Carolyn Y. Muller

Silencing of HPV 18 oncoproteins With RNA interference causes growth inhibition of cervical cancer cells.

Silencing the expression of human papillomavirus (HPV) oncoproteins should have therapeutic benefits for cervical cancer. The authors objective was to study RNA interference of the HPV 18 E6/E7 bicistronic mRNA with E6 small interfering RNA (siRNA) and E7 siRNA and determine the effect of each siRNA on oncoprotein expression, resultant cell growth, and downstream molecular effects. RNA interference was used to knockdown HPV 18 E6 and E7 oncoproteins on the HPV 18 positive cervical cancer cell lines HeLa and C4I. Western blotting was used to assay for each oncoprotein expression and select downstream molecular targets. Cell cycle analyses, cell viability assays, and colony formation assays were performed to determine the effect of treatment by both HPV 18 E6 siRNA and E7 siRNA. The transfection reagent oligofectamine and Tax siRNA were used as negative controls. Transfection with E6 siRNA caused complete loss of E6 but not E7 oncoprotein. However, E7 siRNA induced complete loss of both E6 and E7 oncoproteins. E6 siRNA mediated the reexpression of p53 protein and a moderate decrease in phosphorylated retinoblastoma protein expression (pRb), resulting in decreased colony formation. Transfection with E7 siRNA mediated a robust increase in p53 expression and complete loss of pRb, resulting in a marked decrease in colony formation compared to the E6 siRNA (P =.001). Flow cytometry revealed significantly increased apoptotic cells with E7 siRNA compared to E6 siRNA and control. RNA interference targeting the E7 portion of the bicistronic HPV 18 mRNA can silence both E6 and E7 oncoproteins and is most effective in cervical cancer growth inhibition.

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

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

Myeloperoxidase promotor polymorphism and risk of hepatoblastoma

Myeloperoxidase (MPO) is a major enzyme found in neutrophils. Oxidation by MPO produces free radicals that demonstrate genotoxic properties. A polymorphism (G to A) within the promotor region of the MPO gene reduces transcription and expression. This polymorphism is associated with a protective effect against some cancers in adults including lung cancer. The objective of our study was to investigate the effect of this MPO polymorphism on the risk of hepatoblastoma, the most common cancer of the liver in childhood. By using PCR‐SSCP, we determined the genotype at this polymorphism in 48 cases of Caucasian children with hepatoblastoma and 180 normal controls. Genotypes were confirmed by a second method using Aci I restriction enzyme restriction. We found that A allele was associated with reduced risk of hepatoblastoma of 50% (OR, 0.51; 95%CI, 0.27–0.93) and G/A or A/A genotype reduced the risk by 56% (OR, 0.44; 95%CI, 0.21–0.90). Our data suggest that A allele is a protective factor with regard to the risk of hepatoblastoma, perhaps by altering genotoxic properties of xenobiotic substances which may act as carcinogens.

Frequent Expression of β-human Chorionic Gonadotropin (β-hcg) in Squamous Cell Carcinoma of the Cervix

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

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

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

Laparoscopy in patients following transverse rectus abdominis myocutaneous flap reconstruction

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

CCND1 polymorphism and age of onset of hepatoblastoma.

Cyclin D1, encoded by the gene CCND1, is a major regulator of the cell cycle transition from G1 phase to S phase. A CCND1 polymorphism (G to A) at codon 242, the boundary of exon 4 and intron 4, affects splicing such that exon 5 is not expressed in the A allele. Since exon 5 is involved in rapid turnover, the variant cyclin D1 corresponding to the A allele may have a longer half-life. A previous study demonstrated that in families with hereditary nonpolyposis colorectal cancer, the age of onset of colorectal cancer varied according to variation at this polymorphic site. We examined this CCND1polymorphism in a series hepatoblastoma, a childhood liver cancer that shares other molecular features with colon cancer. We determined in an analysis of 84 children with hepatoblastoma that the G/A exon 4 polymorphism in CCND1 is correlated with the age of onset of hepatoblastomas. The A/A genotype is associated with an earlier age of onset compared to the G/A or G/G genotype. The median age of patients with the G/G genotype was 22 months, compared to 17 months in patients with the G/A genotype and 11 months for the A/A genotype. These findings suggest that the CCND1 A polymorphism may contribute to tumor development in children with hepatoblastoma.

Understanding the mechanisms of FHIT inactivation in cervical cancer for biomarker development.

Objective: Loss of the fragile histidine triad (Fhit) protein has been documented in cervical cancer and dysplasia. The goal of this study was to confirm the utility of homozygous deletions, aberrant methylation, and immunohistochemical evaluations of FHIT as functionally relevant determinants of FHIT expression. Methods: We studied matched DNA, RNA, and protein from nine early-passage cervical cancer cell lines. DNA markers spanning FHIT were used to examine the extent of homozygous deletions for each cell line. 5′ CpG island methylation of FHIT was investigated by methylation-specific polymerase chain reaction (PCR) assays. FHIT transcripts were characterized by reverse transriptase (RT)-PCR. Western blot analysis and immunohistochemistry were performed to characterize Fhit protein expression. Results: Homozygous deletions were found in six of nine cervical cancer cell lines, but only had homozygous deletions involving an exon. All nine lines had both methylated and unmethylated alleles according to methylation-specific PCR. Loss of wild-type FHIT transcripts were found in five of nine lines. By western blot analysis, Fhit protein expression was lost in five of nine lines, producing an exact correlation with RT-PCR results. Immunohistochemical staining was concordant with Fhit protein expression by western blotting in eight of nine cell lines. Conclusion: A perfect correlation was found between FHIT mRNA expression and western blot analysis. Assays for Fhit protein expression and large FHIT homozygous deletions are representative biomarkers of Fhit expression. By contrast, the aberrant methylation assay is not concordant with FHIT gene expression, and we suggest caution in its use as a functionally relevant biomarker for cervical cancer.

Ectopic production and localization of β-human chorionic gonadotropin in lymphoepithelioma-like carcinoma of the cervix : A case report

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

Prevention of Gynecologic Malignancies

describing a small number of patients treated at a single