A study of genotyping for management of human papillomavirus-positive, cytology-negative cervical screening results.

The effective management of women with human papillomavirus (HPV)-positive, cytology-negative results is critical to the introduction of HPV testing into cervical screening. HPV typing has been recommended for colposcopy triage, but it is not clear which combinations of high-risk HPV types provide clinically useful information. This study included 18,810 women with Hybrid Capture 2 (HC2)-positive, cytology-negative results and who were age ≥30 years from Kaiser Permanente Northern California. The median follow-up was 475 days (interquartile range [IQR], 0 to 1,077 days; maximum, 2,217 days). The baseline specimens from 482 cases of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) and 3,517 random HC2-positive noncases were genotyped using 2 PCR-based methods. Using the case-control sampling fractions, the 3-year cumulative risks of CIN3+ were calculated for each individual high-risk HPV type. The 3-year cumulative risk of CIN3+ among all women with HC2-positive, cytology-negative results was 4.6%. HPV16 status conferred the greatest type-specific risk stratification; women with HC2-positive/HPV16-positive results had a 10.6% risk of CIN3+, while women with HC-2 positive/HPV16-negative results had a much lower risk of 2.4%. The next most informative HPV types and their risks in HPV-positive women were HPV33 (5.9%) and HPV18 (5.9%). With regard to the etiologic fraction, 20 of 71 cases of cervical adenocarcinoma in situ (AIS) and adenocarcinoma in the cohort were positive for HPV18. HPV16 genotyping provides risk stratification useful for guiding clinical management; the risk among HPV16-positive women clearly exceeds the U.S. consensus risk threshold for immediate colposcopy referral. HPV18 is of particular interest because of its association with difficult-to-detect glandular lesions. There is a less clear clinical value of distinguishing the other high-risk HPV types.

Human leukocyte antigen class I and II alleles and risk of cervical neoplasia: results from a population-based study in Costa Rica.

To examine human leukocyte antigen (HLA) involvement in the development of all grades of cervical neoplasia, a nested case-control study of 10,077 women in Guanacaste, Costa Rica, was conducted. Participants had invasive cervical cancer, high-grade squamous intraepithelial lesions (HSILs; n=166), or low-grade squamous intraepithelial lesions (LSILs); were positive for human papillomavirus (HPV) with no evidence of cervical neoplasia (n=320); or were HPV negative with no evidence of cervical neoplasia but with a history of high-risk sexual behavior (n=173). Compared with women who were HPV negative, women with HLA-DRB1*1301 were associated with decreased risk for cancer/HSILs (odds ratio [OR], 0.4; 95% confidence interval [CI], 0.2-0.7) and for LSILs/HPV (OR, 0.6; 95% CI, 0.3-0.9). Women with both HLA-B*07 and HLA-DQB1*0302 had an 8.2-fold increased risk for cancer/HSILs (95% CI, 1.8-37.2) and a 5.3-fold increased risk for LSILs/HPV (95% CI, 1.2-23.7). These results support the hypothesis that multiple risk alleles are needed in order to increase risk for cervical neoplasia, but a single protective allele may be sufficient for protection.

HLA class II DR-DQ and increased risk of cervical cancer among Senegalese women.

To examine Senegalese women to confirm and extend associations between HLA class II types and cervical cancer previously observed among African-American, Caucasian, Hispanic, and Japanese ethnic populations, 55 Senegalese women with invasive cervical carcinoma were compared with age-matched (human papillomavirus) HPV-positive (n = 83) and HPV-negative (n = 107) control women. PCR-based HPV and HLA typing methods were used. Data were analyzed using a global randomization test and conditional logistic regression. Although this study failed to confirm a previously reported association between cervical cancer and DQB1*03 alleles, the DRB1*1101-DQB1*0301 haplotype was detected more frequently among cervical carcinoma cases than among controls (adjusted odds ratio, 2.6; 95% confidence interval, 1.0-7.1). Furthermore, as reported by others, we observed a negative association of borderline statistical significance between DRB1*13 and cervical carcinoma (adjusted odds ratio, 0.5; 95% confidence interval, 0.2-1.1). Observations from this study confirm earlier findings of a negative association between DRB1*13 and cervical cancer and suggest that specific DRB1-DQB1 haplotype combinations, rather than individual DQB1*03 alleles, increase the risk for cervical cancer.

Determinants of genital human papillomavirus detection in a US population.

This study investigated the association of selected demographic and behavioral characteristics with the detection of low-risk, high-risk, and uncharacterized genital human papillomavirus (HPV) in women attending clinic for routine nonreferral gynecologic health care. Cervical specimens obtained from 3863 women 18-40 years old (mean, 28 years) with no history of high-grade cervical disease were analyzed for 38 HPV types. Overall, HPV prevalence was 39.2%. The prevalence of high-risk, low-risk, and uncharacterized HPV types was 26.7%, 14.7%, and 13.0%, respectively. As expected, the characteristics most strongly associated with overall HPV detection were age and numbers of lifetime and recent sex partners. Low-risk, high-risk, and uncharacterized HPV detection increased with increasing numbers of sex partners. There was a decline in high-risk and low-risk HPV detection with increasing age but little change in uncharacterized HPV detection. These results suggest that the uncharacterized HPV types have a different natural history than either low-risk or high-risk HPV types.

Improved amplification of genital human papillomaviruses.

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

Familial and sporadic cases of nasopharyngeal carcinoma in Taiwan.

BACKGROUND: Nasopharyngeal carcinoma (NPC) has a striking geographic/ethnic distribution, with especially high rates among southern Chinese. Previous studies have indicated that a family history of NPC is associated with increased risk and noted familial clustering in low-risk populations. MATERIALS AND METHODS: We investigated differences between sporadic and familial cases of NPC in a case-control study of 375 histologically confirmed NPC cases (99% response rate) and 328 age-, sex-, and geographically-matched controls (88% response rate). All participants answered a detailed risk factor interview and donated blood for EBV and CYP 2E1 testing. RESULTS: Subjects with a first degree relative with NPC had on odds ratio (OR) of 7.6 (95% confidence interval (CI) = 2.3-25), while those with a family history of any other cancer had only a slightly elevated risk of disease (OR = 1.4; 95% CI = .93-2.2). Of the cases, 25 (6.7%) were familial--having at least one first degree relative with NPC. No significant difference was seen between familial and sporadic cases with respect to sex, age, ethnicity, histology or stage. There was a nonsignificant (p = 0.16) increase in T1N2 tumors among familial cases, suggesting a more aggressive tumor. Family history of other cancers, EBV serologies, or the distribution of the RsaI c2 form of the allele of cytochrome P450 2E1 were also not significantly different between the two groups. CONCLUSIONS: In conclusion, while genetic factors are likely to play an important role in NPC pathogenesis, our results provide little evidence that a familial form of NPC exists with characteristics notably distinct from sporadic cases.

Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.

Comparison of human leukocyte antigen DR-DQ disease associations found with cervical dysplasia and invasive cervical carcinoma.

BACKGROUND: Human leukocyte antigen (HLA) molecules, whose biological role is in the regulation of immune responses to foreign antigens and in discrimination of self from non-self antigens, are encoded by a series of closely linked genetic loci found on chromosome 6. Although the evidence for a link between HLA and cervical cancer has been controversial, it has been recently reported that certain HLA class II haplotypes (linked class II alleles) are positively associated with invasive cervical cancer, while other class II haplotypes are negatively associated or protective. Since HLA associations between human papillomavirus type 16 (HPV16)-mediated cancer cases and non-HPV16-mediated cancer cases have been found to be different, this suggests that specific HLA class II haplotypes may influence the immune response to HPV infection and may affect the risk of acquiring invasive cervical carcinoma. PURPOSE: This study was conducted to determine if the same HLA class II haplotypes that are associated with invasive cervical carcinoma are also associated with cervical dysplasia (presumed precursors of invasive cervical cancer). METHODS: We have examined HLA DR-DQ haplotypes among 128 Hispanic women from New Mexico with biopsy-confirmed cervical dysplasia in a case-control study using sensitive DNA-based polymerase chain reaction amplification and sequence-specific oligonucleotide probe hybridization methods to detect the presence and type of HPV and to detect allelic polymorphism in the HLA DRB1 and DQB1 loci. RESULTS: Dysplasia cases were divided into two groups for comparison to controls: severe dysplasia/carcinoma in situ (CIS), and slight/moderate dysplasia. The frequency distribution of HLA class II haplotypes among the HPV16-positive severe dysplasia/CIS cases had a statistically significant (two-tailed P < .005) difference compared with controls, whereas haplotypes among the severe dysplasia/CIS cases containing HPV types other than HPV16 did not show statistically significant frequency differences. DR-DQ haplotypes previously found to be associated with HPV16-invasive cervical carcinomas were also associated with HPV16-positive severe dysplasia/CIS. However, no statistically significant haplotype frequency difference was observed between slight/moderate dysplasia cases and controls. In addition, we noted a DQA1-DQB1 haplotype negatively associated with severe dysplasia/CIS but not with invasive cervical cancers. CONCLUSIONS: Our results strongly suggest that certain HLA haplotypes confer an increased risk for severe cervical dysplasia and invasive cervical carcinoma following HPV16 infection. IMPLICATIONS: Further molecular studies are needed to identify HLA alleles or haplotypes that may provide increased susceptibility to HPV-associated cervical disease.

HLA DR-DQ associations with cervical carcinoma show papillomavirus-type specificity.

Cervical carcinoma is now known to be associated with human papillomaviruses (HPV), but the evidence for a link with specific HLA loci is controversial. The role of genetic variation at the HLA class II loci and among HPV types in cervical carcinoma was investigated by PCR DNA amplification and oligonucleotide probe typing of paraffin-embedded invasive cervical cancer tissue from Hispanic patients and of cervical swabs from Hispanic controls. Certain HLA class II haplotypes (such as DRB1*1501-DQB1*0602) were associated significantly, while DR13 haplotypes were negatively associated with cervical carcinoma. These associations are HPV16-type specific. These results suggest that specific HLA class II haplotypes may influence the immune response to specific HPV-encoded epitopes and affect the risk of cervical neoplasia.

Limitations in plasticity of the T-cell receptor repertoire.

How constrained is T-cell recognition? Is a truncated T-cell receptor (TCR) repertoire, missing half of its V beta components (where V indicates variable), still broad enough to produce an antigen-specific T-cell response to all determinants? These questions can be answered for certain T-cell antigenic determinants whose response in the wild type is limited to specific gene segments. Our results show that mice with such a deletion in their TCR V beta genes (V beta truncated haplotype, Va beta) are unable to respond to two antigen determinants (sperm whale myoglobin 111-121/I-Ed and myelin basic protein 1-11/I-Au) whose response in the wild type is restricted to the missing V beta (V beta 8.2 in the case of 111-121/I-Ed and V beta 8.2 and V beta 13 in the case of 1-11/I-Au) gene segments. Fundamentally, this restriction could have been attributed to another aspect of immunodominance--that a favored TCR with high affinity would dominate the response, but in its absence, a hierarchy of T cells with lesser efficiency and expressing alternate TCR V genes could take over. However, from our experiments it has become evident that there is an absolute limit to the flexibility inherent in the TCR repertoire. Since it is clear that mouse populations have many ambient deletion ligands (such as self-superantigens) that can result in the loss of multiple V beta gene segments during normal T-cell development, these deletions can have serious consequences, such as unresponsiveness to the antigen as a whole--a hole in the repertoire--if a dominant determinant of that antigen normally shows restricted TCR V beta gene usage.

Investigation of a chronic feed-passage problem on a broiler farm in northwest Arkansas.

A commercial broiler farm with a history of poor feed conversion and chronic feed-passage problems was chosen for investigation. Chickens were taken from the broiler flock at specified intervals during growout and tested by virus isolation and enzyme-linked immunosorbent assay (ELISA) for avian reovirus. Abnormal tissue pathology was first seen in the broilers at 9 days of age and continued sporadically throughout the growout period. Antireovirus antibody levels began to increase at 24 days of age. Avian reovirus and avian adenovirus was recovered at different intervals starting at 17 and 31 days of age, respectively. One-day-old specific-pathogen-free chicks housed in filtered-air positive-pressure isolation units were inoculated with two inocula recovered from the field study. Avian reovirus was recovered from the tissues of both treatment groups using chick kidney cells. Significant weight differences were seen in one of the two treatment groups. This avian reovirus was given the name SS-412.

Morphometric and cytochemical analysis of lysosomes in rat Peyer's patch follicle epithelium: their reduction in volume fraction and acid phosphatase content in M cells compared to adjacent enterocytes.

M cells are specialized epithelial cells over lymphoid follicles in Peyer's patches which take up viruses, bacteria, and antigenic macromolecules from the intestinal lumen. Unlike ordinary enterocytes which sequester pinocytosed material in lysosomes, M cells transport such material across the epithelium to antigen-processing areas in lymphoid follicle domes, suggesting a difference in lysosomal activity or a different route for movement of endocytic vesicles. Ileal Peyer's patches in rats were examined by electron microscopy to identify lysosomes by acid phosphatase activity. Acid phosphatase was found in dense bodies in enterocytes but not in M cells. Stereological analysis showed the volume fraction occupied by dense bodies in M cells to be 16 times less than in enterocytes (P less than .0005), even though the volume fractions of cytoplasm occupied by mitochondria in M cells and enterocytes were not significantly different. The small volume fraction of dense bodies and the absence of acid phosphatase activity in M cells thus correlate with absence of lysosomal degradation of luminal microorganisms during transport into lymphoid follicles by M cells and may provide not only a complete array of microbial antigens for initiation of immune responses, but also a route through the mucosal barrier for microorganisms which can evade local containment mechanisms.

Separation and spectrophotometric determination of trace quantities of lithium in high-purity beryllium and beryllium oxide.

A spectrophotometric method is described for the determination of trace quantities of lithium in beryllium metal and its oxide. Lithium is selectively separated from beryllium by extraction from 1M potassium hydroxide solution into 0.1M dipivaloylmethane in diethyl ether. Fluoride, added before the extraction, successfully masks the beryllium; as little as 3 microg of lithium can be separated from as much as 1 g of beryllium. The lithium is then back-extracted into 0.1M hydrochloric acid and is determined spectrophotometrically with o-(2-hydroxy-3,6-disulpho-1-naphthylazo) benzene arsonic acid, Thoron. In an acetone-water medium Beer's law is obeyed over the range 0.1-1.0 microg ml. The method has been applied successfully to the determination of lithium in concentrations as low as 3 ppm; the relative standard deviation for the determination of 200 ppm is 3%.