PRESENCE OF URINARY UREAPLASMA UREALYTICUM OR UREAPLASMA PARVUM IS NOT ASSOCIATED WITH THE OCCURRENCE OF CHRONIC PROSTATITIS/CHRONIC PELVIC PAIN SYNDROME Yu Seo and Gil Lee Yu SeoYu Seo More articles by this author and Gil LeeGil Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1637AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Ureaplasmas are the smallest human parasites. Its slow growth in infected host cells allow for the possibility of being causal pathogen for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Therefore, we examined the presence of Ureaplasma urealyticum (UU) and Ureaplsma pavum (UP) in 696 urine samples from 669 men who attended a urology outpatient clinic with various reasons by using an automated sequencing method. METHODS 696 urine samples that are nonbacterial (from prostate and urine ordinary cultures) and nongonorrheal and nonchlamydial (based on PCR) were randomly selected from stored samples. Clinical information of individual subject is presented in Table 1. We amplified the UU/UP urease complex components with consensus primers. Finally, we read the genetic sequences and classified between the UU and UP from ureaplasma spp. RESULTS The overall rates of UU and UP were 3.88% (27/696) and 6.46% (45/696), respectively. In univariate analysis, only UU infection significantly increases the likehood of urinary WBC count ≥5 only (odds ratio [OR]; 7.377, 95% confidence interval [CI]: 2.244-24.248, P=0.001) (Table 1). However, the presence of both UU and UP in urine do not increase the risk of having the CP/CPPS symptoms and signs. The mean NIH-CPSI scores in total of pain Items, urinary and quality of life items do not differ among the negative, UU positive, and UP positive groups (Table 2). CONCLUSIONS We find that only UU can induce asymptomatic pyuria. However, the presence of urinary UU or UP is not definitively associated with the occurrence of CP/CPPS. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e489-e490 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yu Seo More articles by this author Gil Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

S-genotyping of 25 sweet cherry (Prunus avium L.) cultivars from the Czech Republic

ABSTRACTSweet cherry (Prunus avium L.) is a self-incompatible species. Determination of the S-genotypes of cherry cultivars is crucial for breeding and to select appropriate cultivars for cross-fertilisation and fruit set. In this study, we characterised the S-genotypes of 25 sweet cherry cultivars, some of which had being bred at the Research and Breeding Institute of Pomology (RBIP), Holovousy, Czech Republic, and others were European cultivars in the RBIP collection. S-genotyping was carried out by polymerase chain reaction using consensus primers for the S-RNase and SFB genes, and capillary electrophoresis. Nine different known S-haplotypes (S1, S2, S3, S4, S5, S6, S9, S13, and S16) were identified and the cultivars were assigned to 12 incompatibility groups. One local cultivar, ‘Ptačka z Plzně’, originated from a wild forest seedling and used as a pollinator, was assigned to Group 0 of universal donors. The pedigree of some cultivars was confirmed by their S-genotype. This study represents the first comprehensive S-genotype screening of sweet cherry genetic resources in the Czech Republic and will be useful for the design of crossing programmes and orchard management of these sweet cherry cultivars.

Prevalence of human papillomavirus genotypes among women with cervical cancer in Ghana.

BackgroundHuman Papillomavirus (HPV) infections have been shown to be a necessary risk factor for the development of cervical cancer. However, HPV genotype distribution varies geographically, both in type and relative prevalence. In order to ensure a successful introduction of available vaccines, there is the need to identify pre-vaccination HPV genotype prevalence in Ghana and the extent of single and multiple-infections.MethodsParaffin-embedded cervical tissues of 256 confirmed cervical cancer cases diagnosed at the Korle-Bu Teaching Hospital during the period January 2004 to December 2006 were selected after hematoxylin and eosin staining and confirmation. Following a heat-proteinase K-based tissue lysis, HPV was detected and typed by a nested-multiplex PCR assay using an E6/E7 consensus primer and type-specific primers.ResultsOf the 256 cases, 230 (89.8 %, 95 % CI 85.7–93.4 %) were positive for HPV DNA. HPV18 (47.4 %), HPV59 (42.2 %), HPV45 (37.4 %) and HPV16 (9.0 %) were the four common HPV genotypes detected. A total of 110 (47.8 %) of the 230 HPV DNA positive tissues, were infected by a single HPV genotype while the other 120 (52.2 %) were infected by multiple HPV genotypes. A significant association was determined between each of the following HPV genotypes and multiple-infection; HPV18 (OR = 6.97; 95 % CI, 3.89–12.50), HPV59 (OR = 9.56; 95 % CI, 5.57–20.02) and HPV45 (OR = 1.94; 95 % CI, 1.12–3.35).ConclusionThe prevalence of the following high risk HPV genotypes (HPV18, HPV59, HPV45) were relatively high among the cases of cervical cancers reported at this hospital in Ghana during the study period. Additionally, there was a high frequency of HPV multiple-infections among these cases.

High Risk Human Papillomavirus Infection of the Foreskin in Asymptomatic Men and Patients with Phimosis

There has been increasing interest in understanding the natural history of HPV and the diseases that it causes in men. HPV infection is strongly associated with penile cancer, lack of neonatal circumcision and phimosis. We investigated the incidence of HPV infection in asymptomatic men and patients with phimosis. We assessed 110 asymptomatic men and 30 patients who underwent circumcision due to phimosis. DNA was extracted from swabbed samples collected from asymptomatic men and from foreskin samples collected at circumcision. Polymerase chain reaction using consensus primers for detecting HPV-MY09/11 was performed to detect generic HPV DNA. HPV genotyping was done by polymerase chain reaction amplification with primers for the E6 gene DNA sequences HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45 and HPV58. HPV was present in 46.66% of patients with phimosis, of whom 50% had high risk HPV genotypes. Of asymptomatic cases 16.36% were HPV positive but only 1 sample showed high risk HPV. We detected a significantly high rate of HPV genital infection in patients presenting with phimosis compared with asymptomatic men (p = 0.00167). The prevalence of high risk HPV genotypes in patients with phimosis was also statistically significant (p= 0.0004). We found a robust association between phimosis and the genital HPV prevalence in men and a significant frequency of high risk HPV. Other studies are needed to investigate the occurrence of factors that can increase the incidence of penile carcinoma and determine its impact on female genital infection in cervical cancer.

Prevalence of human papillomavirus (HPV), distribution of HPV types, and risk factors for infection in HPV-positive women.

The aim of this study was to describe the prevalence of human papillomavirus (HPV), the distribution of different HPV types, and the putative risk factors for infection among HPV-positive women from the State of Alagoas, Northeast Brazil. We analyzed data from 515 patients attending public and private health centers. HPV DNA from cervical samples was extracted and HPV genotyping was performed by polymerase chain reaction using MY09/11 consensus primers followed by direct sequencing. The chi-squared test for independence was used to assess statistical differences between the HPV groups. HPV DNA was found in 111 (21.55%) cervical samples. Twenty genotypes were detected: HPV6, 11, 16, 31, 33, 35, 39, 52, 53, 54, 58, 61, 62, 66, 70, 72, 81, 82, 83, and 84. In addition, multiple sexual partners (P = 0.002) and the use of oral contraceptives (P = 0.015) were associated with the presence of HPV. These findings may be relevant to the design of screening and vaccination strategies targeting specific groups of women in Northeast Brazil.

Family-specific vs. universal PCR primers for the study of mitochondrial DNA in plants

Mitochondrial genomes (mtDNAs or mitogenomes) of seed plants are characterized by a notoriously unstable organization on account of which available so-called universal or consensus primers may fail to fulfil their foreseen function - amplification of various mtDNA regions in a broad range of plant taxa. Thus, the primers developed for groups assumed to have similar organization of their mitogenomes, such as families, may facilitate a broader usage of more variable non-coding portions of these genomes in group members. Using in silico PCR method and six available complete mitogenomes of Fabaceae, it has been demonstrated that only three out of 36 published universal primer and three Medicago sativa-specific primer pairs that amplify various mtDNA regions are suitable for six representatives of the Fabaceae family upon minor modifications, and develop 21 Fabaceae-specific primer pairs for amplification of all 14 cis-splicing introns in genes of NADH subunits (nad genes) which represent the most commonly used non-coding mtDNA regions in various studies in plants. Using the same method and six available complete mitogenomes of representatives of related families Cucurbitaceae, Euphorbiaceae and Rosaceae and a model plant, Arabidopsis thaliana, it has further been demonstrated that applicability of newly developed primer pairs for amplification of nad introns in more or less related taxa was dependent not only on species evolutionary distances but also on their genome sizes. A reported set of 24 primer pairs is a valuable resource which may facilitate a broader usage of mtDNA variability in future studies at both intra- and inter-specific levels in Fabaceae, which is the third largest family of flowering plants rarely studied at the mtDNA level, and in other more or less related taxa.

Prevalence of human papillomavirus infection & cervical abnormalities in HIV-positive women in eastern India.

Background & objectives:India has the third highest burden of HIV and highest number of cervical cancer in the world. A cross-sectional study was performed to determine the prevalence and types of human papillomavirus (HPV) infection, and the factors associated with HPV infection and abnormal cervical cytology in HIV-positive women attending the Antiretroviral Therapy (ART) Centre in a tertiary care hospital in eastern India.Methods:We screened 216 HIV- positive women with Papanicolau smear cytology and HPV testing. HPV DNA was detected by using consensus primers followed by sequencing.Results:Of the 216 HIV-positive women screened, 58 (26.85%) were HPV-positive; 56 (25.9%) were of high-risk (HR) HPV type. The most prevalent HPV type was HPV-16 (7.9%); non 16 and 18 HPV types were present in 17.6 per cent patients. Age ≤ 35 yr [(OR), 2.56 (1.26-5.19)], illiteracy [OR, 2.30 (1.19-4.46)], rural residence [OR, 3.99 (1.27-12.56)] and CD4 ≤350/μl [OR, 2.46 (1.26-4.83)] were associated with increased risk of acquisition of HPV. One hundred thirty nine (74.33%) patients had normal/ negative for intraepithelial lesions (NILM) cytology, three (1.60%) had atypical squamous cells of undetermined significance (ASCUS), 32 (17.11%) had low-grade squamous intraepithelial lesions (LSIL), 10 (5.35%) had high-grade squamous intraepithelial lesions (HSIL) and three (1.60%) had carcinoma cervix. WHO clinical Stage III and IV [OR, 2.83 (1.07-7.49)] and CD4 ≤350/μl [OR, 2.84 (1.30-6.20)] were risk factors for abnormal cytology.Interpretation &conclusions:Our study showed 26.85 per cent HPV positivity in HIV infected women in this region, with HPV-16 as the commonest genotype. Abnormal cervical cytology was seen in about 25 per cent women. Regular Pap smear screening as recommended by the National AIDS Control Organization will help in early detection of cervical abnormalities in HIV- positive women.

Real-time quantitative polymerase chain reaction detection of minimal residual disease in acute lymphoblastic leukemia: a single-center experience

Background Monitoring of minimal residual disease (MRD) has become a frontline clinical practice in the treatment of virtually all childhood acute lymphoblastic leukemia (ALL) cases and in many cases of adult patients with ALL. The MRD diagnostics has proven to be the strongest prognostic factor allowing for risk group assignment into different treatment arms. The MRD techniques need to be sensitive (≤10–4), which means, the ability to detect one malignant cell among 10 000 normal cells; broadly applicable; accurate; reliable; fast; and affordable. Aim The objective of this study is to evaluate the analysis of immunoglobulin heavy chain (IGH) or T-cell receptor (TCR) gene rearrangements as targets for MRD assessment in ALL, allowing early detection of relapsed cases, compare with the results of morphological evaluation of the same cases and to risk stratify patients with ALL according to the MRD assessment as a prognostic marker independent and superior to other conventional risk factors. Patients and methods Overall, 30 patients (15 males and 15 females) with age ranged from 1 to 25 years old were included in this study. Patients were subjected to full medical history, clinical examination, laboratory examinations such as complete blood count, bone marrow aspirate smear examination, cytochemistry, and immunophenotyping. The molecular studies done by real-time PCR were performed using consensus primers and allele-specific primers for (IGH) or (TCR) gene rearrangements as targets to detect MRD. The ALL cases were assessed by real-time quantitative PCR at the time of diagnosis and at the end of induction chemotherapy, and comparative cycle threshold (Ct) relative quantification method was used for quantitative gene expression. Results The results showed discrepancy between the morphologic examination for ALL assessment depending on the 5% blast index as an indicator of remission or nonremission; morphological nonremissions (>5% blast) were detected in eight (26.7%) cases whereas 22 (73.3%) cases showed morphological remissions ( Conclusion Approximately 81% of ALL cases previously diagnosed as being in the remission state depending on the morphologic assessment only tend to harbor MRD as evident by positive MRD assessment using real-time PCR. The molecular assessment of MRD allows early detection of relapse with chance of intervention and tailoring of treatment according to the patients' need. It was found that there was no relation between MRD results and risk stratification in ALL according to age and white blood cell at diagnosis; this confirms that detection of MRD of leukemic cells can be considered a superior prognostic marker of relapse, independent from conventional prognostic factors. The risk classification in ALL based on MRD results could be used to guide the final treatment strategy and predict early relapse; this should allow prediction of response and relapse while patients are still in the state of clinical remission and morphological remission too.

Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor.

The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5+/6+) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients.

Combined SYBR Green real-time polymerase chain reaction and microarray method for the simultaneous determination of human papillomavirus loads and genotypes

ObjectiveThe aim of this study was to describe the principle of the Cheil HPV DNA Chip assay and evaluate its accuracy. In order to quantify the human papillomavirus (HPV) load and identify HPV genotypes simultaneously, this assay combined the two methods: SYBR Green quantitative real-time polymerase chain reaction (PCR) and DNA microarray.MethodsWe designed novel consensus primer sets that target the conserved region of the HPV L1 gene for quantifying and detecting a broad range of HPV types by quantitative real-time PCR. Subsequently, using the PCR products, DNA microarray was performed with 36 HPV type-specific probes. To validate this method, direct sequencing and correlation analysis among HPV genotype, viral load, and cytological abnormality was performed by Cohen’s kappa values, two-sided McNemar chi-square test, Kruskal-Wallis test, and odds ratios.ResultsThe kappa value of the Cheil HPV DNA Chip was 0.963 (95% confidence interval, 0.919 to 0.98), which was significantly higher than the value of 0.527 (95% confidence interval, 0.447 to 0.59) obtained using a conventional HPV DNA Chip. HPV16 (χ2=62.28, P<0.01), HPV33 (χ2=7.18, P<0.01), and HPV58 (χ2=9.52, P<0.01), which are classified as high-risk HPVs, were detected at significant levels in samples with high-grade lesions. And viral loads tended to be higher in groups with high odds ratios.ConclusionThe Cheil HPV DNA Chip is an effective diagnostic assay for simultaneously detecting HPV genotypes and loads in cervical samples.

Prevalence of genital human papillomavirus among rural and urban populations in southern Yunnan province, China.

This study was designed to investigate and compare the HPV prevalence, genotypes distribution and associated risk factors in rural and urban women living in Xishuang Banna district, in the province of Yunnan. A total of 177 and 190 women from rural and urban areas were engaged, respectively. HPV DNA was amplified using the L1 consensus primers system (MY09/11 and GP5/6) and HPV GenoArray test was conducted for genotyping. Proportions were compared by chi-square test, and logistic regression was used to evaluate risk factors. A total of 54 women were positive for HPV DNA. Among rural women, 23 women were positive for HPV infection, of which 21 showed a single infection and 2 had a multiple infection. HPV-16 (10/23) was the most prevalent genotype followed by HPV-52 (5/23), and HPV-58 (5/23). Urban women had a higher infection rate for overall HPV (31/54) and for multiple genotype infection (8/31). HPV-52 (9/31) was the most prevalent genotype followed by HPV-39 (7/31) and HPV-68 (5/31). The age-specific HPV prevalence was also different between rural and urban women. In urban area, women with age <35 years had the highest HPV prevalence, which declined thereafter as age advanced. However, in rural women the highest HPV prevalence was observed in an older age group (>56 years). Ethnicity, smoking and parity were significantly associated with HPV infection among urban women. Our study demonstrates that HPV prevalence and genotype distribution varies among women from rural and urban areas in the south of Yunnan.

Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer.

Background:Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs.Objectives:The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay.Patients and Methods:Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails.Results:Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%.Conclusions:The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner.

Infection with multiple types of human papillomavirus in sexually active young women

Study design: cross. Study objective: estimate the frequency and distribution types of human papillomavirus (HPV) in sexually active young women. Methods: samples of vaginal and endocervical canal of 158 women, aged between 18 and 35 years were collected through self-collection technique. The HPV DNA was extracted and amplified by PCR end point using the consensus primers PGMY09/11 and genotyping was performed by PCR specific type (TS-PCR) and by analysis of the fragments obtained from the use of restriction enzymes (RFLP). Results: HPV DNA was detected in 23% of participants, 92% were high-risk HPV genotypes (HR), being the prevalent HPV45, followed by HPV16 and 31 infections with multiple HPV types were detected in 35% of samples, indicating infection by more than two types in 22.2% of the genotyped. Higher frequency of HPV DNA was detected in women ≤ 25 old. Conclusion: this demonstrated a high frequency of HPV infection in sexually active young women, as well as a high prevalence of infection with multiple types of high oncogenic risk