Thermographic mapping of the skin surface in biometric evaluation of cellulite treatment effectiveness.

BACKGROUND: Cellulite is one of the worst tolerated aesthetic imperfections. Edema that accompanies cellulite causes disorders of blood flow what may be observed as changes in the skin surface temperature. The aim of this paper was to develop a new method based on the analysis and processing of thermal images of the skin for biometric evaluation of severity of cellulite and monitoring its treatment. METHODS: The observations of the treatment effects were conducted on 10 females (33.4 ± 6.4 years). Thermal images of the volunteers' thighs were captured before starting the therapy (T0 ). In the following stages: T1 , T2 , and T3 , thermal images were captured 2 weeks after the first, second and third Alidya treatment administration, respectively. Profiled algorithms were developed to determine the mean Grey Level Co-occurrence Matrix (GLCM) contrast in the acquired thermograms. RESULTS: The mean GLCM contrast for the phase T0 was 70.91, and for the stages T1 , T2 , and T3 : 57.78, 41.80, and 38.53, respectively. CONCLUSION: The use of proposed method (GLCM contrast) enables biometric evaluation of the effectiveness of cellulite treatment. Traditionally used parameters of infrared analysis such as local points of the maximum and minimum temperature or the median temperatures are not useful in thermal, biometric evaluation of anti-cellulite preparations.

A randomised phase II trial of selumetinib vs selumetinib plus temsirolimus for soft-tissue sarcomas.

BACKGROUND: The MEK inhibitor, selumetinib, suppresses soft-tissue sarcoma (STS) cell proliferation in vitro. Mammalian target of rapamycin inhibitors possess modest activity against STS; however, resistance develops via MAPK pathway feedback activation. The combination of selumetinib and temsirolimus synergistically inhibits STS cell line growth. Therefore, a randomized phase II trial of selumetinib vs selumetinib plus temsirolimus was conducted. METHODS: Seventy-one adults with advanced STS who received ⩽ 2 prior chemotherapeutics were randomized to selumetinib 75 mg p.o. bid and allowed to crossover upon progression, or to selumetinib 50 mg p.o. bid plus temsirolimus 20 mg i.v. weekly, with primary endpoint of progression-free survival (PFS). RESULTS: There was no difference in PFS between the two arms for the overall cohort (median 1.9 vs 2.1 months); an improved median PFS was observed in the combination arm (N = 11) over single agent (N = 10) in the prespecified leiomyosarcoma stratum (median 3.7 vs 1.8 months; P = 0.01). Four-month PFS rate was 50% (95% confidence interval 0.19-0.81) with the combination vs 0% with selumetinib alone in the leiomyosarcoma cohort. Most common grade 3/4 adverse events with the combination were mucositis (29%), lymphopenia (26%), neutropenia and anaemia (20% each). CONCLUSIONS: While single-agent selumetinib has no significant activity in STS, the combination may be active for leiomyosarcomas.

Genomic Analyses of Germline and Somatic Variation in High-Grade Serous Ovarian Cancer.

Background High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 577 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations. Results Approximately one-third of tumors had loss-of-function germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM , and PALB2 . Loss-of-function germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP , and NF1 . In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of loss-of-function variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536 , and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. Conclusions From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 577 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Genomic analyses of germline and somatic variation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 557 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations (SCNA). RESULTS: Approximately one-third of tumors had loss-of-function (LOF) germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM, and PALB2. LOF germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP, and NF1. In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of LOF variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536, and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. CONCLUSIONS: From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 557 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Human papillomavirus type 18: association with poor prognosis in early stage cervical cancer.

BACKGROUND: Cervical carcinoma is a leading cause of mortality from cancer among women worldwide, accounting for approximately 160,000 deaths annually. Prognosis in patients with this disease is dependent on several well-established clinical features (stage of disease and age of patient) and pathologic features (lymph node status, grade of tumor, and depth of invasion). Although the features associated with poor clinical outcome have been well studied, molecular markers such as human papillomavirus (HPV) type that may reflect the underlying biologic basis for clinical behavior are poorly understood. PURPOSE: To test the hypothesis that differences in survival among patients with cervical carcinoma are associated with HPV DNA type, we conducted a historical cohort study of patients treated at our institutions over a 10-year period. METHODS: Fresh primary tumor tissue samples from 291 women with all stages of cervical carcinoma diagnosed from April 1983 through August 1993 were rapidly frozen and stored at -70 degrees C until analysis. High-molecular-weight DNA was extracted and purified by homogenization, proteinase K digestion, phenol extraction, ammonium acetate salt displacement, ethanol precipitation, and ribonuclease treatment. HPV nucleotide sequences were amplified from tumor DNA samples by polymerase chain reaction with the use of both consensus L1 (MY09/MY11) primers that recognize more than 25 HPV types and modifications of type-specific primers developed for HPV types 16, 18, and 6. Clinical data were abstracted from hospital, office, and tumor registry records. Univariate analysis was conducted using Student's t test and chi-squared tests. Survival curves were estimated by use of the Kaplan-Meier method; differences between groups were examined by the logrank test. Multivariate survival analysis was performed according to the Cox proportional hazards model. RESULTS: HPV DNA was detected in 247 (85%) of 291 tumors: HPV16 in 52%, HPV18 in 20%, other HPV types in 13%, and no HPV DNA in 15%. Eighty-eight percent of squamous tumors contained HPV DNA compared with 79% of adenocarcinomas, the latter harboring predominantly HPV18. Women 45 years old or younger with a history of cigarette smoking tended to have HPV DNA in their tumors, but the HPV type was not associated with established prognostic factors such as stage, grade, lymph node metastasis, or depth of stromal invasion. After a median follow-up of 38.9 months, among potential prognostic factors of patient age, histologic cell type, grade, and HPV DNA status, only stage was predictive of survival in the entire study population. However, among the 171 patients treated with type III radical hysterectomy (removal of uterus and upper vagina along with other tissues extending to the pelvic wall) and pelvic lymphadenectomy (removal of all lymphatic tissue in the pelvis), multivariate analysis determined that lymph node status (adjusted risk ratio [RR] = 3.12; 95% confidence interval [CI] = 1.35-7.21), depth of stromal invasion (adjusted RR = 3.14; 95% Cl = 1.05-9.34), and the presence of HPV18 DNA (adjusted RR = 2.59; 95% CI = 1.08-6.22) were statistically significant predictors of survival. CONCLUSION: HPV18 DNA type is an independent prognostic factor in patients with cervical carcinomas treated with radical hysterectomy and pelvic lymphadenectomy. IMPLICATIONS: The use of molecular markers such as HPV DNA type may allow the identification of patients with early stage cervical cancer at high risk for disease recurrence.

Carcinoma of the vulva: HPV and p53 mutations.

Recent evidence suggests that squamous cell carcinoma of the vulva may have more than one etiology, with only some tumors associated with human papillomavirus (HPV). Cells infected with HPV produce a viral protein (E6) which binds to and causes rapid degradation of p53, possibly contributing to cellular transformation. In several human malignancies, point mutations of p53 alter activity of the p53 protein contributing to cellular transformation. We tested, for the first time, the possibility that HPV-negative tumors of the vulva may have a high incidence of inactivating mutations of p53; while HPV-containing vulvar tumors rarely would have p53 mutations. Twenty-one tumors of the vulva were evaluated for the presence of HPV sequences by amplication with the polymerase chain reaction (PCR) and Southern blotting. These were evaluated for p53 mutations by single strand conformation polymorphism and sequencing of PCR products. HPV DNA sequences were found in 12 of 21 (57%) cancers of the vulva; only one of these 12 (8%) HPV-positive samples had a missense mutation of p53. In contrast, four of nine (44%) HPV-negative vulvar tumors had point mutations of p53. The p53 mutations were found in only metastatic lesion and the only recurrent tumor samples suggesting that the acquisition of p53 mutations may be associated with neoplastic progression. In conclusion, alterations in p53 activity appear to be important in the development of carcinoma of the vulva.

p53 mutations in HPV-negative cervical carcinoma.

Human papillomavirus (HPV) infection has been strongly linked to the development of cervical carcinoma. Two viral oncoproteins, E6 and E7, produced by HPV, have been shown to immortalize primary human genital epithelial cells by interacting with the protein products of cellular tumor suppressor genes p53 and Rb, respectively. E6 binds to the cellular p53 protein promoting p53 degradation and inactivity. This mechanism has been suggested to contribute to the oncogenesis of HPV-positive anogenital cancers. In HPV-negative cervical carcinoma, p53 mutation is thought to be the possible mechanism of oncogenesis. We have studied 257 cervical carcinoma specimens for HPV infection by Southern blot analysis and polymerase chain reaction (PCR). Of 257 samples, 39 were HPV-negative. We have further studied 21 HPV-negative specimens for p53 mutations utilizing PCR amplification of genomic DNA followed by single-stranded conformation polymorphism (SSCP) analysis and DNA sequencing. We found only two missense point mutations of p53 gene. In summary, although inactivation of p53 mediated either by E6 or by mutations may be an important key step in the development of cervical carcinoma, our study suggests that other mechanisms may also be involved in development of cervical cancer.

A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive.

Eighteen women with high-grade cervical or vulvar intraepithelial neoplasia who were positive for human papillomavirus (HPV) 16 and were HLA-A2 positive were treated with escalating doses of a vaccine consisting of a 9-amino acid peptide from amino acids 12-20 encoded by the E7 gene emulsified with incomplete Freund's adjuvant. Starting with the eleventh patient, an 8-amino acid peptide 86-93 linked to a helper T-cell epitope peptide with a covalently linked lipid tail was added. Patients with colposcopically and biopsy-proven cervical intraepithelial neoplasia/vulvar intraepithelial neoplasia II/III received four immunizations of increasing doses of the vaccine each 3 weeks apart, followed by a repeat colposcopy and definitive removal of dysplastic tissue 3 weeks after the fourth immunization. Patients were skin tested with the E7 12-20 peptide as well as control candida, mumps, and saline prior to and after the series of immunizations. Peripheral blood mononuclear cells were obtained by leucopheresis prior to and after the series of immunizations for analyses of CTL reactivity to the E7 12-20 and 86-93 epitope sequences. The presence of HPV 16 was assessed by DNA PCR on cervical scrapings and the biopsy specimens after vaccination. Pathology specimens were analyzed before and after vaccination for the presence of dysplasia, and the intralesional infiltrate of CD4/CD8 T-cells and dendritic cells was measured by immunohistochemical staining. Only 3 of 18 patients cleared their dysplasia after vaccine, but an increased S100+ dendritic cell infiltrate was observed in 6 of 6 patients tested. Cytokine release and cytolysis assays to measure E7-specific reactivity revealed increases in 10 of 16 patients tested. No positive delayed type hypersensitivity skin test reactivity was shown in any patient to HPV E7 12-20 before or after vaccinations. Virological assays showed that 12 of 18 patients cleared the virus from cervical scrapings by the fourth vaccine injection, but all biopsy samples were still positive by in situ RNA hybridization after vaccination. Six patients had partial colposcopically measured regression of their cervical intraepithelial neoplastic lesions in addition to the three complete responders. The data establish that a HPV-16 peptide vaccine may have important biological and clinical effects and suggest that future refinements of an HPV vaccine strategy to boost antigen-specific immunity should be explored.

A phase I radioimmunotherapy trial evaluating 90yttrium-labeled anti-carcinoembryonic antigen (CEA) chimeric T84.66 in patients with metastatic CEA-producing malignancies.

Chimeric T84.66 (cT84.66) is a genetically engineered human/murine chimeric IgG, with high affinity and specificity to carcinoembryonic antigen (CEA). The purpose of this Phase I dose escalation therapy trial was to evaluate the toxicities, biodistribution, pharmacokinetics, tumor targeting, immunogenicity, and organ and tumor absorbed dose estimates of cT84.66 labeled with 90Y. Patients with metastatic CEA-producing malignancies were first administered 5 mCi 111In-labeled DTPA-cT84.66 (5 mg), followed by administration of the therapy dose of 90Y-labeled DTPA-cT84.66 1 week later. The therapy infusion was immediately followed by a 72-h administration of DTPA at 250 mg/m2/24 h. Dose levels of administered activity ranged from 5 to 22 mCi/m2 with three to six patients per level. Serial nuclear scans, blood samples, and 24-h urine collections were performed out to 5 days after infusion. Human antichimeric antibody response was assayed out to 6 months. Patients were administered up to 3 cycles of therapy every 6 weeks. Radiation absorbed doses to organs were estimated using a five compartment model and MIRDOSE3. Twenty-two patients received at least one cycle of therapy, with one individual receiving two cycles and two receiving three cycles of therapy. All were heavily pretreated and had progressive disease prior to entry in this trial. Reversible leukopenia and thrombocytopenia were the primary dose-limiting toxicities observed. Maximum tolerated dose was reached at 22 mCi/ m2. In general, patients with liver metastases demonstrated more rapid blood clearance of the antibody. Thirteen patients developed an immune response to the antibody. Average radiation doses to marrow, liver, and whole body were 2.6, 29, and 1.9 cGy/mCi 90Y, respectively. Dose estimates to tumor ranged from 66 to 1670 cGy (8.7 to 52.2 cGy/mCi 90Y) for each cycle of therapy delivered. Although no major responses were observed, three patients demonstrated stable disease of 12-28 weeks duration and two demonstrated a mixed response. In addition, a 41-100% reduction in tumor size was observed with five tumor lesions. 90Y-labeled cT84.66 was well tolerated, with reversible thrombocytopenia and leukopenia being dose limiting. Patients with extensive hepatic involvement by tumor demonstrated unfavorable biodistribution for therapy with rapid blood clearance and poor tumor targeting. Average tumor doses when compared with red marrow doses indicated a favorable therapeutic ratio. Stable disease and mixed responses were observed in this heavily pretreated population with progressive disease. This trial represents an important step toward further improving the therapeutic potential of this agent through refinements in the characteristics of the antibody and the treatment strategies used. Future trials will focus on the use of peripheral stem cell support to allow for higher administered activities and the use of combined modality strategies with radiation-enhancing chemotherapy drugs. Further efforts to reduce immunogenicity through humanization of the antibody are also planned. Finally, novel engineered, lower molecular weight, faster clearing constructs derived from cT84.66 continue to be evaluated in preclinical models as potential agents for radioimmunotherapy.

Initial clinical experience evaluating Yttrium-90-chimeric T84.66 anticarcinoembryonic antigen antibody and autologous hematopoietic stem cell support in patients with carcinoembryonic antigen-producing metastatic breast cancer.

cT84.66 is a human/murine IgG1 with high affinity and specificity for carcinoembryonic antigen (CEA). An earlier Phase I trial defined the maximum tolerated dose for 90Y-diethylenetriaminepentaacetic acid (DTPA)-cT84.66 at 22 mCi/m2. Dose-limiting toxicities were reversible leukopenia and thrombocytopenia. The purpose of this Phase I trial was to evaluate the feasibility and toxicities of administering higher activities of 90Y-DTPA-cT84.66 with stem cell support in patients with CEA-producing breast cancer. Patients with CEA-producing breast cancer refractory to standard therapies underwent peripheral stem cell collection followed by infusion of 111indium-DTPA-cT84.66. Those patients demonstrating tumor targeting received a single therapy dose of 90Y-DTPA-cT84.66, followed by Ca-DTPA infusion for 72 h posttherapy. Stem cells were reinfused following a divided schedule. To date, seven patients have been accrued to this trial. Each patient received an imaging dose of (111)In-cT84.66. Six patients demonstrated tumor imaging and received a single cycle of 90Y-cT84.66 at 15 mCi/m2 (three patients) and 22.5 mCi/m2 (three patients). One patient did not demonstrate tumor imaging and was not treated. At these administered activities, 90Y-cT84.66 was well tolerated. No dose-limiting toxicities have been observed. All patients demonstrated hematopoietic recovery after stem cell infusion. One patient demonstrated stable disease for 4 months; one patient had stable disease and reduction of bone pain for 3 months; and a third patient experienced >50% reduction of an ovarian metastasis, resolution of malignant pleural effusion, stable pleural metastases, and stable bone scan for 14 months. Preliminary results from this ongoing Phase I trial are promising and demonstrate the feasibility and potential for antitumor effects of stem cell supported 90Y-cT84.66 therapy in patients with CEA-producing breast cancers.

Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor.

The interaction of growth factors, such as epidermal growth factor (EGF) with their receptors, on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acids, such as arachidonic acid, which can be further metabolized by the lipoxygenase (LO) pathway. Several LO products have been shown to stimulate oncogenes and have mitogenic and chemotactic effects. In this study, we have evaluated the regulation of 12-LO activity and expression in breast cancer cells and tissues. Leukocyte-type 12-LO messenger RNA (mRNA) expression was studied by a specific RT-PCR method in matched, normal, uninvolved and cancer-involved breast tissue RNA samples from six patients. In each of these six patients, the cancer-involved section showed a much higher level of 12-LO mRNA than the corresponding normal section. 12-LO mRNA levels also were greater in two breast cancer cell lines, MCF-7 and COH-BR1, compared with the nontumorigenic breast epithelial cell line, MCF-10F. The growth of the MCF-7 cells was significantly inhibited by two specific LO blockers but not by a cyclooxygenase blocker. Treatment of serum-starved MCF-7 cells with EGF for 4 h led to a dose-dependent increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid. EGF treatment also increased the levels of the leukocyte-type 12-LO protein expression at 24 h. These results suggest that activation of the 12-LO pathway may play a key role in basal and EGF-induced breast cancer cell growth.

A randomized, double blind, Phase III trial using oral beta-carotene supplementation for women with high-grade cervical intraepithelial neoplasia.

To evaluate the effect of daily beta-carotene (30 mg) versus placebo over a 2-year period on cervical intraepithelial neoplasia (CIN) 2 and 3 lesions. Human papillomavirus (HPV) typing was done to determine whether lesion regression was related to HPV. Micronutrient levels were measured to determine whether levels were predictive of regression. Variables that influence the risk of HPV infection and CIN, such as cigarette smoking and sexual behavior, were evaluated. Women were randomized to beta-carotene or placebo, with cytology and colposcopy every 3 months. Cervical biopsies were performed before treatment and after 6 and 24 months to evaluate response. Persistence of or progression to CIN 3 resulted in removal from the study, whereas treatment continued for 2 years on all others. The presence and type of HPV was determined by PCR. Response was defined as an improvement in CIN by 2 grades. Mantel-Haenszel chi(2) test was used to analyze response to treatment. Fisher's exact test was used to determine the effect of HPV and CIN grade on response Wilcoxon's rank-sum tests were used to compare micronutrient levels between groups. Twenty-one of 124 enrolled women were not randomized because they either moved, became pregnant, voluntarily withdrew, or the pathological review of their initial cervical biopsies did not confirm CIN 2 or 3. Of the remaining 103 women, 33 experienced lesion regression, 45 had persistent or progressive disease, and 25 women did not complete the study and were considered nonresponders in the final analysis. The overall regression rate (32%) was similar between treatment arms and when stratified for CIN grade. Data on 99 women with HPV typing showed that 77% were HPV-positive and 23% HPV-negative at enrollment. HPV-positive lesions were subdivided into indeterminate-, low-, and high-risk categories; the response rate was highest for women with no HPV detected (61%), lower for indeterminate/low-risk (30%), and lowest for high-risk (18%; P =.001). CIN regression was negatively correlated with retinol levels. In conclusion, beta-carotene does not enhance the regression of high-grade CIN, especially in HPV-positive subjects.

Cerebellar cell degeneration in the leaner mutant mouse.

Leaner is an autosomal recessive mutation of the mouse which results in a severe ataxia accompanied by cellular losses in the cerebellar cortex. The purpose of this report is to construct a developmental profile of these losses. Of the three cerebellar cell types studied, the granule cells are the first to show obvious degenerative changes. Pycnotic cells are numerous in the internal granule cell layer at postnatal day 10, and, while they are found throughout the cortex, they are more concentrated in the anterior folia. Initially, there is a strong tendency for the pycnotic cells to be located in the deep half of the internal granule cell layer. By four postnatal months the rate of loss has slowed but the finding of occasional pycnotic cells in animals up to one year old suggests it continues for the life of the animal. Quantitative analysis of Purkinje and Golgi cells in leaner cerebella reveals a progressive loss of these cells as well. The number of Golgi cells falls uniformly to around half of the wild-type number. By contrast, the Purkinje cells show much more extensive degeneration. Further, the rate of cell death shows a regional variation; it is significantly more rapid in anterior folia. Overall, the number of Purkinje cells in leaner falls to about one-fifth of the wild-type number. The loss of both Purkinje and Golgi cells occurs late relative to the major events of cerebellar maturation. Significant cell loss is not observed until the end of the first postnatal month. For the next 4 to 6 weeks there is extensive cell death but, rather than abating, it appears to continue at a low rate for the life of the animal. It is hoped that this developmental sketch of the leaner defect will stimulate others to approach leaner and its alleles, tottering and rolling, as models for heterogeneity of disease expression.

Polymorphism of the hpv-16 e6 gene of cervical-carcinoma.

Polymorphism of the human papillomavirus type 16 (HPV-16) genome has been reported to occur within the noncoding regulatory long control region (LCR) and in the E7 and L1 genes. The current study focuses on the HPV-16 E6 oncogene which interacts with the antioncogenic regulator p53. Seventy-eight HPV-16-positive DNA samples derived from cervical carcinomas were screened for the presence of polymorphism in the HPV-16 E6 gene by polymerase chain reaction (PCR) linked single stranded conformational polymorphism (SSCP) analysis. Nine DNA samples had heterozygous mutations within the same region of the E6 gene 3' terminus; T to C transitions at HPV-16 position 511 (silent) and one of the nine also had a 513 mutation (Met to Thr). These mutations correlated with the clinical aggressiveness of the tumor, suggesting that the presence of these mutations may be due to genomic instability of advanced cervical carcinoma.

Mutational analysis of the hMSH2 gene in a wide variety of tumors.

The hMSH2 gene participates in DNA mismatch repair and its mutation can result in genetic instability of the human genome which is an important feature of tumorigenesis. In this study, genetic alterations of the hMSH2 gene were examined in 43 ovarian, 36 non-small cell lung (NSCL), 31 poorly differentiated gastric, 15 endometrial, and 11 colon cancers, nine gastric cancer cell lines, 41 adult T-cell leukemias (ATLs), two ATL cell lines, and 37 non-Hodgkin's lymphomas (NHLs), using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. Microsatellite instability (MSI) was also investigated for ovarian, NSCL, and colon cancers. The incidence of MSI was 1/36 (3%) for NSCL, 2/23 (9%) for ovarian, and 1/11 (9%) for colon cancers. Missense base changes of the hMSH2 gene were identified in two gastric cancer patients (ATG to ATA resulting in Met changing to Ile at codon 688 in exon 13 and ACA to GCA resulting in Thr changing to Ala at codon 803 in exon 14). These mutations were found in samples with no MSI. One ovarian and one gastric cancer, and six ATL samples showed two types of polymorphisms of hMSH2 (CTT to TTT resulting in Leu changing to Phe at codon 390 in exon 7 and CAG to AAG resulting in Gin to Arg at codon 419 in exon 7). Our data suggest that MSI and hMSH2 mutations are uncommon in sporadic tumors.

Human papillomavirus type 6 in squamous cell carcinoma of the bladder and cervix.

Human papillomavirus (HPV) types 16 and 18 are the most frequent genotypes identified in genital malignancies, while HPV types 6 and 11 are found predominantly in condylomas and low-grade dysplasias. It is thought that HPV types 16 and 18 represent high-risk genotypes, while HPV types 6 and 11 rarely, if ever, participate in the development of malignant tumors. In a series of over 300 invasive tumors of the lower genital tract analyzed for the presence of HPV three have been found to contain HPV type 6 DNA: two invasive squamous cell carcinomas of the cervix and one squamous cell carcinoma of the bladder. Human papillomavirus type 6 was the only HPV type detected in these tumor DNAs by Southern blot hybridization and by the polymerase chain reaction using both consensus and type-specific primers. In situ hybridization using whole genomic RNA probes localized viral DNA to tumor cells. Although extensive virologic and epidemiologic studies conducted in the last decade indicate that HPV types 16 and 18 are more likely to be associated with high-grade dysplasias and invasive cancer, HPV type 6 may not be as innocuous as previously supposed.

Human papillomaviruses and cervical cancer: analysis of histopathologic features associated with different viral types.

The histopathologic features of 41 cervical carcinomas were correlated with the presence of human papillomavirus (HPV). Southern blots of DNA extracted from the tumors were hybridized with 32P-labeled type specific probes for HPV 6, 11, 16, 18, and 31. HPV was found in 26/41 (63%) of the tumors. The HPV types were: HPV 16 in 17 tumors (41%), HPV 18 in six tumors (15%) and HPV 31 in two tumors (5%). No tumor hybridized to either HPV 6 or HPV 11. HPV was identified in all histologic subtypes of cervical carcinoma; however, different HPV types were associated with specific histologic features. HPV 18 was identified in four of eight adenocarcinomas, while HPV 16 was found in only one. HPV 16 was most strongly associated with the keratinizing tumors. It was found in 10/13 (77%) of the large cell keratinizing (LCK) and in only 4/16 (25%) of the large cell nonkeratinizing cervical carcinomas (LCNK). A mucoepidermoid with extensive keratinization and pearl formation also contained HPV 16. One of three additional adenosquamous carcinomas had HPV 31, as did one LCNK tumor. In one LCK tumor, a HPV was identified that hybridized to both HPV 16 and 18. The LCNK group contained the highest percentage of tumors in which no papillomavirus DNA was identified (9/16 lacked HPV DNA). No papillomavirus was detected in six tumors from other sites or in five cervical specimens with no histologic evidence of HPV infection. These data indicate that HPV is involved in all major histologic types of cervical carcinoma, and suggest that the different HPV types transform slightly different cell populations, or that transformation by HPV 18 tends to induce adeno-differentiation while HPV 16 leads to squamous maturation.

Clinical and histologic features of vulvar carcinomas analyzed for human papillomavirus status: evidence that squamous cell carcinoma of the vulva has more than one etiology.

The association between the human papillomavirus (HPV) and malignant neoplasms of the uterine cervix is well established; however, its role in the pathogenesis of vulvar cancer has not been well defined. This study correlates the clinical and histopathologic features of 21 invasive carcinomas of the vulva with the presence of HPV DNA as detected by Southern blot and polymerase chain reaction (PCR) analysis. By one or both techniques, HPV DNA was detected in 10 of the 21 tumors analyzed; all HPVs containing tumors hybridized with HPV-16 probes, although PCR also detected HPV-6 in two of the HPV-16-containing tumors. No HPV-18 DNA was detected in any tumor by PCR or Southern blot hybridization. Both the invasive cancer and the surrounding intraepithelial disease tended to display histopathologic features that usually could distinguish HPV-associated cancers from those without HPV DNA. The intraepithelial lesions associated with HPV-containing tumors were of the bowenoid type with koilocytosis, while tumors lacking HPV generally demonstrated a simplex type of intraepithelial lesion. Invasive tumors with no viral DNA were more frequently keratinizing than the HPV-containing cancers. Race, parity, hormonal therapy, and alcohol use did not affect the HPV status; however, HPV DNA was more prevalent in the tumors of younger women and in those with a history of tobacco use. Human papillomavirus status had no impact on the stage of disease or its prognosis. These findings identify two subsets of vulvar carcinoma cases based on HPV hybridization data and the histopathologic characteristics of the tumor.

The use of molecular probes to distinguish new primary tumors from recurrent tumors in gynecologic malignancies.

This is the first report using DNA molecular probe technology to distinguish between recurrent tumor and a second primary malignancy in a patient. Tumor DNA was extracted from squamous cell carcinoma of the cervix at the time of radical hysterectomy. Eighteen months later a squamous cell cancer was found in a vaginal apex biopsy from which DNA was extracted. Tumor DNA from both lesions was subjected to restriction enzyme digestion and DNA molecular hybridization with human papillomavirus (HPV) probes. Although both lesions were positive for HPV 16, their respective restriction enzyme patterns had different HPV genetic arrangements, thereby demonstrating their distinctness.

Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription-polymerase chain reaction.

The expression of aromatase in human breast tumors has been studied by the reverse-transcription polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from the exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis has revealed that exons I.3 and PII are the two major exon Is present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells. The RT-PCR analysis also detected two products, I.3A (334 bp in length) and I.3B (222 bp in length), when it was carried out using a primer derived from exon I.3 and a reverse primer derived from exon II. The nucleotide sequences of these products have been determined and indicate that I.3A contains a region which was previously thought to be an intron. In addition, RT-PCR analyses of RNA isolated from eight pairs of breast tumor and neighboring normal tissue specimens were performed to evaluate the exon I usage and the distribution of I.3A- and I.3B-containing aromatase RNA messages in breast tumor and neighboring normal tissues. The results suggest that I.3B- and I.3A-containing messages are mainly present in breast tumor and neighboring normal tissues, respectively. Finally, the exon I/promoter usage for aromatase expression in eight cell lines (skin fibroblast, MCF-7, MDA-MB-231, T-47D, SK-BR-3, JAR, OVCAR-3, and human adipose stromal cells) was examined by primer-directed RT-PCR analyses. These studies provide a basis for further evaluation of the control mechanism of aromatase expression and estrogen biosynthesis in breast tumors.