Successful term pregnancy following MR-guided focused ultrasound treatment of uterine leiomyoma.

OBJECTIVE: Term vaginal delivery after magnetic resonance-guided focused ultrasound therapy (MRgFU) for symptomatic uterine leiomyoma. STUDY DESIGN: A 38-year-old nulligravida underwent MRgFU treatment per study protocol for a solitary 9 x 10 x 10 cm uterine myoma and conceived 18 months following the procedure. She was counseled on the unknown implications of MRgFU during subsequent pregnancy. Myoma size increased significantly during gestation. At 39 weeks, she underwent indicated labor induction with vacuum-assisted vaginal delivery of a healthy male infant. CONCLUSION: In one pregnancy following MRgFU, there were no associated antepartum or intrapartum obstetrical complications.

MR guided focused ultrasound: technical acceptance measures for a clinical system.

Magnetic resonance (MR) guided focused ultrasound (MRgFUS) is a hybrid technique which offers efficient and safe focused ultrasound (FUS) treatments of uterine fibroids under MR guidance and monitoring. As a therapy device, MRgFUS requires systematic testing over a wide range of operational parameters prior to use in the clinical environment. We present technical acceptance tests and data for the first clinical MRgFUS system, ExAblate 2000 (InSightec Inc., Haifa, Israel), that has been FDA approved for treating uterine fibroids. These tests characterize MRgFUS by employing MR temperature measurements in tissue mimicking phantoms. The coronal scan plane is empirically demonstrated to be most reliable for measuring temperature elevations resulting from high intensity ultrasound (US) pulses ('sonications') and shows high sensitivity to changes in sonication parameters. Temperatures measured in the coronal plane were used as a measure of US energy deposited within the focal spot for a range of sonication parameters used in clinical treatments: spot type, spot length, output power, sonication duration, US frequency, and depth of sonication. In addition, MR images acquired during sonications were used to measure effective diameters and lengths of available sonication spot types and lengths. At a constant 60 W output power, the effective spot type diameters were measured to vary between 4.7 +/- 0.3 mm and 6.6 +/- 0.4 mm; treatment temperatures were found to decrease with increasing spot diameter. Prescribing different spot lengths was found to have no effect on the measured length or on measured temperatures. Tests of MRgFUS positioning accuracy determined errors in the direction parallel to the propagation of the US beam to be significantly greater than those in the perpendicular direction; most sonication spots were erroneously positioned towards the FUS transducer. The tests reported here have been demonstrated to be sufficiently sensitive to detect water leakage inside the FUS transducer. The data presented could be used for comparison by those conducting acceptance tests on other clinical MRgFUS systems.

Integrations of the hepatitis B virus (HBV) and human papillomavirus (HPV) into the human telomerase reverse transcriptase (hTERT) gene in liver and cervical cancers.

Chronic infections with the hepatitis B virus (HBV) and high-risk human papillomaviruses (HPVs) are important risk factors for hepatocellular carcinoma (HCC) and cervical cancer (CC), respectively. HBV and HPV are DNA viruses that almost invariably integrate into the host genome in invasive tumors. The viral integration sites occur throughout the genome, leading to the presumption that there are no preferred sites of integration. A number of viral integrations have been shown to occur within the vicinity of important cancer-related genes. In studies of HBV-induced HCC and HPV-induced CC, we have identified two HBV and three HPV integrations into the human telomerase reverse transcriptase (hTERT) gene. Detailed characterization of the integrations revealed that four integrations occurred within the hTERT promoter and upstream region and the fifth integration occurred in intron 3 of the hTERT gene. None of the integrations altered the hTERT coding sequence and all resulted in juxtaposition of viral enhancers near hTERT, with potential activation of hTERT expression. Our work supports the hypothesis that the sites of oncogenic viral integration are nonrandom and that genes at the sites of viral integration may play important roles in carcinogenesis.

Epstein-Barr virus DNA is not increased in tonsillar carcinoma.

Epstein-Barr virus (EBV) is a known oncogenic virus associated with a wide variety of cancers, including nasopharyngeal carcinoma. Waldeyer's ring, a collection of lymphoid tissues, includes the nasopharynx, pharyngeal, and lingual tonsils. To determine if EBV plays a causative role in carcinomas arising from other tissues in Waldeyer's ring, we examined pharyngeal tonsillar carcinomas for evidence of EBV infection. As previously reported, DNA was extracted from 53 consecutive tonsil cancers, as well as from age- and gender-matched non-cancerous tonsillectomy specimens. Three different sets of primers for discrete exons of EBV were then used to determine if active or latent EBV infection was expressed in the extracted DNA using the polymerase chain reaction (PCR). All positive bands were then sequenced to confirm the presence of amplified EBV fragments. None of the samples showed evidence for active EBV infection. In primers demonstrating latent infection, 1 of 53 (1.9%) of tumors were positive, versus 6 of 53 (11.3%) of the controls. These results indicate that EBV expression is not increased in DNA from tonsil cancers and that EBV infection does not have a causal relationship with tonsil cancer.

Human papillomavirus in cutaneous squamous cell carcinoma and cervix of a patient with psoriasis and extensive ultraviolet radiation exposure.

"High-risk" human papillomaviruses (HPVs) are associated with intraepithelial neoplasia and cancer of the uterine cervix. HPV has also been found in nonmelanoma skin cancer (NMSC), especially in squamous cell carcinomas (SCCs) of immunosuppressed patients. Recently, lesions of psoriasis have been shown to harbor HPV, and patients with psoriasis often have a history of extensive therapy with ultraviolet radiation (UVR). UVR is the major known risk factor in the occurrence of NMSC, in which HPV may be a cofactor for SCC. We report an otherwise healthy, nonimmunosuppressed patient with psoriasis who had a history of extensive exposure to UVR and experienced multiple SCCs on UV-exposed body sites. By the polymerase chain reaction method, we detected HPV in 5 of 9 SCCs. Automated sequencing showed HPV types 12 and 17. Only 1 of 3 normal skin specimens was HPV positive (HPV type 17). This positive specimen was from UV-exposed skin; one of the two HPV-negative, normal skin specimens was located on a body site not exposed to sun. In addition, HPV type 62 was found in a brush specimen of the uterine cervix. This case report suggests an association between psoriasis, HPV infection, and UVR exposure, in onset of SCC.

Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at the cytogenetic level. To explore this relationship at the molecular level, we used a PCR-based method to rapidly isolate cellular sequences flanking the sites of HPV16 integrations in primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on metaphases derived from cells cultured in the presence of aphidicolin. Our data demonstrate that HPV16 integrations in cervical tumors frequently occur within CFSs at the molecular level. In addition, we have determined the precise molecular locations of the CFSs FRA6C and FRA17B.

Differential distribution of sequence variations in HPV-16 E6.

OBJECTIVE: The E6 regions of the oncogenic human papillomaviruses (HPVs) are important in carcinogenesis and immune recognition. We examined the E6 DNA sequence from HPV-16-associated cervical cancers to determine the frequency and degree of variation from the consensus sequence in selected populations. METHODS: Samples positive for HPV-16 were analyzed using polymerase chain reaction followed by automated DNA sequencing: 62 from U.S. women, 20 each from Italian and Indian women, and 21 from Thai women. RESULTS: Of 151 codons, 18 contained 24 base substitutions, reflecting the overall conserved nature of this region. The HPV-16 E6 region from U. S. women showed considerably more sequence variation than that from European and Asian women. Five patterns common to U.S. and European and Asian samples accounted for 78% of all tumor-associated viruses. The E6 regions known to be involved in p53 binding and degradation are involved with a surprising degree of sequence variation, whereas the carboxy end of the molecule is highly conserved. CONCLUSIONS: The area of greatest sequence variation includes a proposed human leukocyte antigen interaction site. A novel large deletion in one sample results in loss of all functional regions of E6. These findings were analyzed for possible significance with regard to immune selection and functional importance of the carboxy end of the E6 protein.

Large plantar wart caused by human papillomavirus-66 and resolution by topical cidofovir therapy.

Warts can be difficult to diagnose and to treat in the setting of human immunodeficiency virus (HIV) infection. A 37-year-old woman with a background of HIV presented with a large verrucous plaque involving her right foot. Human papillomavirus (HPV)-66 was identified in the lesional skin biopsy sample and in scrapings obtained from her cervix. The wart rapidly responded to topical cidofovir therapy. HPV-66 is a novel HPV type to be associated with verruca vulgaris. Topical cidofovir should be further investigated as an alternative treatment modality for verruca vulgaris.

Cervical cancer in older women: a molecular analysis of human papillomavirus types, HLA types, and p53 mutations.

OBJECTIVE: The purpose of this study was to evaluate cervical cancers in older women to determine whether they differed from tumors in younger women with respect to human papillomavirus types, frequencies of p53 mutations, and presence of a proposed high-risk HLA-DR2 haplotype. STUDY DESIGN: Cervical tissue was obtained from women undergoing surgical treatment of in situ or invasive carcinoma of the cervix. Viral and genomic deoxyribonucleic acid was extracted. The presence of human papillomavirus deoxyribonucleic acid was detected by polymerase chain reaction amplification. Viral subtypes were assigned by means of a combination of type-specific amplification and automated sequencing of the L1 region. The presence of p53 mutations was evaluated by direct sequencing of exons 5 through 9. The HLA-DR locus was screened for the presence of the high-risk DRB1*1501 allele by means of selective polymerase chain reaction amplification followed by agarose gel electrophoresis of HLA-DR2 types. RESULTS: Tumors from 39 women 62 to 85 years old were analyzed. Tumors from 104 younger women formed a reference group. Human papillomavirus 16 was found in 41% and 54% and human papillomavirus 18 was found in 10% and 12% of the tissue samples from older and younger women, respectively. The overall distributions of human papillomavirus types did not differ statistically between the groups. One of the 25 older patients tested had a p53 mutation. This tumor also had a positive test result for human papillomavirus 18. The DR*1501 allele was present in 33% of the older patients and 28% of the younger patients. The expected frequency of this allele in white Americans is 19.8%. The increased frequency of this allele among both older and younger women with cervical cancer was statistically significant (P < .05). CONCLUSIONS: We hypothesized that cervical cancer in older women might differ from that in younger women with respect to human papillomavirus types, natural host immunity, or the frequency of nonviral origins of the cancer. The findings show, however, that tumors from older women are extremely similar to those from younger women with respect to the human papillomavirus types present and the infrequent occurrence of p53 mutations. In addition we found that an HLA-DR allele that is associated with a risk of cervical cancer in younger women is also associated with risk in older women. These findings are most consistent with a model similar to that in younger women but with an unusually long latency for the transforming effect of the virus in some hosts.

Nonrandom patterns of simple and cryptic triplet repeats in coding and noncoding sequences.

Triplet repeats of the sequence purine, purine, and pyrimidine [RRY(i)] are frequent and often polymorphic in humans. Some RRY(i) are composed predominantly of a continuous repeat of one sequence [simple RRY(i)], but the majority are cryptic RRY(i) that are not obvious until the bases are classified into R or Y before the full extent of the repeat becomes apparent. RRY(i) can be divided into 18 classes based on predominant nucleotides. These classes are highly nonrandom in abundance and in location within genes. In humans, simple or cryptic RRY(i), in which AAT or AAC triplets predominate, are preferentially located 3' of Alu repeats. RRY(i) with a predominance of AGC or GGC show a dramatic enrichment in coding sequence, and GGC also shows a dramatic enrichment in 5' untranslated regions of genes. Characterization of RRY(i) present in coding regions identify 10 protein motifs (An, Dn, Hn, Pn, Qn, Tn, GnS0-3Gm, (G/S)n, (S/G/N)n, and (L/P)n). Six of the protein motifs appear predominantly in DNA-binding proteins/transcription factors. Alignment of homologous protein sequences from other mammals reveals that both simple and cryptic RRY(i) are a major source of deletions or insertions in the genes that contain them. Cryptic RRY(i) may be candidates for triplet repeat genetic diseases and, when mutated in somatic cells, may contribute to carcinogenesis.

The pattern of spontaneous germ-line mutation: relative rates of mutation at or near CpG dinucleotides in the factor IX gene.

Mutations at CpG dinucleotides were delineated in the factor IX gene of 38 hemophilia B patients. When transitions at CpG were considered with those previously reported by us and those compiled in the factor IX mutation database, the following patterns emerged. Many CpG sites were mutated with high frequency, while two CpG sites were infrequently mutated (R29-->Q and R116-->TGA). Of the 6 possible nonsense mutations and the 14 missense mutations that would produce a nonconservative change at conserved amino acids, all have been observed to cause hemophilia B except A-10-->T and R338-->Q. By contrast, none of the 6 missense changes at nonconserved amino acids have been observed to cause hemophilia B. At those CpG sites that are frequently mutated, the rate of transitions is estimated to be 20-fold higher than transitions at non-CpG sites. Point mutations in close proximity to CpG dinucleotides did not seem elevated.

"Cryptic" repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: analysis of coding cRRY(i) in the proopiomelanocortin (POMC) and TATA-binding protein (TBP) genes.

Triplets of the form of purine, purine, pyrimidine (RRY(i)) are enhanced in frequency in the genomes of primates, rodents, and bacteria. Some RRY(i) are "cryptic" repeats (cRRY(i)) in which no one tandem run of a trinucleotide predominates. A search of human GenBank sequence revealed that the sequences of cRRY(i) are highly nonrandom. Three randomly chosen human cRRY(i) were sequenced in search of polymorphic alleles. Multiple polymorphic alleles were found in cRRY(i) in the coding regions of the genes for proopiomelanocortin (POMC) and TATA-binding protein (TBP). The highly polymorphic TBP cRRY(i) was characterized in detail. Direct sequencing of 157 unrelated human alleles demonstrated the presence of 20 different alleles which resulted in 29-40 consecutive glutamines in the amino-terminal region of TBP. These alleles are differentially distributed among the races. PCR was used to screen 1,846 additional alleles in order to characterize more fully the range of variation in the population. Three additional alleles were discovered, but there was no example of a substantial sequence amplification as is seen in the repeat sequences associated with X-linked spinal and bulbar muscular atrophy, myotonic dystrophy, or the fragile-X syndrome. The structure of the TBP cRRY(i) is conserved in the five monkey species examined. In the chimpanzee, examination of four individuals revealed that the cRRY(i) was highly polymorphic, but the pattern of polymorphism differed from that in humans. The TBP cRRY(i) displays both similarities with and differences from the previously described RRY(i) in the coding sequence of the androgen receptor. Our data suggest how simple tandem repeats could evolve from cryptic repeats.

Germline mutations in the factor IX gene: a comparison of the pattern in Caucasians and non-Caucasians.

DNA samples of patients from around the world have been sequenced to precisely define the mutations in the factor IX gene resulting in hemophilia B. This study compares the patterns of independent mutation between 127 Caucasian and 44 non-Caucasian patients with hemophilia B. Caucasians and non-Caucasians are found to have similar patterns of transitions and transversions (both at CpG and at non-CpG sites) as well as insertions, deletions, microdeletions, and complex changes (chi 2 = 2.71, p = 0.922). An analysis of subgroups of transitions and transversions shows similar patterns among Caucasians and non-Caucasians (chi 2 = 2.98, p = 0.83). If the subset of non-Caucasian samples (24) in which the mutation is known to have occurred outside of the United States, Canada, and Europe (UCE) is compared to the Caucasian subset, the above conclusions are unchanged. The invariant nature of this pattern of mutation is most simply compatible with a predominance of endogenous processes or common mutagen exposure rather than mutagen exposure specifically associated with non-Caucasian status or non-Western lifestyle.