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.

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.

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.

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.

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.

The accuracy of urinary cytology in daily practice.

BACKGROUND: Most studies of urinary cytology have been research analyses designed to test the method itself, and many claim that the high diagnostic yields in these studies cannot be achieved in daily practice. The authors examined the clinical and pathologic records in three hospital pathology practice settings--academic, community, and cancer referral settings--to determine the diagnostic yield of urinary cytology under daily clinical conditions. METHODS: Records of consecutive urinary cytology specimens from 1672 patients reported from the years 1990-1994 were reviewed and correlated with histologic and clinical information. Initial analyses were based on the records themselves, without review of pathologic specimens. Subsequently, a subset of specimens was reviewed to determine reasons for noncorrelations. RESULTS: Results confirmed that the diagnostic sensitivity and specificity of urinary cytology for high grade transitional cell neoplasms, as reported in the daily practice of pathology, are very high (79% and >95%, respectively). Disaggregated cells from low grade transitional cell neoplasms usually lack recognizable features of neoplasia, and attempts to classify such lesions cytologically result in low diagnostic yield, with an overall sensitivity of 26%. Of these 1672 patients, 707 had insufficient clinical information for analysis, despite diligent and persistent efforts to acquire it. CONCLUSIONS: The diagnostic yield of consultations based on urinary cytology in the daily practice of pathology is high, regardless of whether the practice setting is referral-based or community-based. The available information indicates that in approximately 79% of patients with high grade transitional cell neoplasms, the neoplasms can be detected using urinary cytology. Conversely, a negative result is predictive of no cancer in more than 90% of cases. Sensitivity for detecting low grade urothelial lesions is low; however, most of these are easily detected cystoscopically. The authors' inability to acquire sufficient information to determine diagnostic yield in a large percentage of their cases was disturbing to them. Not only did this deficiency render their analyses incomplete, but lack of easily accessible follow-up and the apparent low priority for quality assurance activities among pathologists in all types of practice settings is likely to significantly reduce the feedback required for pathologists to acquire and maintain expertise in this very difficult area.

Effects of tamoxifen on the cytology of the uterine cervix in breast cancer patients.

Tamoxifen, a nonsteroidal antiestrogen, is the endocrine therapy of choice for all stages of breast cancer. Because tamoxifen is well tolerated and has minimal side effects, it is currently being evaluated in large scale trials as a chemopreventive agent for women at risk for developing breast cancer. The potential adverse effects of tamoxifen, specifically the development of proliferative lesions of the endometrium, coupled with the prospect of its wider use, places new emphasis on recognizing tamoxifen-associated histologic and cytologic changes in the female genital tract. The current study evaluated cervical smears from 52 breast cancer patients treated with tamoxifen compared with 21 smears from breast cancer patients who had not received tamoxifen. Cytologic diagnoses were classified according to the Bethesda system. The presence of blood, inflammation, and hormonal effect were also assessed. No squamous intraepithelial lesions were identified. A total of 21 of 38 smears (55%) from patients receiving tamoxifen alone and 11 of 14 smears (78%) from women who received tamoxifen in combination with adjuvant cytotoxic chemotherapy showed atypias compared with only 6 of the 21 breast cancer patients (28%) who did not have hormonal therapy. The number of smears showing atypia was equally divided into changes interpreted as benign reactive and atypical squamous cells of undetermined significance (ASCUS). Of the 19 patients whose smears were classified as ASCUS, 13 patients had a subsequent cervical biopsy and none showed dysplasia or diagnostic human papilloma virus changes. Tamoxifen therapy was not associated with an increase in the presence of blood or inflammation, and no discernible alteration in the hormonal state was seen in the cervical smears. We conclude that the use of tamoxifen may be associated with benign squamous atypia in cervical smears and that the atypia is not associated with intraepithelial lesions.

Enrichment for DNA mismatch repair-deficient cells during treatment with cisplatin.

In addition to playing a role in tumorigenesis, loss of DNA mismatch repair results in low-level intrinsic resistance to cisplatin and carboplatin. We used a mismatch repair-deficient (clone B) and -proficient (clone B/rev) pair of Chinese hamster ovary sublines to determine the ability of cisplatin to enrich for repair-deficient cells during growth in vitro and in vivo. Clone B cells were 1.8-fold resistant to cisplatin as measured by a clonogenic assay. These cells were molecularly engineered to express constitutively the green fluorescent protein, and changes in the fraction of these repair-deficient cells were monitored by flow cytometric analysis. A single 1-hr exposure to cisplatin at an IC50 concentration enriched populations initially containing either 5 or 10% clone B cells by 81 and 75%, respectively, when measured at 5 days. Enrichment increased as a function of drug concentration to 158 and 169%, respectively, following an IC90 exposure. When grown as a xenograft, a single LD10 dose of cisplatin enriched the tumors by 48% from 4.6 to 6.8% repair-deficient cells (p = 0.04). To determine whether similar enrichment occurs during the treatment of human ovarian cancer patients, paired tumor samples were obtained from 38 patients before and after treatment with a minimum of 3 cycles of platinum drug-based primary chemotherapy and analyzed immunohistochemically for changes in the fraction of tumor cells expressing hMHL1. Following treatment there was a reduction in hMLH1 staining in 66% of the cases (p = 0.0005). Our results demonstrate that, despite the fact that loss of mismatch repair yields only modest levels of cisplatin resistance, even a single exposure to cisplatin produces quite a marked enrichment for repair-deficient cells in vitro and in vivo. Our results are consistent with the concept that treatment with cisplatin or carboplatin selects for preexisting mismatch repair-deficient cells, and that this contributes to the frequent development of clinical resistance.

Loss of heterozygosity in squamous cell carcinomas of the head and neck defines a tumor suppressor gene region on 11q13.

Tumor suppressor genes APC, RB1, and DCC, as well as genes localized to 3p and 11q, have been implicated in the development of a number of human tumors. To determine whether allelic deletions occur at these loci in squamous cell carcinomas (SSCs) of the head and neck, 25 primary, 1 metastatic, and 3 recurrent tumors, along with the corresponding constitutional tissues, were analyzed by using a battery of polymorphic DNA markers. For two primary tumors, we also analyzed subsequent metastatic tumors of the lung. Polymerase chain reaction-based restriction fragment length polymorphism studies demonstrated loss of heterozygosity for the APC gene in 2 of 12 (17%), the RB1 gene in 5 of 22 (23%), and the DCC gene in 5 of 13 (38%) informative cases. Alleles on chromosomes 3p, 11q13, and 18q21.1 were lost in 7 of 20 (35%), 9 of 23 (39%), and 4 of 17 (24%) informative cases, respectively. A breakpoint was identified within the chromosomal region 3p13-21.2 in a SCC of the tongue. Breakpoints within 11q13 were identified in 2 additional tumors. Thus, allelic deletions of DCC, 3p, and 11q13 appear to be common in head and neck cancers, suggesting that these genes play a critical and complex role in the development of these tumors. Furthermore, the present study provides definitive evidence for a tumor suppressor gene at chromosome band 11q13 and localizes this gene to the INT2-D11S533 interval for future cloning and sequencing.

Detection of human papillomavirus DNA and oncoprotein overexpression are associated with distinct morphological patterns of tonsillar squamous cell carcinoma.

Human papillomavirus (HPV) DNA has been detected in approximately 15% of squamous cell carcinomas (SCCs) of the head and neck. Recent studies have shown a predilection of HPV for certain anatomical sites, especially the tonsillar region, with viral DNA identified in approximately 60% of SCCs of the Waldeyer's tonsillar ring. This study was undertaken to determine whether there are differences in morphology or in oncogene expression in SCC of the tonsil with and without detectable HPV DNA. Twenty-two SCCs of the tonsil were analyzed for the presence of HPV DNA by polymerase chain reaction (PCR) using both a consensus primer set (My09/My11) and type-specific primers. Viral transcription was established in both primary and metastatic tumors by RNA in situ hybridization. The morphology of invasive SCC was classified into three subtypes: well keratinized (K-SCC), intermediate keratinized (I-SCC), and poorly keratinized (P-SCC). Expression of p53, pRB, and cyclin D1 (bcl-1) were studied by immunohistochemistry. In these cases (6 K-SCCs, 2 I-SCCs, and 14 P-SCCs), HPV DNA was detected in 14 (64%), with 11 containing HPV-16 (10 P-SCCs, 1 I-SCCs, and 0 K-SCCs) and 1 each containing HPV-33, HPV-59, and an unclassified HPV type (all P-SCCs). Viral oncoprotein E6/E7 transcription was demonstrated in 7 of 7 HPV-16-positive tumors. Cyclin D1 protein overexpression was detected in the majority of HPV-negative tumors (7 of 8 cases), whereas it was minimal or absent in 13 HPV-positive tumors. Overexpression of p53 protein was detected in 3 HPV-negative K-SCCs. In the HPV-positive tumors, fewer malignant cells expressed pRB and the staining was less intense than in the HPV-negative cancers. HPV DNA and E6/E7 expression, especially HPV-16, is detected in the majority of tonsillar SCCs and is almost exclusively associated with a poorly keratinized tumor histology. Decreased expression of cyclin D1, pRB, and p53 in tumors with HPV DNA is consistent with the known effects of the viral oncoproteins on the cellular protein. The morphology of the HPV-positive tumors suggests that HPV may have a predilection for a population of nonkeratinizing squamous cells or that the virally transformed cells inhibit keratinization of the tumor cells. Well keratinized tonsillar SCCs lack HPV DNA and are associated with overexpression of cyclin D1 protein and/or p53, suggesting that mechanisms that alter the cell cycle regulatory proteins, either by interaction with viral oncoproteins or by changes in the cellular proteins themselves, is critical for tumorigenesis of tonsillar SCC.

Colposcopic regression patterns in high-grade cervical intraepithelial neoplasia.

OBJECTIVE: To evaluate the serial changes in colposcopic and cervicographic findings of women with cervical intraepithelial neoplasia (CIN) II and III enrolled in a phase III randomized comparison of oral beta carotene and placebo. METHODS: All subjects treated with beta carotene or placebo for at least 6 months were included if they met the criteria of persistent or progressive disease (no change or worsening of CIN grade) or disease regression (improvement of two grades or more). These two groups were compared for changes in colposcopic and cervicographic patterns. Colposcopically directed biopsies and cervicography were done at enrollment and after 6 months. Quarterly Papanicolaou smears and colposcopic assessments also were performed. Findings of mosaic pattern, punctation, and white epithelium were graded and diagrammed at colposcopic examinations. Cervicographic measurements of the centripetal movement of metaplastic epithelium were recorded. Data were analyzed by chi 2 analysis and Fisher exact tests. RESULTS: Data were available for 23 subjects with regression and 16 with persistent lesions. Small lesions were significantly more likely to regress than large ones. Lesions without coarse punctation were significantly more likely to regress than lesions with coarse punctation, and lesions with mild acetowhite changes were more likely to regress than those with dense white epithelium. A pattern of centripetal movement of the metaplastic epithelium toward the cervical os was noted in lesions that regressed, but not in those that persisted or progressed. CONCLUSION: This study describes the centripetal growth of metaplastic squamous epithelium associated with the regression of CIN II and III. This observation contributes to our understanding of the process of disease regression in CIN and may be useful in identifying individuals for conservative management. Failure to identify this pattern correlates with persistent or progressive disease.

Molecular analysis of the INK4 family of genes in prostate carcinomas.

PURPOSE: A newly recognized class of INK4 family of cyclin-dependent kinase inhibitors CDKIs) include its prototype, p16 (INK4A/MTS1/CDKN2), and three others, p15 (INK4B/MTS2), p18 (INK4C), and p19 (INK4D). The putative tumor suppressor gene, p16 is frequently altered in certain neoplasms and many cell lines. The potential role of INK4 CDKIs in pathogenesis of prostate carcinoma was studied. MATERIALS AND METHODS: Thirty-two primary prostate cancer samples and two prostate cancer cell lines were examined for alterations of the p16, p15, p18, and p19 genes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and Southern blot analysis. RESULTS: Alteration of the p16 gene was found in one of 32 primary prostate cancer samples by PCR-SSCP. DNA sequencing of the sample showed a 24-basepair insertion in exon 1 of the p16 gene at codon 11. No other mutations were found in p15, p18, or p19 genes by PCR-SSCP. Furthermore, none of the p16, p15, p18, or p19 genes had alterations by Southern blot analysis. CONCLUSIONS: These results indicate that structural abnormalities of the INK4 CDKIs is a rare event in prostate carcinoma, and the loss of function of INK4 CDKIs by other mechanisms, such as methylation should be further explored.

Human papillomavirus (HPV) in head and neck cancer. An association of HPV 16 with squamous cell carcinoma of Waldeyer's tonsillar ring.

BACKGROUND: Certain strains of human papillomavirus (HPV) have been shown to be etiologically related to the development of uterine cervical and other genital cancers, but their role in the development of malignancies at other sites is less well established. Previous studies have shown HPV DNA in tumors of the head and neck, but its prevalence has varied depending on the detection methods and the types of tumor and/or tissue examined. This study was undertaken to estimate the frequency of HPV DNA in squamous cell carcinoma (SCC) at different sites of the esophagus, head and neck and to compare the clinical behavior of HPV positive and negative tumors. METHODS: DNA was extracted from frozen tissue of 167 SCCs of the esophagus, head and neck. The DNA was screened for HPV sequences by polymerase chain reaction with two sets of consensus primers, one to a conserved region in the L1 gene (MY09/ MY11) and the other to a conserved region in the E1 open reading frame (IU/IWDO). The products were run on agarose gels, detected by ethidium bromide staining, and then the gels were subjected to Southern blot analysis and hybridized with probes specific to HPV 6, 16, and 18. All tumors found to be HPV positive with the consensus primers were amplified with type specific primers, and in selected cases the presence of HPV DNA was confirmed by restriction enzyme digestion of the tumor DNA with conventional Southern blot analysis. RESULTS: Overall, HPV sequences were found in 25 of 167 tumors (15%), but HPV was detected most frequently in tumors in Waldeyer's tonsillar ring. In that area, 9 of 15 (60%) were HPV positive. No HPV DNA was detected in 11 esophageal SCCs, 7 tumors of the pharynx/hypopharynx, or 6 pyriform sinus carcinomas. HPV DNA was detected in the following tumor sites: 1 of 28 (3.6%) in the larynx, 1 of 10 (10%) in the oral cavity, 5 of 39 (12.8%) in the tongue, 2 of 15 (13.5%) in the floor of the mouth, 3 of 21 (14.3%) supraglottic, and 1 of 7 (14.3%) in the lip. A high incidence of HPV DNA was also found in metastatic tumors located in cervical lymph nodes for which no primary site was clinically identified (3 of 8, 37.5%). With respect to age, gender, and tobacco and alcohol consumption, analysis of clinical data obtained by retrospective review showed no difference between patients with HPV DNA in their tumors and those in which no HPV was detected. However, HPV positive patients had larger tumors (P = 0.09) and a higher incidence of lymph node metastasis (P = 0.003). In spite of the higher stage of disease at presentation in HPV positive patients, there was no significant difference in 3-year survival rates between HPV positive patients and HPV negative patients (43.1% vs. 48.8%, respectively). Median follow-up was 27 months. CONCLUSIONS: In the head and neck, HPV-associated SCC had site specificity with the viral DNA frequently found in tumors in Waldeyer's tonsillar ring. Patients with HPV positive tumors presented with a higher stage of disease than patients with HPV negative tumors, but there was no significant difference in the 3-year survival rates between these two groups of patients.

A study of biomarkers in cervical carcinoma and clinical correlation of the novel biomarker MN.

The MN protein is a newly described biomarker found to be overexpressed in most cervical carcinomas. This study was an effort to evaluate the prognostic importance of tumor MN expression, HPV status, and the presence of other biomarkers in cervical cancers. Tumor DNA and protein for study were extracted from archived frozen tissue. Tumor tissues and controls were evaluated by Western blot analysis for MN, intestinal alkaline phosphatase (IAP), c-myc, and p53 protein overexpression. Immunohistochemistry was performed for MN quantification and the study of expression patterns in histologic subtypes of cervical cancer. HPV data were obtained by PCR amplification of extracted DNA using consensus and type-specific primers. Clinical data were obtained from the patients' records and from the cancer registry. Clinical and molecular data were correlated by chi2, Fisher's exact test, and logistic regression. The results demonstrate that IAP is not overexpressed in clinical specimens of cervical carcinoma, although in somatic cell hybrid experiments, overexpression of IAP correlates with the malignant state. None of 47 tumors, including those which were HPV negative, overexpressed p53. c-myc protein overexpression occurred in 11 of 52 tumors, most of which contained HPV 16, but this was not significantly different from the tumors as a whole. There was no apparent association between MN protein expression and the overexpression of c-myc protein. MN was overexpressed in all cancers and quantitatively varied with the histologic subtype. Specifically, lower expression of MN correlated with adenosquamous and less-differentiated histology (P < 0.01 for grade 3 tumors). Low expression of MN protein also correlated with HPV negativity (P < 0.05). In stage IB and IIA cancers, low expression of MN was associated with deeper cervical stromal invasion (P < 0.03). Further, low expression of MN correlated with lymph node metastases in small (<3.5 cm) IB and IIA cervical cancers (P < 0.04). These data suggest that MN is emerging as a potentially important new biomarker for cervical carcinoma. The overexpression commonly seen in cervical cancer is possibly associated with loss of a critical tumor suppressor gene located on chromosome 11. Low expression of MN antigen appears to correlate with several adverse prognostic features and further prospective study is warranted.

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

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

HPV6 variants from malignant tumors with sequence alterations in the regulatory region do not reveal differences in the activities of the oncogene promoters but do contain amino acid exchanges in the E6 and E7 proteins.

Human papillomavirus type 6 (HPV6) causes benign epithelial proliferations of the anogenital and aerodigestive tract, which usually tend to regress spontaneously. The low incidence of HPV6 in carcinomas and the rare progression of the benign tumors has led to the classification of HPV6 as "low-risk" virus. A series of reports, however, described the isolation of HPV6 variants from malignant tumors characterized by sequence rearrangements in the noncoding regulatory region (NCR). It was speculated that these sequence alterations play a role in tumor progression by enhancing the promoter activity and thereby increasing the expression of the viral oncogenes E6 and E7. To elucidate if HPV6 isolates from malignancies do regularly exhibit sequence alterations in the regulatory region we first determined and complied the sequences of the NCRs of a number of isolates from benign and malignant lesions. This analysis revealed in general a high degree of sequence conservation between the individual isolates. Most of the isolates, however, differed, independently of origin, by a major and one or two minor insertions from the prototype HPV6b sequence. When tested in a functional assay these altered NCR sequences did not result in significantly different activities of the promoters responsible for the expression of the E6 and E7 genes. Further analysis of the E6 and E7 coding region revealed a surprisingly high sequence variability within the E6 ORF and allowed the detection of amino acid exchanges unique for isolates from carcinomas.

p16 (CDKN2/cyclin-dependent kinase-4 inhibitor/multiple tumor suppressor-1) gene is not altered in uterine cervical carcinomas or cell lines.

The p16 (CDKN2/cyclin-dependent kinase-4 inhibitor/multiple tumor suppressor-1) gene is frequently altered in cell lines and some types of cancers. To assess whether alterations of this gene are important in the pathogenesis of cervical cancer, we examined 41 primary tumors and 8 cell lines, using Southern blot and polymerase chain reaction-single strand conformation polymorphism analyses. We did not detect any deletions or mutations, which indicates that inactivation of the p16CDKN2 gene is not critical in the tumorigenesis of cervical cancer or in establishment of cervical cancer cell lines.