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.

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.

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.

Loss of Rassf1a enhances p53-mediated tumor predisposition and accelerates progression to aneuploidy.

Loss of RASSF1A leads to several mitotic abnormalities, including cytokinesis failure and tetraploidization. Uncontrolled proliferation of tetraploid cells is known to trigger genomic instability and tumor development and is normally prevented through activation of a p53-dependent tetraploidy checkpoint. RASSF1A is the most commonly silenced and p53 is the most frequently mutated tumor suppressor gene in human cancer. However, their mutual contribution to tumorigenesis has never been investigated in animal models. Here, we explore whether concomitant loss of RASSF1A and p53 will result in increased levels of aneuploidy, genomic instability and tumorigenesis. We have intercrossed Rassf1a-knockout mice with mice lacking the p53 gene and generated a combination of single- and compound-mutant animals. Rassf1a(-/-) p53(-/-) mice were viable and fertile and developed normally. However, these mice were remarkably tumor prone and succumbed to malignancies significantly faster than single-mutant littermates, with a median survival time of 136 days (versus 158 days in p53(-/-) mice, P=0.0207, and >600 days in Rassf1a(-/-) animals, P<0.0001). Rassf1a-null mice with one functional p53 allele displayed a more moderate, yet tumor-prone phenotype, characterized by increased tumor multiplicity as compared with single knockouts. On cell-cycle profiling and cytogenetic analysis, cells derived from Rassf1a(-/-) p53(-/-) mice exhibited several mitotic defects associated with high levels of tetraploidy/aneuploidy. Conversely, cells with a proficient p53 allele could better cope with the mitotic failures imposed by Rassf1a loss. Altogether, we provide the first experimental evidence for a pivotal role of Rassf1a as an early 'gatekeeper' gene, whose loss of function deteriorates cellular fitness by enhancing tetraploidization. Concomitant loss of p53, which causes unrestrained propagation of tetraploids into aneuploid cells, further undermines genomic stability and accelerates tumorigenesis.

A phase II study of Hsp-7 (SGN-00101) in women with high-grade cervical intraepithelial neoplasia.

OBJECTIVE: Approximately 2 million women worldwide are infected with high-risk human papillomaviruses (HPV), resulting in a substantial risk for the development of invasive lower genital malignancies. This study was undertaken to determine the effects of vaccination with a protein encoding a bacterial heat shock protein fused to sequences from the oncogenic E7 protein of HPV-16 in women with high-grade cervical intraepithelial neoplasia. Endpoints included lesion regression, immune response, and viral clearance. METHODS: Twenty-one women were prospectively entered into an IRB-approved Phase II study. All women had biopsy-proven high-grade cervical intraepithelial neoplasia and persistent post-biopsy lesions visible by colposcopy. Four injections of HPV-16 Hsp E7 fusion protein at a dose of 500 mug were given 3 weeks apart after which Loop Electrosurgical Excision of the Transformation Zone (LLETZ) was performed. Immune parameters were evaluated pre-vaccine and at the time of LLETZ, and HPV testing was performed at intervals before and after LLETZ. Study subjects were followed for 1 year after LLETZ. RESULTS: Seven of 20 women (35%) evaluable for response had complete regression of their intraepithelial neoplasia at the time of LLETZ, 1 (5%) had regression to CIN I, 11 (55%) had stable disease and 1 (5%) had progression due to enlargement of her lesion. Immune responses were seen in 9 of the 17 women tested; 5 of the 7 complete responders had an immune response. Only 5 of 21 women had HPV-16 or -18. HPV clearance was not associated with lesion regression. CONCLUSION: Hsp-7 (SGN-00101), at this dose and schedule induced lesion regression in women with high-grade intraepithelial neoplasia. The fact that regression was correlated with immune response suggests that enhancing the immunogenicity of this vaccine may lead to improvement in the rate of lesion eradication.

Phase II trial of imatinib mesylate in recurrent, biomarker positive, ovarian cancer (Southwest Oncology Group Protocol S0211).

Platinum-resistant ovarian cancer continues to be a difficult therapeutic problem. Clearly, molecularly targeted agents should be evaluated in this patient population. Patients were eligible for this phase II study with stage III or IV ovarian cancer, whose tumor expressed Kit (CD117) or platelet-derived growth factor receptor (PDGFR) and with relapse of measurable disease within 6 months of completing frontline, platinum- and taxane-based chemotherapy. Patients were treated daily with 400 mg of imatinib mesylate orally. It was assumed that the agent would be of no further interest if the population response rate was less than 10%. A two-stage design was used for patient accrual. A total of 34 patients were registered to the study. Of these, 15 were found to be ineligible or not evaluable (8 because their tumor samples were negative for both DC117 and PDGFR). Of 19 evaluable patients, 2 (11%) tested positively for c-Kit and 17 (89%) tested positively for PDGFR. There were no objective responders. Thirteen patients (68%) had increasing disease or symptomatic deterioration, and six (32%) went off protocol during the first month due to adverse events. Median progression-free survival was 2 months (95% CI 1-3 months) and median overall survival was 10 months (95% CI 6-18 months). Eleven percent of patients experienced grade 4 hematologic/metabolic toxicity and 37% experienced grade 3 nonhematologic toxicity. We conclude that imatinib mesylate as a single agent does not appear to have useful clinical activity in c-Kit and/or PDGFR positive, recurrent ovarian cancer in heavily pretreated patients with ovarian cancer.

Uterine effects of tamoxifen: a prospective study.

The purpose of the study was to evaluate tamoxifen-associated changes in the vagina and uterus in postmenopausal breast cancer patients. Between June 1994 and December 1998, 45 patients enrolled in a prospective study before commencing tamoxifen therapy. Patients with endometrial thickness >5 mm or neoplasia were excluded. Transvaginal ultrasonography, vaginal maturation indexes (VMI), and endometrial biopsy were performed at baseline and repeated at 6 months (n= 42), 1 year (n= 39), 2 years (n= 32), 3 years (n= 26), 4 years (n= 19), and 5 years (n= 15). For the 39 patients followed for 1 year, VMI (% parabasal/intermediate/superficial) was 21/71/8 at baseline compared with 1/90/9 at 1 year (P value = 0.0008/0.001/0.78). At baseline, mean endometrial thickness and uterine volume were 2.6 mm and 64 cm(3), respectively, compared with 5.8 mm and 84 cm(3) at 1 year (P= 0.0002, 0.002). At baseline, 80% of patients had atrophic endometrium and 9% proliferative endometrium compared with 61% and 26% at 1 year, respectively (P= 0.04). No cases of endometrial hyperplasia or adenocarcinoma were detected. Findings observed at 6 months persisted through 5 years of follow-up. Tamoxifen exerts a weak estrogenic effect on the vagina and uterus in highly prescreened postmenopausal women without preexisting endometrial pathology.

Long-term follow-up of a phase II trial of oral altretamine for consolidation of clinical complete remission in women with stage III epithelial ovarian cancer in the Southwest Oncology Group.

OBJECTIVE: This report provides follow-up progression-free survival (PFS) and median survival data for women who achieved clinical complete remission (cCR) from stage III ovarian cancer after first-line therapy and were treated with altretamine consolidation therapy. METHODS: Patients who enrolled in the SWOG 9326 study from September 1993 to July 1997 were required to have documented cCR from stage III ovarian cancer following front-line platinum-based therapy. Treatment consisted of 6 months of oral altretamine at 260 mg/m(2)/day for 14 consecutive days of a 28-day cycle. RESULTS: Ninety-seven of 112 enrolled patients were evaluable for efficacy. This report presents median 6.2-year follow-up, dating from study registration. Median PFS was 28 (95% CI: 19-43) months. Median PFS for patients with optimal disease was 45 (95% CI: 27-48) months and for patients with suboptimal disease was 17 (95% CI: 12-26) months. Twenty-six of 61 (43%) patients with optimally debulked lesions and 5 of 36 (14%) patients with suboptimally debulked lesions remained disease free. Median survival of patients with optimally debulked disease has not been reached; median survival of patients with suboptimally debulked disease was 39 (95% CI: 19-51) months. No treatment-related adverse events were reported during the follow-up period. CONCLUSIONS: Consolidation therapy with oral altretamine was generally well tolerated and associated with prolonged progression-free and overall survival in the Phase II setting.

HPV infection and number of lifetime sexual partners are strong predictors for 'natural' regression of CIN 2 and 3.

The aim of this paper was to evaluate the factors that predict regression of untreated CIN 2 and 3. A total of 93 patients with colposcopic persistent CIN 2 and 3 lesions after biopsy were followed for 6 months. Human papillomavirus (HPV) types were determined by polymerase chain reaction at enrolment. We analysed the biologic and demographic predictors of natural regression using univariate and multivariate methods. The overall regression rate was 52% (48 out of 93), including 58% (22 out of 38) of CIN 2 and 47% (26 out of 55) of CIN 3 lesions (P=0.31 for difference). Human papillomavirus was detected in 84% (78 out of 93) of patients. In univariate analysis, 80% (12 out of 15) of lesions without HPV regressed compared to 46% (36 out of 78) of lesions with HPV infection (P=0.016). Women without HPV and those who had a resolution of HPV had a four-fold higher chance of regression than those with persistent HPV (relative odds=3.5, 95% CI=1.4-8.6). Women with five or fewer lifetime sexual partners had higher rates of regression than women with more than five partners (P=0.003). In multivariate analysis, HPV status and number of sexual partners remained as significant independent predictors of regression. In conclusion, HPV status and number of lifetime sexual partners were strongly predictive of regression of untreated CIN 2 and 3.

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.

Phase II trial of oral altretamine for consolidation of clinical complete remission in women with stage III epithelial ovarian cancer: a Southwest Oncology Group trial (SWOG-9326).

OBJECTIVE: The aim of this study was to evaluate the 2-year survival rate in a group of women in complete clinical remission (cCR) from Stage III ovarian cancer following front-line therapy who were then treated with a 6-month course of altretamine. METHODS: Patients were documented to be in cCR by physical examination, computed tomography or magnetic resonance imaging scan, and serum CA-125. Treatment consisted of altretamine (Hexalen) 260 mg/m(2)/day po divided into four doses taken after meals and at bedtime for 14 of 28 days for six cycles. Based on previous experience in the Southwest Oncology Group, the treatment would be considered promising if the 2-year survival rate was > or = 65% as measured from study registration. RESULTS: From 9/1/93 and 7/1/97, 112 patients were registered and 97 were fully evaluable. The majority of patients had optimally debulked (< or = 1 cm: 63%), high-grade (Grade 3: 82%) tumors. The 2-year survival rate in this study was 75% (95% CI: 66-84%). For those patients with optimal disease, the 2-year survival rate was 82% (95% CI: 72-92%) and for those with suboptimal disease it was 64% (95% CI: 48-79%). Four patients (4%) experienced Grade 4 and 21 patients (22%) experienced Grade 3 toxicities consisting primarily of nausea/vomiting, neutropenia, fatigue, anxiety, and paresthesias. CONCLUSIONS: The 2-year survival rate in this study warrants further evaluation of consolidation therapy for women in clinical complete remission following front-line chemotherapy for Stage III ovarian cancer. Caution is advised in the interpretation of these data, however, because of the nonrandomized nature of the trial and the unknown contribution of front-line use of paclitaxel to the durability of clinical complete response.

Antecedent manipulations in a tangible condition: effects of stimulus preference on aggression.

After a functional analysis indicated that aggression of an 8-year-old boy with autism was maintained by access to preferred items, antecedent manipulations involving the relative preference of restricted and noncontingently available stimuli were conducted. Restricting highly preferred items evoked the highest rates of aggression regardless of the preference level of the noncontingently available alternative items. Restricting less preferred stimuli was associated with moderate rates of aggression even when the alternative items were more preferred.

Using antecedent manipulations to distinguish between task and social variables associated with problem behaviors exhibited by children of typical development.

In the antecedent functional assessment literature, researchers have introduced task demands and social attention simultaneously while varying the level of task difficulty. Though research has demonstrated situations in which a combination of social and task antecedents occasion socially avoidant responses from children with disabilities, no current studies have been offered to assess the impact of high levels of adult attention devoid of task demands on problem behaviors exhibited by children of typical development. A multiple element design was used to assess the specific effects of task and social antecedents on the problem behaviors of four children of typical development. Results identified two children whose behavior was associated with a combination of difficult task demands and attention in the form of commands and redirections and two children whose behavior was associated only with high levels of adult attention that did not include commands or redirections. These results suggest that antecedent functional assessment procedures can assess the impact of high levels of attention without the presence of task demands.

Recurrence of pulmonary mucinous cystic tumor of borderline malignancy.

Cystic mucinous tumors of the lung are recently described neoplasms whose histology is different from most lung adenocarcinomas, and represent a spectrum of malignant potential. Little is known of the behavior of the more malignant subtype. We present a cystic mucinous tumor of borderline malignancy that recurred locally following initial limited resection, and was treated with lobectomy.

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.

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.

Pulmonary toxicity of induction chemotherapy prior to standard or high-dose chemotherapy with autologous hematopoietic support.

We closely followed the pulmonary function of 150 consecutive high-risk breast cancer patients who underwent standard induction CAF (cyclophosphamide, doxorubicin, 5-fluorouracil) chemotherapy, followed by randomization to either standard-dose CPB (cyclophosphamide, cisplatin, bischloroethylnitrosourea [BCNU]) chemotherapy (SDC) or to high-dose CPB chemotherapy (HDC) with autologous bone marrow transplantation (ABMT) and peripheral blood progenitor cell support (PBPCS). Previously, we have described a delayed pulmonary toxicity syndrome (DPTS) which characterizes the pulmonary dysfunction after HDC and ABMT in this patient population. However, little is known concerning the role induction chemotherapy plays in its development. We found that after three cycles of induction CAF, the mean diffusing capacity of the lungs for carbon monoxide (DL(CO)) significantly decreased by 12.6%. Additionally, in patients receiving HDC, the mean DL(CO) further decreased to a nadir of 55.2 +/- 14.1% which was significantly lower than those receiving SDC (nadir: 80.7 +/- 12.3%). DPTS occurred in 72% of patients receiving HDC as compared with only 4% of patients receiving SDC. All individuals diagnosed with DPTS were treated with prednisone and the 2-yr follow-up of pulmonary function revealed a gradual improvement in mean DL(CO) such that there were no differences between HDC and SDC groups at the end of the study. No mortality was attributable to pulmonary toxicity in either group. After induction chemotherapy, but before HDC, bronchoalveolar lavage (BAL) demonstrated significant elevations in interleukin-6 (IL-6), IL-8, neutrophils, and lymphocytes. We conclude that induction CAF produces asymptomatic pulmonary dysfunction and inflammation which may prime the lungs for further injury by HDC and predispose to the development of DPTS. Fortunately, in this specific ABMT patient population, the early and judicious use of prednisone appears to improve pulmonary function in patients who develop DPTS.

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.

Does human papillomavirus have a role in cancers of the uterine corpus?

OBJECTIVE: Thepurpose of this study was to determine the role of the human papillomavirus (HPV) in invasive uterine corpus cancer by characterizing the frequency of HPV DNA in malignant uterine tumors. METHODS: Hysterectomy specimens from 66 women with uterine carcinoma were analyzed. Tumor specimens were frozen at -80 degrees C at the time of surgical resection. DNA was later extracted and examined for HPV DNA using type-specific PCR primers for HPV 6, 16, and 18 and consensus primers MY09/MY11, which detect DNA from 33 other common HPV types. Isolation procedures were undertaken to prevent contamination. RESULTS: The histologic diagnoses of the 66 uterine cancer cases included 58 endometrial adenocarcinomas, 4 adenosquamous carcinomas, 3 malignant mixed mesodermal tumors, and 1 squamous cell carcinoma. HPV was detected by both type-specific and consensus primers in only 2 of the uterine specimens. None of the typical endometrioid adenocarcinoma specimens contained HPV DNA. HPV 16 was detected in 1 of the adenosquamous carcinoma samples and HPV 18 was detected in the squamous carcinoma specimen. CONCLUSION: HPV DNA is not found in malignancies of the uterine corpus without malignant squamous elements when the risk of contamination is minimized. For these tumors, HPV appears to be unrelated to the neoplastic transformation process.