Perifosine plus docetaxel in patients with platinum and taxane resistant or refractory high-grade epithelial ovarian cancer.

OBJECTIVES: On the basis of reversal of taxane resistance with AKT inhibition, we initiated a phase I trial of the AKT inhibitor perifosine with docetaxel in taxane and platinum-resistant or refractory epithelial ovarian cancer. METHODS: Patients with pathologically confirmed high-grade epithelial ovarian cancer (taxane resistant, n=10; taxane refractory, n=11) were enrolled. Peripheral blood samples and tumor biopsies were obtained and (18)F-FDG-PET and DCE-MRI scans were performed for pharmacodynamic and imaging studies. RESULTS: Patients received a total of 42 treatment cycles. No dose-limiting toxicity was observed. The median progression-free survival and overall survival were 1.9 months and 4.5 months, respectively. One patient with a PTEN mutation achieved a partial remission (PR) for 7.5 months, and another patient with a PIK3CA mutation had stable disease (SD) for 4 months. Two other patients without apparent PI3K pathway aberrations achieved SD. Two patients with KRAS mutations demonstrated rapid progression. Decreased phosphorylated S6 correlated with (18)F-FDG-PET responses. CONCLUSIONS: Patients tolerated perifosine 150 mg PO daily plus docetaxel at 75 mg/m(2) every 4 weeks. Further clinical evaluation of effects of perifosine with docetaxel on biological markers and efficacy in patients with ovarian cancer with defined PI3K pathway mutational status is warranted.

Optical technologies and molecular imaging for cervical neoplasia: a program project update.

There is an urgent global need for effective and affordable approaches to cervical cancer screening and diagnosis. In developing nations, cervical malignancies remain the leading cause of cancer-related deaths in women. This reality may be difficult to accept given that these deaths are largely preventable; where cervical screening programs have been implemented, cervical cancer-related deaths have decreased dramatically. In developed countries, the challenges of cervical disease stem from high costs and overtreatment. The National Cancer Institute-funded Program Project is evaluating the applicability of optical technologies in cervical cancer. The mandate of the project is to create tools for disease detection and diagnosis that are inexpensive, require minimal expertise, are more accurate than existing modalities, and can be feasibly implemented in a variety of clinical settings. This article presents the status and long-term goals of the project.

A new approach for identifying patients with ovarian epithelial neoplasms based on high-resolution mass spectrometry.

We investigated the serum profiles of patients with ovarian neoplasm using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) to obtain serum "fingerprints" for use in identifying patients with these neoplasms. We used HPLC-HRMS to analyze serum samples from patients with ovarian neoplasms and control subjects. Serum samples from 145 patients were analyzed, including 85 with ovarian epithelial neoplasms. We also compared the results of this serum-fingerprinting approach with the results of the CA-125 test and imaging. Fingerprinting successfully permitted the separation of control patients and patients with ovarian neoplasms. The sensitivity and specificity of the test were between 96% and 100%. When the results of this test were concordant with the results of the CA-125 test, 99% of serum samples were correctly classified as being from a patient with an ovarian neoplasm or with no ovarian neoplasm. We found that a metabolite of molecular weight 472 is the main metabolite in the separation of patients with ovarian neoplasms from control subjects. HPLC-HRMS serum profiling could become a screening test for ovarian neoplasms.

Ultrastaging improves detection of metastases in sentinel lymph nodes of uterine cervix squamous cell carcinoma.

The technique of sentinel lymph node (SLN) detection is increasingly being applied in patients with uterine cervix carcinoma. This study presents the pathologic findings of SLNs in 48 such patients. The institutional pathology files were searched for all patients with a diagnosis of cervical squamous cell carcinoma who had SLNs reported. Patient age, follow-up, tumor size, presence/absence of lymphatic invasion, number and status of SLNs and non-SLNs, location of SLNs, and size of metastases in SLNs were recorded. All SLNs were sectioned in 2-mm slices perpendicular to the long axis and submitted entirely for microscopic examination. For all SLNs negative on the initial hematoxylin and eosin (H&E) stained slides, an ultrastaging protocol was performed consisting of 5 sets of slides at 40-mum intervals (1 H&E slide+2 unstained slides), representing an additional 5 intervals. Lymph nodes negative by the additional H&E intervals had immunohistochemistry for cytokeratin performed on 1 unstained slide. Forty-eight patients ranging from 25 to 62 years of age had a total of 208 SLNs removed. Fifteen (31%) patients had positive SLNs with 1 to 5 positive SLNs per case. The metastasis size ranged from a single cell to 27 mm. Twelve patients had metastasis detected by routine processing in 23 SLNs, whereas ultrastaging detected metastases in 3 SLNs of 3 additional patients. In 2 patients with metastasis detected by ultrastaging, the metastasis was detected by wide H&E intervals (level 2 for 1 patient; level 3 for 1 patient); in 1 patient, the metastasis was detected only by immunohistochemistry and consisted of a single cell. Of the 15 patients with positive SLNs, 3 patients had a total of 6 positive non-SLNs. All of the patients with a positive SLN are currently living. Thirty-three (69%) patients had negative SLNs. Of these, 1 patient had a single positive non-SLN for a false negative rate of 6.25%. Negative SLN predicts negative non-SLN. For most patients with a positive SLN, the SLN will be the only metastasis detected; a minority of patients with a positive SLN may have a positive non-SLN.

Tumor suppressor 101F6 and ascorbate synergistically and selectively inhibit non-small cell lung cancer growth by caspase-independent apoptosis and autophagy.

101F6 is a candidate tumor suppressor gene harbored on chromosome 3p21.3, a region with frequent and early allele loss and genetic alterations in many human cancers. We previously showed that enforced expression of wild-type 101F6 by adenoviral vector-mediated gene transfer significantly inhibited tumor cell growth in 3p21.3-deficient non-small cell lung cancer (NSCLC) cells in vitro and in vivo. The molecular mechanism of 101F6-mediated tumor suppression is largely unknown. A computer-aided structural and functional model predicts the 101F6 protein to be a member of the cytochrome b561 protein family that is involved in the regeneration of the antioxidant ascorbate. 101F6 protein is expressed in normal lung bronchial epithelial cells and fibroblasts but is lost in most lung cancers. Treatment with 101F6 nanoparticle-mediated gene transfer in combination with a subpharmacologic dose (200-500 micromol/L) of ascorbate synergistically and selectively inhibited lung cancer cell growth in vitro. Systemic injection of 101F6 nanoparticles plus the i.p. injection of ascorbate synergistically inhibited both tumor formation and growth in human NSCLC H322 orthotopic lung cancer mouse models (P<0.001). Furthermore, exogenous expression of 101F6 enhanced intracellular uptake of ascorbate, leading to an accumulation of cytotoxic H(2)O(2) and a synergistic killing of tumor cells through caspase-independent apoptotic and autophagic pathways. The antitumor synergism showed by the combination treatment with systemic administration of 101F6 nanoparticles and ascorbate on lung cancer offers an attractive therapeutic strategy for future clinical trials in cancer prevention and treatment.

Urinary mesothelin provides greater sensitivity for early stage ovarian cancer than serum mesothelin, urinary hCG free beta subunit and urinary hCG beta core fragment.

OBJECTIVES: Early detection of ovarian cancer should improve overall survival. Multiple serum markers have been evaluated as possible tests to detect early stage disease, but few urine markers have been studied. Mesothelin has been detected in serum from patients with ovarian cancer, but has not been previously reported in urine. METHODS: Mesothelin was assayed in the serum and in the urine from 28 patients with early stage (I/II) invasive epithelial ovarian cancers, 111 with advanced stage (III/IV) invasive disease and 19 with tumors of low malignant potential. Marker values have been compared to those in healthy controls and 115 patients with benign pelvic masses. Thresholds were set to include 95% of mesothelin values for 127 sera and 89 urines from healthy women. Urine values were considered: (1) as assayed; (2) normalized using the ratio of serum to urine creatinine; and (3) normalized using the Cockroft-Gault formula for glomerular filtration rate (GFR). Urines were also assayed for human chorionic gonadotropin (hCG) free beta subunit and beta subunit core fragment and similarly normalized. RESULTS: Optimal sensitivity for early stage disease was obtained when urine mesothelin was normalized using GFR. A greater fraction of patients with early stage disease was detected with the mesothelin urine assay (42%) than with the serum assay (12%). Similarly, 75% of patients with advanced ovarian cancer had elevated mesothelin in urine compared to 48% in serum. Serum and urine levels of mesothelin correlated for early (p=0.02) and late (p<0.001) disease. Urine mesothelin exhibited greater sensitivity for early stage ovarian cancer than did hCG free beta subunit or beta subunit core fragment and complementarity was not observed. CONCLUSION: Urine mesothelin deserves further evaluation as a biomarker for detection of early stage ovarian cancer in combination with other urinary markers.

Kappa statistics to measure interrater and intrarater agreement for 1790 cervical biopsy specimens among twelve pathologists: qualitative histopathologic analysis and methodologic issues.

BACKGROUND: As part of a program project to evaluate emerging optical technologies for cervical neoplasia, we performed fluorescence and reflectance spectroscopic examinations of patients with abnormal Papanicolaou smears. Biopsy specimens were taken from each area and measured optically, and study pathologists performed qualitative histopathologic readings. Several methodologic issues arose in this analysis: (1) the interpathologist and intrapathologist agreement between institutions for the 1790 biopsy specimens; (2) the interinstitutional agreement among the two institutions conducting the trials on 117 randomly chosen biopsy specimens; (3) the interinstitutional agreement among the two institutions and a third expert gynecologic pathologist to ensure the expert readings were comparable to those outside both institutions on 117 randomly chosen biopsy specimens; and (4) an additional three reviews of the 106 difficult biopsy specimens by all three institutions. METHODS: All 1790 specimens from 850 patients were reviewed three times at each institution in blinded fashion; those for which the first and second reviews were identical were not reviewed a third time. A randomly selected sample of 117 specimens was randomly ordered and read by study pathologists at The University of Texas M. D. Anderson Cancer Center, British Columbia Cancer Agency (BCCA), and Brigham and Women's Hospital (BWH). The 106 difficult cases were treated in the same manner as the randomized and random-ordered cases. Generalized, unweighted, and weighted kappas and their 95% confidence intervals were used to assess agreement. Binary comparisons were used to compare diagnostic categories. FINDINGS: The kappas for the three readings of the overall data set using eight-category World Health Organization (WHO) criteria were as follows: 0.66 for the generalized, 0.72 for weighted, and ranged from 0.59 to 0.94 unweighted binary categories; those read using four-category Bethesda criteria: 0.70 for generalized, 0.69 for weighted, and 0.56-0.94 for unweighted binary categories. For the pool versus the study pathologist readings, the eight-category kappa was 0.51 for generalized, 0.72 for weighted, and 0.56-0.82 for unweighted binary categories; for those read using Bethesda criteria: 0.70 for generalized, 0.70 for weighted, and 0.59-0.82 for the unweighted binary categories. The interpathologist and intrapathologist readings were fair by Landis standards at the low end of the diagnostic scale (atypia, human papillomavirus, and CIN1) and substantial to almost perfect at the high end (CIN2, CIN3, and CIS). The randomly selected and randomly ordered sample of 117 specimens read with the WHO system yielded a generalized kappa of 0.45; among the three institutions (M. D. Anderson Cancer Center vs. BCCA, M. D. Anderson vs. BWH, and BCCA vs. BWH), the unweighted kappas were 0.46, 0.41, and 0.49 and the weighted were 0.65, 0.66, and 0.68, respectively; for the Bethesda, a generalized kappa of 0.65, unweighted kappas of 0.66, 0.65, and 0.47, and weighted of 0.74, 0.72, and 0.74. The difficult specimens read with the WHO system yielded a generalized kappa of 0.23; among the three institutions the unweighted kappas were 0.20, 0.30, and 0.37, and the weighted were 0.17, 0.34, and 0.31; for the Bethesda, a generalized kappa of 0.25; among the three institutions, the unweighted kappas were 0.21, 0.32, and 0.37, and the weighted were: 0.07, 0.21, and 0.37, respectively. INTERPRETATION: Kappas in this expert group of pathologists were in the moderate, substantial, and almost perfect ranges for the overall and randomized samples. The randomized sample was representative of the larger sample. The kappa of the specimens for which disagreements arose was, predictably, in the slight range. Our findings will aid both the correlations with optical measurements using fluorescence and reflectance spectroscopy and the quantitative histopathologic analysis of these study specimens.

Patterns of gene expression in different histotypes of epithelial ovarian cancer correlate with those in normal fallopian tube, endometrium, and colon.

PURPOSE: Epithelial ovarian cancers are thought to arise from flattened epithelial cells that cover the ovarian surface or that line inclusion cysts. During malignant transformation, different histotypes arise that resemble epithelial cells from normal fallopian tube, endometrium, and intestine. This study compares gene expression in serous, endometrioid, clear cell, and mucinous ovarian cancers with that in the normal tissues that they resemble. EXPERIMENTAL DESIGN: Expression of 63,000 probe sets was measured in 50 ovarian cancers, in 5 pools of normal ovarian epithelial brushings, and in mucosal scrapings from 4 normal fallopian tube, 5 endometrium, and 4 colon specimens. Using rank-sum analysis, genes whose expressions best differentiated the ovarian cancer histotypes and normal ovarian epithelium were used to determine whether a correlation based on gene expression existed between ovarian cancer histotypes and the normal tissues they resemble. RESULTS: When compared with normal ovarian epithelial brushings, alterations in serous tumors correlated with those in normal fallopian tube (P = 0.0042) but not in other normal tissues. Similarly, mucinous cancers correlated with those in normal colonic mucosa (P = 0.0003), and both endometrioid and clear cell histotypes correlated with changes in normal endometrium (P = 0.0172 and 0.0002, respectively). Mucinous cancers displayed the greatest number of alterations in gene expression when compared with normal ovarian epithelial cells. CONCLUSION: Studies at a molecular level show distinct expression profiles of different histologies of ovarian cancer and support the long-held belief that histotypes of ovarian cancers come to resemble normal fallopian tube, endometrial, and colonic epithelium. Several potential molecular markers for mucinous ovarian cancers have been identified.

Atypical PKCiota contributes to poor prognosis through loss of apical-basal polarity and cyclin E overexpression in ovarian cancer.

We show that atypical PKCiota, which plays a critical role in the establishment and maintenance of epithelial cell polarity, is genomically amplified and overexpressed in serous epithelial ovarian cancers. Furthermore, PKCiota protein is markedly increased or mislocalized in all serous ovarian cancers. An increased PKCiota DNA copy number is associated with decreased progression-free survival in serous epithelial ovarian cancers. In a Drosophila in vivo epithelial tissue model, overexpression of persistently active atypical PKC results in defects in apical-basal polarity, increased Cyclin E protein expression, and increased proliferation. Similar to the Drosophila model, increased PKCiota proteins levels are associated with increased Cyclin E protein expression and proliferation in ovarian cancers. In nonserous ovarian cancers, increased PKCiota protein levels, particularly in the presence of Cyclin E, are associated with markedly decreased overall survival. These results implicate PKCiota as a potential oncogene in ovarian cancer regulating epithelial cell polarity and proliferation and suggest that PKCiota is a novel target for therapy.

Phase II trial of capecitabine in recurrent squamous cell carcinoma of the cervix.

PURPOSE: To determine the efficacy and safety of capecitabine in women with inoperable, recurrent, or metastatic squamous cell cervical cancer. PATIENTS AND METHODS: In a phase II IRB approved trial, capecitabine was given at a dosage of 2000 mg/m2/day orally in a divided dose daily for 14 days followed by a 7-day rest period. A standard dose modification scheme was used with one allowed dose reduction or dose escalation. National Cancer Institute criteria for progression, response, and toxicity were utilized. Quality of life data were obtained using the Memorial Symptom Assessment Scale and Functional Assessment for Cancer Therapy, which included a subscale for cervical cancer. RESULTS: Twenty of 23 enrolled patients were evaluable for response. Stable disease was noted in 5 patients, with a median duration of response of 3.5 months (range, 3-6.5 months). No partial or complete responses were seen. Common grade 3 toxicities were fatigue (30.4%); abdominal pain, constipation, hand-foot syndrome, nausea, and vomiting (8.7% each); as well as dyspnea, headache, and coagulopathy (4.3% each). There were no grade 4 toxicities. All patients with previous exposure to infused 5-FU had evidence of progression. No statistically significant changes in quality of life were noted from baseline to post-cycle 2. CONCLUSION: Single-agent capecitabine in patients with recurrent cervical cancer resulted in no objective responses. Although capecitabine is a well-tolerated regimen, as a single agent, it offers minimal benefit in a poor-prognosis cervical cancer population.

Combined reflectance and fluorescence spectroscopy for in vivo detection of cervical pre-cancer.

Optical technologies, such as reflectance and fluorescence spectroscopy, have shown the potential to provide improved point-of-care detection methods for cervical neoplasia that are sensitive, specific, and cost-effective. Our specific goals are to analyze the diagnostic potential of reflectance and fluorescence spectra, alone and in combination, to discriminate normal and precancerous cervical tissue in vivo and to identify which classification features contain significant diagnostic information. Reflectance spectra are measured at four source-detector separations and fluorescence emission spectra are measured at 16 excitation wavelengths, from 324 sites in 161 patients. These 20 spectral features are permuted in all possible combinations of one, two, and three; and classification algorithms are developed to evaluate the diagnostic performance of each combination. Algorithms based on fluorescence spectra alone yield better diagnostic performance than those based on reflectance spectra alone. The combination of fluorescence and reflectance do not significantly improve diagnostic performance compared to fluorescence alone, except in the case of discriminating high-grade precancers from columnar normal tissue. In general, fluorescence emission spectra at 330- to 360-nm and 460- to 470-nm excitation provide the best diagnostic performance for separating all pairs of tissue categories.

Synergistic tumor suppression by coexpression of FHIT and p53 coincides with FHIT-mediated MDM2 inactivation and p53 stabilization in human non-small cell lung cancer cells.

Aberrations of the tumor suppressor genes FHIT and p53 are frequently associated with a wide range of human cancers, including lung cancer. We studied the combined effects of FHIT and p53 proteins on tumor cell proliferation and apoptosis in human non-small cell lung carcinoma (NSCLC) cells in vitro and on tumor growth in animal models by adenoviral vector-mediated cotransfer of wild-type FHIT and p53 genes. We found that the coexpression of FHIT and p53 synergistically inhibited tumor cell proliferation in NSCLC cells in vitro and suppressed the growth of human tumor xenografts in nude mice. Furthermore, we found that this synergistic inhibition of tumor cell growth corresponded with the FHIT-mediated inactivation of MDM2, which thereby blocked the association of MDM2 with p53, thus stabilizing the p53 protein. Our results therefore reveal a novel molecular mechanism consisting of FHIT-mediated tumor suppression and the interaction of FHIT with other cellular components in the pathways regulating p53 activity. These findings show that combination treatment with synergistic tumor-suppressing gene therapy such as Ad-FHIT and Ad-p53 may be an effective therapeutic strategy for NSCLC and other cancers.

Myristoylation of the fus1 protein is required for tumor suppression in human lung cancer cells.

FUS1 is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region that is deleted in many cancers. Using surface-enhanced laser desorption/ionization mass spectrometric analysis on an anti-Fus1-antibody-capture ProteinChip array, we identified wild-type Fus1 as an N-myristoylated protein. N-myristoylation is a protein modification process in which a 14-carbon myristoyl group is cotranslationally and covalently added to the NH2-terminal glycine residue of the nascent polypeptide. Loss of expression or a defect of myristoylation of the Fus1 protein was observed in human primary lung cancer and cancer cell lines. A myristoylation-deficient mutant of the Fus1 protein abrogated its ability to inhibit tumor cell-induced clonogenicity in vitro, to induce apoptosis in lung tumor cells, and to suppress the growth of tumor xenografts and lung metastases in vivo and rendered it susceptible to rapid proteasome-dependent degradation. Our results show that myristoylation is required for Fus1-mediated tumor-suppressing activity and suggest a novel mechanism for the inactivation of tumor suppressors in lung cancer and a role for deficient posttranslational modification in tumor suppressor-gene-mediated carcinogenesis.

Selection of potential markers for epithelial ovarian cancer with gene expression arrays and recursive descent partition analysis.

PURPOSE: Advanced-stage epithelial ovarian cancer has a poor prognosis with long-term survival in less than 30% of patients. When the disease is detected in stage I, more than 90% of patients can be cured by conventional therapy. Screening for early-stage disease with individual serum tumor markers, such as CA125, is limited by the fact that no single marker is up-regulated and shed in adequate amounts by all ovarian cancers. Consequently, use of multiple markers in combination might detect a larger fraction of early-stage ovarian cancers. EXPERIMENTAL DESIGN: To identify potential candidates for novel markers, we have used Affymetrix human genome arrays (U95 series) to analyze differences in gene expression of 41,441 known genes and expressed sequence tags between five pools of normal ovarian surface epithelial cells (OSE) and 42 epithelial ovarian cancers of different stages, grades, and histotypes. Recursive descent partition analysis (RDPA) was performed with 102 probe sets representing 86 genes that were up-regulated at least 3-fold in epithelial ovarian cancers when compared with normal OSE. In addition, a panel of 11 genes known to encode potential tumor markers [mucin 1, transmembrane (MUC1), mucin 16 (CA125), mesothelin, WAP four-disulfide core domain 2 (HE4), kallikrein 6, kallikrein 10, matrix metalloproteinase 2, prostasin, osteopontin, tetranectin, and inhibin] were similarly analyzed. RESULTS: The 3-fold up-regulated genes were examined and four genes [Notch homologue 3 (NOTCH3), E2F transcription factor 3 (E2F3), GTPase activating protein (RACGAP1), and hematological and neurological expressed 1 (HN1)] distinguished all tumor samples from normal OSE. The 3-fold up-regulated genes were analyzed using RDPA, and the combination of elevated claudin 3 (CLDN3) and elevated vascular endothelial growth factor (VEGF) distinguished the cancers from normal OSE. The 11 known markers were analyzed using RDPA, and a combination of HE4, CA125, and MUC1 expression could distinguish tumor from normal specimens. Expression at the mRNA level in the candidate markers was examined via semiquantitative reverse transcription-PCR and was found to correlate well with the array data. Immunohistochemistry was performed to identify expression of the genes at the protein level in 158 ovarian cancers of different histotypes. A combination of CLDN3, CA125, and MUC1 stained 157 (99.4%) of 158 cancers, and all of the tumors were detected with a combination of CLDN3, CA125, MUC1, and VEGF. CONCLUSIONS: Our data are consistent with the possibility that a limited number of markers in combination might identify >99% of epithelial ovarian cancers despite the heterogeneity of the disease.

Grading ovarian serous carcinoma using a two-tier system.

In this study, we evaluate a two-tier system for grading ovarian serous carcinoma. This system is based primarily on the assessment of nuclear atypia with the mitotic rate used as a secondary feature. The study included 50 cases of low-grade ovarian serous carcinoma and 50 cases of high-grade ovarian serous carcinoma retrieved from the files of the Department of Pathology at the University of Texas M. D. Anderson Cancer Center from a 28-year period. Cases assigned to the low-grade category were characterized by the presence of mild to moderate nuclear atypia. As a secondary feature, they tended to show up to 12 mitoses per 10 high power fields (HPFs), whereas those in the high-grade category had marked nuclear atypia and as a secondary feature more than 12 mitoses per 10 HPFs. For comparison, the tumors were also graded using the Shimizu/Silverberg and the FIGO grading systems. Patients in the low-grade ovarian serous carcinoma group ranged in age from 19 to 75 years (mean 41.7 years) while patients in the high-grade ovarian serous carcinoma group ranged in age from 27 to 76 years (mean 55 years). All of the cases except one were advanced FIGO stage. Using the Shimizu/Silverberg system, the low-grade ovarian serous carcinoma cases were distributed as follows: grade 1, 47 cases; grade 2, 3 cases. Using the FIGO grading system, 35 cases were grade 1 and 15 cases were grade 2. Regarding the high-grade ovarian serous carcinoma group using the Shimizu/Silverberg system, 14 of the cases were grade 2 and 36 cases were grade 3. Using the FIGO grading system, 1 case was grade 1, 38 cases were grade 2, and 11 cases were grade 3. Most of the patients in both groups were treated with total abdominal hysterectomy and bilateral salpingo-oophorectomy and also received cisplatinum-based chemotherapy. On follow-up, 37 patients in the low-grade ovarian serous carcinoma group had died of disease at a median 4.2 years after diagnosis compared with 46 patients in the high-grade ovarian serous carcinoma group who died of disease at a median of 1.7 years. Eight patients in the low-grade ovarian serous carcinoma group and 4 patients in the high-grade ovarian serous carcinoma group were alive with disease at median follow-ups of 4.3 and 3.85 years, respectively. Four patients with low-grade serous carcinoma were alive without evidence of disease after a follow-up that ranged from 4.4 to 22.6 years (median 6.85 years), and one died of other causes 14 years after the diagnosis of her ovarian tumor. On multivariate analysis, residual tumor and tumor grade based on the M. D. Anderson two-tier system for grading ovarian serous carcinoma were found to be significant independent prognostic factors (P = 0.003 and 0.04, respectively). Of interest, 60% of the low-grade ovarian serous carcinomas in this study were associated with a serous neoplasm of low malignant potential, whereas this association was present in only 2% of the high-grade ovarian serous carcinomas. This finding could reflect a difference in the pathogenesis of ovarian serous carcinomas of different grades. In summary, there is usually a good correlation between the two-tier grading system herein presented and the Shimizu/Silverberg and the FIGO grading systems. Because this system is based on defined criteria that are easy to follow and because it involves only two diagnostic categories, it should provide better reproducibility in the grading of ovarian serous carcinoma. However, additional studies are required to validate these statements.

Second malignancies after chemotherapy and radiotherapy for Hodgkin disease.

The purpose of this preliminary study was to determine the incidence of second malignancies after combined-modality therapy for adults with Hodgkin disease and relate it to the details of initial treatment. We retrospectively studied 286 patients ranging in age from 16 to 88 years with stage I or II Hodgkin disease who were treated between 1980 and 1995 with chemotherapy followed 3 to 4 weeks later by radiotherapy. Patients received a median of three cycles of induction chemotherapy. Mitoxantrone, vincristine, vinblastine, and prednisone was used in 161 cases, mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) in 67 cases, Adriamycin, bleomycin, vinblastine, and dacarbazine in 19 cases, lomustine, vinblastine, procarbazine, and prednisone/doxorubicin, bleomycin, dacarbazine, and lomustine in 18 cases, and other chemotherapeutic regimens in the remaining 21 cases. The median radiotherapy dose was 40 Gy given in 20 daily 2-Gy fractions. Median follow-up of surviving patients was 7.4 years. There were 2,230 person-years of observation. Significantly increased relative risks (RR) were observed for acute myeloid leukemia (RR, 69.3; 95% CI, 14.3-202.6) and melanoma (RR, 7.3; 95% CI, 1.5-21.3). The 5-, 10-, and 15-year actuarial risks of acute myeloid leukemia were 0.8%, 1.3%, and 1.3%, respectively. Patients treated with MOPP had the highest 15-year actuarial risk of leukemia (1.6%). The 5-, 10-, and 15-year actuarial risks of solid tumors were 1.9%, 9.3%, and 16.8%, respectively. Consolidative radiotherapy to both sides of the diaphragm resulted in a trend toward an increased risk of solid tumors relative to radiotherapy to only one side of the diaphragm (p = 0.08). In an effort to reduce the risk of second malignancies, we have stopped using the alkylating agents nitrogen mustard and procarbazine and elective paraaortic and splenic radiotherapy after chemotherapy.

Role of decreased levels of lipid phosphate phosphatase-1 in accumulation of lysophosphatidic acid in ovarian cancer.

The levels of lysophosphatidic acid (LPA) are consistently elevated in the ascites of ovarian cancer patients, suggesting that ovarian cancer cells are exposed to an LPA replete environment. LPA stimulates cell proliferation, cell survival, resistance to cisplatin, production and activation of proteases, invasiveness and production of the neovascularizing factors, vascular endothelial growth factor, and interleukin 8. Although ovarian cancer cells can produce LPA, this may not be the major reason for altered LPA levels in ascites. We have demonstrated that the major mechanism of degradation of LPA by ovarian cancer cells is through a lipid phosphate phosphatase (LPP)-like activity. We demonstrate herein that LPP-1 mRNA is decreased in the majority of ovarian cancers. This is recapitulated in ovarian cancer cell lines, where LPP-1 RNA levels are lower than those in normal ovarian epithelium and immortalized ovarian epithelial cells. Introduction of LPP-1 into ovarian cancer cell lines results in increased LPA hydrolysis, which is associated with a marked inhibition of cell proliferation and colony-forming activity and a marked increase in apoptosis. Thus, the LPA-rich environment of the ovarian cancer cell in vivo and the subsequent effects of cellular pathophysiology may be a consequence of both increased LPA production and decreased LPA metabolism by ovarian cancer cells.

The human lipid phosphate phosphatase-3 decreases the growth, survival, and tumorigenesis of ovarian cancer cells: validation of the lysophosphatidic acid signaling cascade as a target for therapy in ovarian cancer.

Lysophosphatidic acid (LPA) is present at elevated concentrations in the ascites and plasma of ovarian cancer patients. Ovarian cancer cells produce and release LPA both constitutively and after stimulation. LPA can induce proliferation, survival, invasiveness, and resistance to chemotherapy of ovarian cancer cells. This suggests that LPA may be critically important for the development or progression of ovarian cancer and is thus a potential target for therapy. In this study, we demonstrate that introduction of the integral membrane protein, human lipid phosphate phosphohydrolase-3 (hLPP-3) enzyme, which hydrolyzes phosphatidic acid, LPA, sphingosine, and ceramide phosphate in vitro with selectivity for LPA, into SKOV3 and OVCAR-3 ovarian cancer cells decreases colony-forming activity, increases apoptosis, and decreases tumor growth in vitro and in vivo. Strikingly, coculture of hLPP-3-expressing cells with nontransfected parental cells decreased the colony-forming activity of the parental cells, compatible with hLPP-3 decreasing levels of an extracellular mediator, likely LPA. Compatible with this contention, the expression of hLPP-3 was associated with increased rates of extracellular LPA hydrolysis. The effects of hLPP-3 on colony-forming activity were substantially reversed by the LPP-resistant LPA analogue, O-methylphosphothionate. The ability of O-methylphosphothionate to ameliorate the effects of hLPP-3, combined with the inability of an enzymatically inactive hLPP-3 to alter cellular function, suggests that the major effect of hLPP-3 was to increase the hydrolysis of extracellular LPA. Thus genetic or pharmacological manipulation of LPA metabolism, receptor activation, or downstream signaling is an attractive approach for therapy of ovarian cancer.

Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway.

ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.

Reflectance spectroscopy for in vivo detection of cervical precancer.

Optical technologies, in particular fluorescence spectroscopy, have shown the potential to provide improved detection methods for cervical neoplasia that are sensitive and cost effective through accurate, objective, instantaneous point-of-care diagnostic tools. The specific goals of this study were to analyze reflectance spectra of normal and neoplastic cervical tissue in vivo and to evaluate the data for use in diagnostic algorithm development. Spectroscopic measurements were obtained at four distinct source-detector separations from 324 sites in 161 patients. As the source-detector separation increases, greater tissue depth is probed. The average spectra of each diagnostic class differed at all source-detector separations, with the greatest differences occurring at the smallest source-detector separations. Algorithms, based on principal-component analysis and Mahalanobis distance classification, were developed and evaluated for all combinations of source-detector separations relative to the gold standard of colposcopically directed biopsy. The diagnostic combination of squamous normal versus high-grade squamous intraepithelial lesions gave good discrimination with a sensitivity of 72% and a specificity of 81%; discrimination of columnar normal versus high-grade squamous intraepithelial lesions also was good, with sensitivity of 72% and specificity of 83%. Thus, reflectance spectroscopy appears promising for in vivo detection of cervical precancer. Strategies that combine fluorescence and reflectance spectroscopy may enhance the discrimination capabilities.