Phase II study of bevacizumab with liposomal doxorubicin for patients with platinum- and taxane-resistant ovarian cancer.

BACKGROUND: Suppression of neoangiogenesis and pegylated liposomal doxorubicin (PLD) each contribute to the management of platinum-resistant/refractory ovarian cancer. The aim of this study is to test the combination of bevacizumab and PLD in women with resistant or refractory ovarian cancer. METHODS: Eligibility criteria were no more than two prior treatments with platinum-containing regimens and one additional regimen, without anthracyclines. Treatment was administered every 3 weeks (bevacizumab 15 mg/kg beginning on cycle 2 and PLD 30 mg/m(2)). The primary end point was progression-free survival (PFS) at 6 months; the secondary end points included side-effects, overall response rates (ORR) and survival (OS). RESULTS: Forty-six patients were enrolled. The average number of courses administered was 7. The median PFS was 6.6 months (range 1-24.6 months) according to Gynecologic Cancer Intergroup Committee (GCIC) criteria and 7.8 months (range 2-13.3 months) according to Response Evaluation Criteria in Solid Tumors (RECIST). The median OS was 33.2 months (range 3-37.5+ months). The ORR was 30.2% [95% confidence interval (CI) 17.2-46.1] and the clinical benefit rate (CBR) was 86.1% (95% CI 72.1-94.7). Adverse events included mucosal and dermal erosions (30% grade 3) and asymptomatic cardiac dysfunction. Additional toxic effects included hypertension, headache, renal dysfunction and proteinuria, wound healing delay, and one episode each of central nervous system (CNS) ischemia and hemolytic uremic syndrome. CONCLUSION: PLD with bevacizumab has improved activity in recurrent ovarian cancer with increased toxicity.

Phase IB study of the combination of docetaxel, gemcitabine, and bevacizumab in patients with advanced or recurrent soft tissue sarcoma: the Axtell regimen.

BACKGROUND: To assess the response of patients with soft tissue sarcoma (STS) to the combination of docetaxel, bevacizumab, and gemcitabine. Vascular endothelial growth factor (VEGF)-A levels and expression of VEGF-A and VEGF receptors 1 and 2 were evaluated. PATIENTS AND METHODS: Thirty-eight chemotherapy-naive patients with STS were enrolled. A dose-finding study for gemcitabine from 1000, 1250, then 1500 mg/m(2) was done in nine patients (three cohorts), followed by an expansion cohort of 27 patients. Dose of docetaxel was 50 mg/m(2), bevacizumab was 5 mg/kg, and gemcitabine was 1500 mg/m(2), every 2 weeks. Serum VEGF-A was measured by enzyme-linked immunosorbent assay and tissue VEGF-A and its receptors by immunohistochemistry. RESULTS: The median follow-up was 36 months. The overall response rate observed was 31.4%, with 5 complete and 6 partial responses, and 18 stable diseases lasting for a median of 6 months. There was no significant hematologic toxicity. The adverse events with the highest grade were attributed to bevacizumab. There was no correlation of VEGF pathway biomarkers with outcome. CONCLUSIONS: The combination of gemcitabine, docetaxel, and bevacizumab is safe and effective in patients with STS. The most concerning adverse events were consequences of bevacizumab administration. The benefit of bevacizumab in this patient population remains unclear.

Absence of epidermal growth factor receptor mutations in cervical cancer.

Epidermal growth factor receptor (EGFR) is overexpressed in the majority of cervical cancers (CCs). Somatic mutations of EGFR have been associated with clinical response to tyrosine kinase inhibitors (TKIs) in lung cancer patients. This study was designed to establish the frequency of EGFR point mutations in patients diagnosed with high-grade squamous intraepithelial lesions (HSIL) and CC. Nine cell lines derived from CC were screened for EGFR mutations in exons 18 through 21. Eighty-nine patient samples derived from invasive CC (n = 80) and HSIL (n = 9) were analyzed for the presence of EGFR mutations in exons 19 and 21. We found no mutations affecting the EGFR kinase domain in exons 18 through 21 in all cell lines tested, and no EGFR mutations were detected in exons 19 and 21 in all 89 human neoplastic samples analyzed. These data indicate that mutations in the EGFR kinase domain are very rare in CC and HSIL. Our results suggest, therefore, that treatment of CC patients with TKIs may not have the same efficacy as seen in patients with lung cancer, and that targeting the EGFR with other inhibitors may be more appropriate.

Adequacy of oophorectomy at the time of gynecologic surgery.

OBJECTIVES: To determine the incidence of incomplete ovarian removal during gynecologic surgery and correlate the risk of inadequate removal with the procedure chosen. METHODS: This is a prospective observational study. Ovaries received during a 4-month period in the participating institutions were independently histologically evaluated. Gross inspection of the ovarian capsule, infundibulopelvic ligament, hilum and utero-ovarian ligament was assessed. Grossly close margins were confirmed histopathologically. Any margin with histologically confirmed ovarian tissue at the margin was interpreted as incompletely removed. Details of each surgical procedure were recorded for comparison. RESULTS: Ovaries (n=174) from 94 patients were collected and 155 were evaluable. The overall incidence of incomplete ovarian removal was 6.5%. Of the 125 ovaries removed abdominally, 23 were laparoscopically assisted and 7 were vaginal; inadequate removal was documented in 5%, 9% and 29%, respectively (P=0.04). There was no relationship of inadequate resection by underlying pathologic diagnosis (P=0.25) or by institution (4.6% university hospital vs. 8.8% community hospital; P=0.29). CONCLUSIONS: Incomplete ovarian removal occurs and is related to surgical approach. A larger study is warranted to evaluate the role of pelvic pathology or surgeon experience as a risk for incomplete oophorectomy.

Germline BRCA1-2 mutations in non-Ashkenazi families with double primary breast and ovarian cancer.

OBJECTIVE: Ashkenazi women with double primary breast and ovarian cancer have a high prevalence (57%) of germline Jewish founder mutations in the BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT) genes. The purpose of this study was to determine the frequency and type of BRCA1-2 mutations in non-Ashkenazi families with at least one member having double primary breast and ovarian cancer. METHODS: Women at increased risk for cancer based upon their family history were enrolled at the University of Texas Southwestern Familial Cancer Registry between 1992 and 2000. Blood samples from patients desiring genetic testing were sent for complete DNA sequencing of the BRCA1 and BRCA2 genes. Families with a member having both breast and ovarian cancer were identified and clinical data were obtained. RESULTS: Sixty-two (7%) of 900 enrolled families were non-Ashkenazi and had at least one member with double primary breast and ovarian cancer. Twenty-one families had members who underwent genetic testing; 41 did not. Thirteen (62%) families had a germline BRCA1 (n = 11) or BRCA2 (n = 2) mutation; only one Jewish founder mutation (185delAG) was detected. Eight (38%) families tested negative. Six (86%) of seven women undergoing genetic testing who themselves had double primary breast and ovarian cancer were BRCA1-2 mutation carriers. CONCLUSIONS: Germline BRCA1-2 mutations are common in non-Ashkenazi families with a member having double primary breast and ovarian cancer. These mutations occurred throughout both genes, emphasizing the need for comprehensive sequencing. One family had the BRCA2 6985delCT mutation, which lies beyond the "ovarian cancer cluster" region.

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers.

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

Intraoperative lymphatic mapping in cervix cancer patients undergoing radical hysterectomy: A pilot study.

OBJECTIVE: Intraoperative lymphatic mapping and sentinel lymph node identification (SLN) have been increasingly evaluated in the treatment of a variety of solid tumors, particularly breast cancer and melanoma. We sought to evaluate the feasibility of these procedures in patients undergoing radical hysterectomy with pelvic lymphadenectomy for treatment of early cervical cancer. METHODS: Twenty patients with normal-appearing lymph nodes underwent intracervical injection of isosulfan blue dye (lymphazurin 1%) at the time of planned radical hysterectomy and bilateral pelvic/low paraortic lymphadenectomy (40 nodal basins). Regional lymphatic tissue was inspected for dye uptake into lymphatic channels and lymph nodes. Tumor characteristics, surgical findings, and specific locations of lymphatic dye uptake were recorded and correlated with final pathology results. RESULTS: Sentinel lymph nodes were identified in 12 of 20 (60%) patients. A total of 23 sentinel nodes were identified in 17 of 40 (43%) nodal basins dissected (range: 0-2 per basin). Successful SLN identification was less likely in patients with tumors >4 cm compared with those with tumors

Chromosome 4 deletions are frequent in invasive cervical cancer and differ between histologic variants.

OBJECTIVE: Patterns of discontinuous deletion of chromosome 4 have been described in histologic variants of lung carcinomas and may represent different "hotspot" targets for gene-environment interactions. Since similar environmental risks exist for cervical cancer, we investigated patterns of discontinuous deletion in two major histologic variants. METHODS: Thirteen archival cases of squamous cell cancer (SCCA) and 11 cases of adenocarcinoma (AC) were precisely microdissected. Matched normal and tumor DNA were used for polymerase chain reaction (PCR) based loss of heterozygosity (LOH) analyses using 19 polymorphic markers spanning chromosome 4. Human papillomavirus (HPV) detection was determined by PCR using general and type-specific primers (HPV 16, 18). Differences in LOH between histologic tumor types and chromosomal regions were determined using Fisher's exact test. RESULTS: Loss at any chromosome 4 locus occurred in 92% of all tumors studied, with the majority of deletions occurring on the long arm of the chromosome. Four discrete minimal regions of discontinuous deletion (R) were identified. For these regions, LOH frequencies were 76% (R1, 4q34-q35), 48% (R2, 4q25-q26), 36% (R3, 4p15.1-p15.3), and 26% (R4, 4p16). Loss in SCCA predominated at 4q (4q34-q35; 83%) and in AC at 4p (4p15.3; 50%). Overall LOH on the p arm was significant in AC (82%) compared to SCCA (31%) (P = 0.02). HPV detection was similar in SCCA (85%) and AC (73%), and HPV 16/18 subtypes were similarly represented in both histologies. CONCLUSIONS: Chromosome 4 deletions are frequent in cervical carcinomas. Different patterns of deletion between SCCA and AC may represent gene regions targeted by different gene-environment interactions in these tumor subtypes.

Laparoscopy in patients following transverse rectus abdominis myocutaneous flap reconstruction.

BACKGROUND: We report our technique and experience performing laparoscopic pelvic surgery on four women after transverse abdominus rectus myocutaneous flap (TRAM). TECHNIQUE: Examination under anesthesia is performed on all patients in the low lithotomy position parallel with the floor. The abdominal aorta is palpated and outlined. A pneumoperitoneum is created either by umbilical or left upper quadrant Veress placement. Patients with an acceptable umbilical location undergo port placement through the incision of the umbilical relocation. Other options include left upper quadrant or paramedian placement avoiding the ligamentum teres vessels. Lateral operative ports (5 mm) are placed with reference to the transverse incision present, the pelvic pathology, and the location of the umbilicus. Techniques of electrocautery, intra- and extracorporeal suturing and knot tying, and clips are preferred to minimize port size. EXPERIENCE: Following unilateral or bilateral TRAM reconstruction, four consecutive breast cancer survivors underwent successful laparoscopic-assisted vaginal hysterectomy with oophorectomy using the periumbilical incision for trocar placement. The only complication was a superficial skin breakdown from an adhesive allergy that required 6 weeks for complete resolution. CONCLUSION: Laparoscopic pelvic surgery is feasible in women after TRAM reconstruction. Knowledge of anatomic and physiologic variations related to the TRAM procedure is necessary in planning a safe operation.

Molecular Papanicolaou tests in the twenty-first century: molecular analyses with fluid-based Papanicolaou technology.

OBJECTIVE: This study was undertaken to demonstrate the feasibility of performing molecular analyses at the deoxyribonucleic acid, ribonucleic acid and protein levels of cervical cytologic examination with a methanol fluid-based Papanicolaou specimen collection system. STUDY DESIGN: Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanicolaou specimen collection buffer after clinical processing. Genomic and human papillomavirus deoxyribonucleic acid polymerase chain reaction and reverse transcriptase-polymerase chain reaction were performed. Messenger ribonucleic acid transcript analysis and human papillomavirus 16 E6 mutational analysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene for E-cadherin. Immunohistochemical staining for protein expression was performed on the processed monolayer slides. RESULTS: Cell pellets from the residual fluid-based cytologic specimen yielded good quality deoxyribonucleic acid and ribonucleic acid. Molecular analyses of genomic deoxyribonucleic acid were successful for the identification of human papillomavirus E6 and p53 polymorphism status by means of restriction enzyme digestion and direct sequencing. Methylation status of the promotor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the template for transcript analysis and mutational analysis of the corresponding complementary deoxyribonucleic acid of the p53 gene. Protein expression analysis was demonstrated by immunohistochemical staining for carcinoembryonic antigen. CONCLUSION: It is feasible to conduct multiple molecular analyses at the deoxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovaginal cell pellets from the residual fluid-based Papanicolaou cytologic specimen. This relatively simple and widely used collection system will allow significant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test.

Ectopic production and localization of beta-human chorionic gonadotropin in lymphoepithelioma-like carcinoma of the cervix: a case report.

A 32-year-old woman underwent a suction curettage for missed abortion. The initial serum human chorionic gonadotropin (beta-hCG) level was 40 IU/ml. The histologic examination of the uterine curettage specimen showed scant strips of a poorly differentiated malignant neoplasm and no chorionic villi. The tumor showed strong immunoreactivity for cytokeratin (AE1/AE3) and beta-hCG but no reactivity for human placental lactogen. The combination of histologic appearance, beta-hCG immunoreactivity, and elevation of serum beta-hCG raised a strong suspicion for epithelioid trophoblastic tumor (ETT). Postcurettage serial serum beta-hCG levels remained in the range of 20 to 45 micrograms/ml. Computerized tomographic scan showed a 1.0-cm circumscribed mass in the upper endocervix. A radical hysterectomy and pelvic lymphadenectomy were performed. Gross examination of the hysterectomy specimen likewise showed a well-circumscribed mass in the upper endocervix. Histologic examination revealed an undifferentiated carcinoma accompanied by intense lymphoplasmacytic infiltrate. A final diagnosis of lymphoepithelioma-like carcinoma (LELC) was rendered. LELC with elevated serum beta-hCG level and immunoreactivity to beta-hCG should be distinguished from ETT in a small endocervical curettage sample.

Allelic loss and microsatellite alterations of chromosome 3p14.2 are more frequent in recurrent cervical dysplasias.

Epidemiological studies have documented the unpredictable clinical progression or recurrence of cervical dysplasia. Recent studies have shown several molecular changes in cervical cancers and their associated dysplasia. We conducted molecular analyses on a retrospectively ascertained cohort of recurrent and nonrecurrent cervical dysplasia cases in an attempt to define molecular biomarkers to predict progressive or recurrent disease. Cases were chosen if long-term follow-up (3-5 years after conization) and biopsy confirmation were available. Paraffin-embedded, postconization cervical tissues from 19 recurrent and 18 nonrecurrent dysplasias were analyzed. Human papillomavirus (HPV) was identified by PCR for general and type-specific (HPV-16 and HPV-18) primers. Allelotyping analysis was performed by multiplex PCR using a panel of 16 microsatellite markers targeting putative tumor suppressor gene regions on chromosomes 3p, 5p, 6p, 9p, 11q, and 17p. The overall rate of HPV infection was similar in both groups. In the allelotyping analysis, loss of heterozygosity at the fragile histidine triad region in 3p14.2 was significantly higher in the recurrent group than in the nonrecurrent group (P = 0.005). Furthermore, microsatellite alterations (MAs) were more frequent in the recurrent group (mean MA index, 0.254) as compared with the nonrecurrent group (mean MA index, 0.085; P = 0.0025). These findings suggest that HPV status alone does not predict recurrence and that loss of heterozygos. ity at the fragile histidine triad region may represent a potential biomarker in predicting recurrence. Frequent MAs in the recurrent group may represent an underlying genomic instability that creates susceptibility for allelic loss, thus increasing the risk for recurrence or progression.

Role of TSG101 in uterine cervix cancer.

OBJECTIVE: TSG101 was first described as a possible tumor suppressor gene in breast cancer. To determine whether TSG101 might play a role in cervical carcinogenesis, we examined a panel of cervical cancer cell lines and primary tumor specimens for transcript abnormalities and mutations in TSG101. METHODS: Total RNA was derived from cell line cultures or primary tumor specimens. We performed nested reverse transcription polymerase chain reaction (RT-PCR) with eight overlapping primer sets, followed by single-strand conformational polymorphism (SSCP) analysis, to screen for mutations in the TSG101 open reading frame. Representative normal and shifted SSCP bands were sequenced. To identify abnormal-sized transcripts, we performed RT-PCR with primers flanking the open reading frame followed by gel electrophoresis. RESULTS: Mutational analysis was performed on cDNAs from 20 primary cervical tumors and 8 cervical carcinoma cell lines. Two polymorphisms were identified, neither of which resulted in an altered amino acid sequence. Transcript analysis was performed on a subset of 16 primary cervix tumors and 6 cervix carcinoma cell lines. The wild-type transcript (1228 bp) was the dominant transcript expressed in all samples. A transcript measuring 330 bp was detected in 5 of 6 cell lines and 11 of 16 primary tumor specimens. CONCLUSION: Our results suggest that mutations in TSG101 rarely occur in carcinomas of the uterine cervix. However, the presence of minor aberrant TSG101 transcripts is a common feature. The relationship between aberrant transcription and carcinogenesis should be further investigated.

Frequent expression of beta-human chorionic gonadotropin (beta-hCG) in squamous cell carcinoma of the cervix.

Human chorionic gonadotropin (beta-hCG) has been detected within tissue homogenates, culture fluid, and sera of patients with squamous cell carcinoma of the cervix. Studies regarding in vivo localization of beta-hCG in squamous cell carcinoma of the cervix are scant and conflicting. Cervical samplings (biopsy and/or curettage specimens) of 63 cases of poorly differentiated invasive squamous cell carcinoma of the cervix were initially stained by the immunoperoxidase technique for the presence of beta-hCG and human placental lactogen (hPL). Based on beta-hCG reactivity, patients were divided into beta-hCG-positive and beta-hCG-negative groups. Thirty-three of the 63 (52%) cases showed localization of beta-hCG in tumor cells. Subsequent specimens of patients, who underwent surgical treatment, were likewise examined for beta-hCG reactivity. These surgical specimens showed focal beta-hCG reactivity in the beta-hCG-positive group only. The beta-hCG reactivity was seen in both high-grade SIL (CIN III), invasive squamous cell carcinoma, and its metastases. The focal beta-hCG reactivity was predominantly confined to the peripheral tumor cells at the stromal-epithelial interface in noninvasive and invasive lesions. Intensity of immunostaining was moderate to strong. The beta-hCG staining was observed in different cancer stages and in various age groups. No hPL reactivity was seen in any cases. Poorly differentiated squamous cell carcinoma of uterine cervix showing immunoreactivity for beta-hCG should be distinguished from choriocarcinoma and other trophoblastic tumors.

Comparison of molecular changes in cervical intraepithelial neoplasia in HIV-positive and HIV-indeterminate subjects.

OBJECTIVE: HIV infection is associated with an increased incidence of cervical malignancy and its precursor lesions (CIN, cervical intraepithelial neoplasia) compared with the general population. We studied the molecular abnormalities in the development of HIV-associated CIN and compared them with those present in CINs arising in HIV-indeterminate subjects ("sporadic CIN"). METHODS: We investigated the presence of human papilloma virus (HPV) sequences, loss of heterozygosity (LOH), and microsatellite alterations (MAs) at five 3p chromosomal regions using 17 polymorphic markers in precisely microdissected archival tissues from 16 HIV-positive CINs and compared them with those present in 39 sporadic CINs. RESULTS: HPV sequences were detected in 36 of 55 (66%) CIN lesions, and high-risk oncogenic strains (HPV 16 and 18) accounted for 15 of them. No differences in the HPV frequencies were found between HIV-associated and sporadic CINs. Allelic losses at one or more chromosome 3p regions were frequently detected in CIN lesions (49%). The overall frequency of 3p LOH and the frequencies at all individual regions were similar in HIV-associated and sporadic CINs. The frequency of MA present in the HIV-associated CIN cases (0.093) was sixfold greater than in sporadic CINs (0.014; P = 0.0001). At least 1 MA was present in 11 (69%) of 16 HIV-associated vs. 5 of 39 (13%) sporadic CIN (P = 0.0006). Molecular changes were independent of the presence of HPV sequences. CONCLUSION: Chromosome 3p deletions are frequently detected in the precursor lesions of cervical carcinoma (CIN) and there are no differences in the 3p LOH frequencies between HIV-associated and sporadic CIN lesions. Microsatellite alterations, which reflect widespread genomic instability, occur at greatly increased frequency in HIV-associated CIN. Although the mechanism underlying the development of increased MAs is unknown, it may play a crucial role in the development of many HIV-associated neoplasias.

Genetic changes during the multistage pathogenesis of human papillomavirus positive and negative vulvar carcinomas.

OBJECTIVE: To identify the molecular alterations found in 30 human papillomavirus (HPV) positive (n = 15) and negative (n = 15) vulvar carcinomas (VC) and their associated preinvasive lesions (VIN [vulvar intraepithelial neoplasia]) and normal epithelium to determine a common molecular pathogenesis of HPV positive and negative VC. METHODS: Loss of heterozygosity (LOH) at seven 3p chromosomal regions (3p12, 3p14.2, 3p14.3-21.1, 3p21.3, 3p22-24, 3p24.3, 3p25), 13q14 (RB) and 17p13.1 (p53) loci, and TP53 gene mutations in microdissected archival tissues were investigated. RESULTS: Fourteen of fifteen HPV positive VC had HPV 16 DNA sequences. The fractional regional loss index (FRL), an index of total allelic loss at all chromosomal regions analyzed, was greater in the HPV negative VCs than in the HPV positive tumors (FRL = 0.55 versus 0.32; P = .048) and was also greater in the HPV negative high-grade VINs as compared with the HPV positive lesions (0.29 versus 0.02; P = .002). Overall, LOH at any 3p region was frequent (80%) in both groups of cancers and in their associated VIN lesions. Although TP53 gene mutations were present in a minority of VCs (20%), allelic losses at the TP53 locus were frequently present, especially in HPV negative VCs, as compared with the HPV positive tumors (62% versus 15%; P = .02). CONCLUSION: A greater number of molecular alterations are found in HPV negative VCs compared with HPV positive tumors. Allelic losses at 3p are common early events in vulvar carcinogenesis in HPV negative cancers detected at a high rate in the corresponding high-grade precursor lesions (VIN II/III). TP53 gene mutations with associated 17p13.1 LOH are more common in HPV negative cancers.

Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas.

Mutations of the human putative protein tyrosine phosphatase (PTEN/MMAC1) gene at chromosome 10q23 have been found frequently in type I endometrial carcinomas. Endometrioid adenocarcinoma is the most frequent histology seen in patients with clinically determined synchronous endometrial and ovarian carcinomas. We report a high incidence of PTEN/MMAC1 mutations and 10q23 loss of heterozygosity (LOH) in patients with synchronous endometrial and ovarian carcinomas. Paraffin-embedded precision microdissected tumors were analyzed for 10 matched synchronous endometrial and ovarian cancers and 11 matched control metastatic endometrial cancers. Single-stranded conformation polymorphism analysis was used to screen for mutations in all tumors and corresponding normal lymphocyte DNA. LOH was determined using a panel of four microsatellite markers within the PTEN/MMAC1 locus. PTEN/MMAC1 mutations were found in 43% (9 of 21) of the endometrial cancers studied, similarly represented in the clinically synchronous group (5 of 10 or 50%) and the advanced metastatic group (4 of 11; 36%; P = 0.53). In two of the five cases of clinically synchronous cancers, identical or progressive PTEN mutations were found in both the endometrial and ovarian cancers, suggesting that the ovarian tumor is a metastasis from the endometrial primary. PTEN/MMAC1 mutations in the advanced endometrial cancers were similar in the corresponding metastases. In one case, the mutation was seen in only one of two metastatic lymph nodes. The LOH analysis demonstrated 55% LOH in at least one PTEN/MMAC1 marker. These findings suggest that the putative tumor suppressor gene PTEN/MMAC1 may be a viable molecular marker to differentiate synchronous versus metastatic disease in a subset of clinically synchronous endometrial and ovarian carcinomas.

In vivo studies of adenovirus-based p53 gene therapy for ovarian cancer.

OBJECTIVES: To test the safety, efficacy, and toxicity of gene therapy using wild-type p53-expressing adenovirus (Ad-CMV-p53) in a nude mouse model with intraperitoneal (i.p.) 2774 human ovarian cancer cell line that contains a p53 mutation. STUDY DESIGN: An initial study of adenovirus tolerance was determined in nude mice by a single i.p. injection of increasing doses of Ad-CMV-p53. Nude mice were implanted with an LD100 dose of 1 x 10(7) cells. To study the efficacy and specificity of Ad-CMV-p53 treatment, the mice received treatment with different adenovirus constructs. One group received Ad-CMV-p53 and another group received a control adenovirus construct, Ad-CMV-beta gal. To study the treatment response to Ad-CMV-p53, the mice were divided into groups and received various treatment schedules of 1 x 10(8) pfu of Ad-CMV-p53. RESULTS: The mice tolerated Ad-CMV-p53 without adverse effects at doses of 1 x 10(8) pfu. The response to Ad-CMV-p53 showed significant survival duration in each dose regimen, with a survival time greater than that of untreated animals (P = 0.0173). However, no statistically significant survival advantage was observed between Ad-CMV-p53- and Ad-CMV-beta gal-treated mice. CONCLUSIONS: These studies show that at the adenovirus dose and administration regimen used, there is effective but not specific 2774 tumor growth inhibition in vivo. Efficient introduction of biologically active genes into tumor cells would greatly facilitate cancer therapy. Thus, although promising, these results caution that much effort will be required to realize the potential for clinical application of adenovirus-based ovarian cancer gene therapy.

Abnormalities of fragile histidine triad genomic and complementary DNAs in cervical cancer: association with human papillomavirus type.

BACKGROUND: Chromosome 3p14.2 contains FRA3B, the most active chromosome breakage site in the human genome. The fragile histidine triad (FHIT) gene, a putative tumor suppressor gene, overlaps FRA3B. Human papillomavirus (HPV), a known cofactor in cervical carcinogenesis, can integrate into FRA3B. We examined abnormalities in FHIT and its RNA transcripts in cervical cancer cell lines and tumors. We also investigated the relationship between loss of heterozygosity (LOH) in FHIT/FRA3B and the presence of oncogenic HPV types. METHODS: Eleven cell lines, 40 tumors (20 fresh and 20 archival), and 10 normal cervical epithelia were examined. Two intragenic polymorphic markers (D3S1300 and D3S4103) and the polymerase chain reaction (PCR) were used to examine FHIT LOH. Reverse transcription-PCR (RT-PCR) analysis and single-strand conformation polymorphism analysis of RT-PCR products were used to characterize FHIT transcripts. Oncogenic HPV types were identified by PCR, using general and type-specific primers. RESULTS: All normal epithelia, 19 of 20 fresh tumors and nine of 11 cell lines expressed wild-type and, occasionally, exon 8-deleted FHIT transcripts. Additional aberrant FHIT transcripts were seen in nine of 20 fresh tumors and in seven of 11 cell lines. DNA sequencing of the aberrant transcripts revealed a variety of insertions and deletions but no point mutations. Three cell lines also had homozygous FHIT deletions. Oncogenic HPV types (i.e., 16, 18, 31, and 33) were detected in 18 of 20 archival tumors, and, in these tumors, LOH within FHIT was identified in nine of 16 informative cases. HPV 16 was found to be associated with LOH in the FHIT/FRA3B region (P = .041). CONCLUSION: FHIT/FRA3B is frequently altered in cervical cancer, demonstrating LOH, occasional homozygous deletions, and frequent aberrant transcripts not found in normal epithelia. However, the presence of wild-type transcripts and the lack of protein-altering point mutations raise questions about FHIT's function as a classic tumor suppressor gene in cervical tissue.

Mutations in the BRCA1-associated RING domain (BARD1) gene in primary breast, ovarian and uterine cancers.

Germline alterations of BRCA1 result in susceptibility to breast and ovarian cancer. The protein encoded by BRCA1 interacts in vivo with the BRCA1-associated RING domain (BARD1) protein. Accordingly, BARD1 is likely to be a critical factor in BRCA1-mediated tumor suppression and may also serve as a target for tumorigenic lesions in some human cancers. We have now determined the genomic structure of BARD1 and performed a mutational analysis of 58 ovarian tumors, 50 breast tumors and 60 uterine tumors. Seven polymorphisms were detected within the 2.34 kb coding sequence of BARD1 . Somatically acquired missense mutations were observed in one breast carcinoma and one endometrial tumor; in at least one of these cases, tumor formation was accompanied by loss of the wild-type BARD1 allele, following the paradigm for known tumor suppressor genes. In addition, a germline alteration of BARD1 was identified in a clear cell ovarian tumor (Gln564His); again, loss of the wild-type BARD1 allele was observed in the malignant cells of this patient. The Gln564His patient was also diagnosed with two other primary cancers: a synchronous lobular breast carcinoma and a stage IA clear cell endometrioid cancer confined to an endometrial polyp 6 years earlier. These findings suggest an occasional role for BARD1 mutations in the development of sporadic and hereditary tumors.