Sharon P. Wilczynski

Human papillomavirus (HPV) in head and neck cancer

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.

Bacteria-Induced Intestinal Cancer in Mice with Disrupted Gpx1 and Gpx2 Genes

Two glutathione peroxidase (GPX) isozymes, GPX-1 and GPX-2 (GPX-GI), are the major enzymes that reduce hydroperoxides in intestinal epithelium. We have previously demonstrated that targeted disruption of both the Gpx1 and Gpx2 genes (GPX-DKO) results in a high incidence of ileocolitis in mice raised under conventional conditions, which include the harboring of Helicobacter species [non-specific-pathogen-free (non-SPF) conditions]. In this study, we have characterized GPX-DKO mice that have microflora-associated intestinal cancers, which are correlated with increased intestinal pathology/inflammation. We found that GPX-DKO mice raised under germ-free conditions have virtually no pathology or tumors. After colonizing germ-free mice with commensal microflora without any known pathogens (SPF), <9% of GPX-DKO mice develop tumors in the ileum or the colon. However, about one-fourth of GPX-DKO mice raised under non-SPF conditions from birth or transferred from SPF conditions at weaning have predominantly ileal tumors. Nearly 30% of tumors are cancerous; most are invasive adenocarcinomas and a few signet-ring cell carcinomas. On the basis of these results, we conclude that GPX-DKO mice are highly susceptible to bacteria-associated inflammation and cancer. The sensitivity exhibited in these mice suggests that peroxidative stress plays an important role in ileal and colonic pathology and inflammation, which can lead to tumorigenesis.

Prognostic significance of human papillomavirus DNA in vulvar carcinoma

Objective To determine the histopathologic, epidemiologic, and prognostic significance of human papillomavirus (HPV) DNA in primary invasive vulvar cancer. Methods From December 1981 through October 1992, primary tumor tissue from 55 newly diagnosed vulvar cancers was evaluated for the presence of HPV DNA. The DNA was extracted from tumor tissue and subjected to the polymerase chain reaction (PCR) using highly conserved consensus L1 primers that detect 25 different HPV genotypes and primers specific for HPV type 6/E6, type 16/E7, and type 18/E6 gene sequences. All PCR products were hybridized to type-specific radiolabeled probes. The association between the presence of HPV DNA and histologic, epidemiologic, and clinical characteristics was analyzed. Results Thirty-three (60%) tumors contained HPV DNA. Patients younger than 70 years of age or who smoked were more likely to have HPV-positive vulvar cancers. Twentyone (95%) of 22 tumors classified as basaloid, warty, or verrucous contained HPV DNA, whereas 12 (39%) of 31 typical squamous tumors contained HPV (P < .001). Two adenocarcinomas were negative for HPV. Tumors with or without HPV DNA did not differ with respect to International Federation of Obstetricians and Gynecologists stage (size and nodal status), tumor grade, or therapy. Using life-table analysis, the absence of HPV DNA and the presence of regional nodal metastasis were predictive of recurrence and death from vulvar cancer. When controlling for lesion size, age, tumor grade, and nodal metastasis using the Cox proportional hazards model, only HPV status remained an independent prognostic factor. Conclusion Human papillomavirus DNA is more common in vulvar cancers of young women who smoke than in older nonsmokers. patients with HPV-negative tumors are at an increased risk of recurrence and death from vulvar cancer.

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

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

Methylation of the RASSF1A Gene in Human Cancers

Abstract Loss of genetic material from chromosome 3p21.3 is one of the most common and earliest events in the pathogenesis of lung cancer and many other solid tumors. The chromosomal area 3p21.3 is thought to harbor at least one important tumor suppressor gene, which, despite many years of investigation, has remained elusive. In our previous studies, we have identified and cloned a gene from the common homozygous deletion area at 3p21.3. The gene, named RASSF1A (Ras ASSociation domain Family 1A), has homology to a mammalian Ras effector. The RASSF1A gene is epigenetically inactivated in a large percentage of human lung cancers, in particular small cell carcinomas. A high frequency of methylation of RASSF1A is found also in breast cancers, renal cell carcinomas, ovarian, gastric and bladder cancers, and in neuroblastomas. The RASSF1A gene is a candidate for a tumor suppressor gene in 3p21.3.

Identification of a 4-microRNA signature for clear cell renal cell carcinoma metastasis and prognosis.

Renal cell carcinoma (RCC) metastasis portends a poor prognosis and cannot be reliably predicted. Early determination of the metastatic potential of RCC may help guide proper treatment. We analyzed microRNA (miRNA) expression in clear cell RCC (ccRCC) for the purpose of developing a miRNA expression signature to determine the risk of metastasis and prognosis. We used the microarray technology to profile miRNA expression of 78 benign kidney and ccRCC samples. Using 28 localized and metastatic ccRCC specimens as the training cohort and the univariate logistic regression and risk score methods, we developed a miRNA signature model in which the expression levels of miR-10b, miR-139-5p, miR-130b and miR-199b-5p were used to determine the status of ccRCC metastasis. We validated the signature in an independent 40-sample testing cohort of different stages of primary ccRCCs using the microarray data. Within the testing cohort patients who had at least 5 years follow-up if no metastasis developed, the signature showed a high sensitivity and specificity. The risk status was proven to be associated with the cancer-specific survival. Using the most stably expressed miRNA among benign and tumorous kidney tissue as the internal reference for normalization, we successfully converted his signature to be a quantitative PCR (qPCR)-based assay, which showed the same high sensitivity and specificity. The 4-miRNA is associated with ccRCC metastasis and prognosis. The signature is ready for and will benefit from further large clinical cohort validation and has the potential for clinical application.

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

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

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

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

Mutations of p53 and human papillomavirus infection in cervical carcinoma

Background. Oncogenic human papillomavirus (HPV) infection has been implicated in the pathogenesis of cervical carcinoma. The HPV oncoproteins E6 and E7 are thought to play a crucial role in this process by their interactions with the p53 protein and the retinoblastoma susceptibility gene product, respectively. The E6 protein binds to and stimulates the degradation of the p53 protein. Mutations involving evolutionary conserved regions of the p53 gene also can alter p53 function. Point mutations of p53 frequently have been identified in a wide variety of human tumors.

Pilot Trial Evaluating an 123I-Labeled 80-Kilodalton Engineered Anticarcinoembryonic Antigen Antibody Fragment (cT84.66 Minibody) in Patients with Colorectal Cancer

Purpose: The chimeric T84.66 (cT84.66) minibody is a novel engineered antibody construct (VL-linker-VH-CH3; 80 kDa) that demonstrates bivalent and high affinity (4 × 1010 m−1) binding to carcinoembryonic antigen (CEA). The variable regions (VL and VH) assemble to form the antigen-combining sites, and the protein forms dimers through self-association of the CH3 domains. In animal models, the minibody demonstrated high tumor uptake, approaching that of some intact antibodies, substantially faster clearance than intact chimeric T84.66, and superior tumor-to-blood ratios compared with the cT84.66 F(ab′)2 fragment, making it attractive for further evaluation as an imaging and therapy agent. The purpose of this pilot clinical study was to determine whether 123I-cT84.66 minibody demonstrated tumor targeting and was well tolerated as well as to begin to evaluate its biodistribution, pharmacokinetics, and immunogenicity in patients with colorectal cancer. Experimental Design: Ten patients with biopsy-proven colorectal cancer (6 newly diagnosed, 1 pelvic recurrence, 3 limited metastatic disease) were entered on this study. Each received 5–10 mCi (1 mg) of 123I-labeled minibody i.v. followed by serial nuclear scans and blood and urine sampling over the next 48–72 h. Surgery was performed immediately after the last nuclear scan. Results: Tumor imaging was observed with 123I-labeled minibody in seven of the eight patients who did not receive neoadjuvant therapy before surgery. Two patients received neoadjuvant radiation and chemotherapy, which significantly reduced tumor size before surgery and minibody infusion. At surgery, no tumor was detected in one patient and only a 2-mm focus was seen in the second patient. 123I-labeled minibody tumor targeting was not seen in either of these pretreated patients. Mean serum residence time of the minibody was 29.8 h (range, 10.9–65.4 h). No drug-related adverse reactions were observed. All 10 patients were evaluated for immune responses to the minibody, with no significant responses observed. Conclusion: This pilot study represents one of the first clinical efforts to evaluate an engineered intermediate-molecular-mass radiolabeled antibody construct directed against CEA. cT84.66 minibody demonstrates tumor targeting to colorectal cancer and a faster clearance in comparison with intact antibodies, making it appropriate for further evaluation as an imaging and therapy agent. The mean residence time of the minibody in patients is longer than predicted from murine models. We therefore plan to further evaluate its biodistribution and pharmacokinetic properties with minibody labeled with a longer-lived radionuclide, such as 111In.