Tang Yuan Chu

Viral load of high-risk human papillomavirus in cervical squamous intraepithelial lesions.

Objectives: This case‐control study was conducted to investigate the role of viral load of high‐risk human papillomaviruses (HPVs) in the development of cervical squamous intraepithelial lesions (SILs) and invasive cancers. Methods: A total of 30 female cases who had histological evidence of low‐grade SIL (n=10) or high‐grade SIL and above (n=20) were identified as the case group at the Tri‐Service General Hospital, Taipei between September 1998 and March 1999. In addition, 80 female controls who had normal cervical cytology were enrolled and individually matched on age (±5 years) and date of recruitment to each case. Cervical swabs collected from study subjects were tested for the positivity and viral load of high‐risk HPVs by Hybrid Capture II assay. Additionally, subjects completed a risk factor questionnaire. Results: Among sex behavioral factors studied, younger age at first intercourse was associated with a significantly elevated risk of cervical SIL and invasive cancers. With respect to HPV infection, high‐risk HPV DNA was present in 70% (21/30) of case and 21% (17/80) of control subjects, resulting in an odds ratio (OR) of 6.6 [95% confidence interval (C.I.)=2.6–17.0]. Moreover, women who had a high viral load were at significantly greater risk for cervical SIL and invasive cancers than those who were infected with a low viral load (OR=18.0, 95% C.I.=3.0–108.5). Conclusions: Among the variables tested, infection with a high viral load of high‐risk HPVs is the strongest determinant for cervical SIL and cervical cancers in Taiwan.

Single nucleotide polymorphism at Fas promoter is associated with cervical carcinogenesis

The causal role of human papillomavirus (HPV) in cervical carcinogenesis is beyond reasonable questioning. The progression from HPV infection, squamous intraepithelial lesions (SIL) to squamous cell carcinomata (SCC), however, is very uncommon and inefficient. Host genetic factors that may confer the susceptibility of disease progression are largely unknown. Apoptosis is an important fail‐safe check for tumor development, in which Fas/FasL interaction contributes substantially. The purpose of our study is to test the hypothesis that an A/G polymorphism at −670 of Fas promoter with different transcriptional activity is associated with the risk for cervical neoplasia. A hospital‐based case‐control study was conducted, in which 104 patients of low grade SIL (LSIL), 131 high grade SIL (HSIL) and 176 SCC as well as age‐matched, 1:1 controls were tested for Fas polymorphism by PCR‐RFLP. HPV genotypes were determined in case groups by MY PCR‐reverse line blot. The frequency of A allele was significantly (p = 0.006) higher in SCC than in control, conferring an odd ratio of 1.5 (95% CI = 1.1–2.0). The distribution of Fas (−670) genotypes also differed significantly between HSIL, SCC and each of their control (p = 0.017 and 0.03, respectively), with the A/A genotype conferring an OR of 1.3 (95% CI = 1.1–1.6) and 1.6 (95% CI = 1.0–2.5), respectively. Remarkably, the frequency of A allele and A/A genotype increased gradually in accordance with the multi‐step carcinogenesis from LSIL, HSIL to SCC (ptest for trend = 0.0066 and 0.0007, respectively). In addition, there was no difference of Fas genotypes between HPV (+) and HPV (−) cases. Fas genotypes, however, differed in LSIL infected with different HPV types (p = 0.033). The present study demonstrated an association between Fas polymorphism and cervical carcinogenesis. We deduced a possible effect of apoptosis of immune cells in this virus‐induced cancer.

Promoter methylation of the secreted frizzled-related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma.

The secreted frizzled‐related protein 1 gene (SFRP1) encodes a Wnt/β‐catenin signaling antagonist and frequently is inactivated by promoter methylation in many tumors. However, the role of SFRP1 in hepatocellular carcinoma (HCC) is not clear. Therefore, the authors investigated whether methylation of the SFRP1 promoter is common in HCC and whether it may influence SFRP1 expression.

Favorable clinical outcome of cervical cancers infected with human papilloma virus type 58 and related types

To determine whether the status of human‐papillomavirus (HPV) infection affects the clinical outcome of cervical carcinoma (CC), HPV genotype was prospectively determined in 94 consecutive CC cases subsequently followed for a median duration of 37.5 months. With a consensus PCR‐RFLP method of HPV genotyping, 81 (86.2%) cancers were positive for HPV DNA. They were classified, according to the phylogenic similarities, into HPV‐16‐related (type 16, n = 45; type 31, n = 2), HPV‐58‐related (type 58, n = 17; type 33, n = 3; type 52, n = 2) and HPV‐18‐related (type 18, n = 8; type 68, n = 1) groups, and analyzed in relation to clinical outcome. The following results were observed: (i) Type‐58‐related HPVs were more prevalent in the old age (older than the median age of 52) group than in the young age group (41% vs. 14.6%, p = 0.045); (ii) 63% (5/8) of patients with advanced stages (III and IV) were HPV‐negative, a figure much higher than that (9.3%, 8/84) of patients with early stages (stage I and II) (p = 0.002); (iii) the occurrence of adenocarcinoma or adenosquamous carcinoma was higher in the HPV‐18‐related group (50%) than in the HPV‐16‐related (33.3%) or the HPV‐58‐related (16.7%) groups (p = 0.024); (iv) the status of lymph‐node metastasis and tumor grade did not correlate with HPV status; (v) 5‐year survival rates were 90.2%, 80% and 74% for HPV‐58‐, HPV‐16‐ and HPV‐18‐related groups, respectively (p = 0.03, after adjustment for tumor stage); (vi) in comparison with the HPV‐16‐related group, the relative risk of death in the HPV‐58‐ and the HPV‐18‐related groups were 0.32 [95% CI, 0.07–1.49] and 1.87 [0.36–14.9] respectively. HPV genotype appears to affect the clinical behavior and outcome of cervical cancer. HPV‐58‐related types are prevalent in the older population, and appear to confer a favorable prognosis. Int. J. Cancer (Pred. Oncol.) 84:553–557, 1999.

Monoclonality and surface lesion-specific microsatellite alterations in premalignant and malignant neoplasia of uterine cervix: a local field effect of genomic instability and clonal evolution

Invasive squamous carcinoma of the uterine cervix (CC) arises from sequential progression of low‐grade (L) and high‐grade (H) squamous intraepithelial lesions (SILs). In clinical observations, these lesions are frequently found as synchronous multiple foci. The nature and evolutionary mechanism of these lesions are largely unknown. We have performed allelotyping of three 3p markers (at 3p14, 3p22–24, and 3p25) on 22 LSILs and 15 HSILs microdissected from patients with multiple (n = 21) or uniform (n = 6) cervical lesions. The results were analyzed together with our previous allelotyping of 57 deeply invasive CCs. Loss of heterozygosity at one of the three markers was observed in 23%, 27%, and 31% of LSILs, HSILs, and CCs, respectively. Frequent and early allelic loss was noted (in 30% of LSILs and 50% of HSILs) at 3p14, which may harbor tumor suppressor genes involved in early stages of cervical carcinogenesis. A high frequency of microsatellite alteration (MA) was found in LSIL (41%) and HSIL (67%) but not in CC (5.3%). In particular, MA was more frequently found in low‐grade lesions in association with invasive cancers (75%, 6/8) than in those associated with SILs (29%, 4/14) (P < 0.05). Together with the finding of a monoclonal origin of premalignant and malignant cervical lesions, the present results allow us to propose a model of local field effect of genomic instability that progressively affects the clonal evolution of SIL of uterine cervix. Genes Chromosomes Cancer 24:127–134, 1999.

Genetic polymorphism of the interferon‐γ gene in cervical carcinogenesis

Beyond human papillomavirus (HPV) infection, host genetic factors may contribute to cervical carcinogenesis. This study aims to test the hypothesis that CA‐dinucleotide repeat polymorphism in the first intron of the interferon‐gamma (IFN‐γ) gene is associated with HPV‐initiated cervical carcinogenesis. A hospital‐based case‐control study including patients with low‐grade squamous intraepithelial lesions (LSILs; n = 93), high‐grade squamous intraepithelial lesions (HSILs; n = 123) and invasive carcinomas (n = 153) of the uterine cervix, as well as 1:1 age‐matched controls, was conducted. The IFN‐γ genotype was determined by PCR and capillary electrophoresis with internal standards. HPV genotype was determined by consensus PCR and reverse line blot hybridization. Genotypes containing the 12 or 14 allele (12 or 14 CA repeats) were significantly more common in patients with HSILs than in controls (46% vs. 22%; OR = 3.0; 95% CI = 1.7–5.2; p < 0.0001). In contrast, genotypes containing 13 and 18 were significantly more common in controls than in patients with HSILs (76% vs. 53%; OR = 0.3; 95% CI = 0.2–0.6; p = 0.0001) or squamous cell carcinomas (74% vs. 63%; OR = 0.6; 95% CI = 0.4–1.0; p = 0.037). The frequency of the 12 and 14 genotypes increased significantly in accordance with the severity of cervical carcinogenesis (ptest for trend = 0.0002), whereas the 13 and 18 genotypes showed the opposite trend (ptest for trend = 0.007). Comparing IFN‐γ genotype and HPV status, 18‐containing genotypes were more frequently found in HPV+ LSILs, and 12‐containing genotypes were less frequently found in HPV+ HSILs. Compared with non‐13 genotypes, 13 genotype HSILs were more frequently infected with HPV58 (70% vs. 45%) and less frequently infected with HPV18 (0% vs. 16%; p= 0.007). Genetic polymorphism of the IFN‐γ gene is associated with individual susceptibility to cervical carcinogenesis. This polymorphism correlates with HPV infection in a disease‐ and type‐specific manner.

Frequent aberration of the transforming growth factor-β receptor II gene in cell lines but no apparent mutation in pre-invasive and invasive carcinomas of the uterine cervix

The type II transforming growth factor‐β (TGF‐β) receptor (RII) gene located at 3p22 plays an important role in regulating growth and differentiation of epithelium, including that of the uterine cervix. Loss‐of‐function mutations of RII have frequently been found in gastrointestinal cancers, with a replication‐error (RER) phenotype characterized by the presence of microsatellite instability (MI). In this study, genomic PCR, SSCP and DNA sequencing were conducted to investigate the coding sequences of the RII gene in cell lines (n = 5) and tissues (n = 15) of squamous carcinomas of the uterine cervix. Intragenic deletions were noted in 2 of 5 cervical‐cancer cell lines (ME180 and HeLa cells). However, no mutation, other than DNA polymorphisms, was found in 15 cervical cancers with either alleleic loss at 3p22 (n = 11) or MI (n = 4). Further analysis of squamous intraepithelial lesions (SIL) with (n = 12) or without (n = 4) MI for the (A)10 change, a prototypic mutation found in over 90% of RER‐positive colon cancers, also showed no aberration. Our study concludes that the RII gene is frequently disrupted in cervical‐cancer cell lines, but is rarely mutated in CC and SIL tissues, including those showing MI or alleleic loss at 3p22. The underlined mechanism of genomic instability in CC and SIL may thus differ from that of colorectal cancer. The allelic loss at 3p22‐24 in CC does not involve the coding sequence of the RII gene. The non‐coding sequence of RII or an unidentified gene may be responsible for it. Int. J. Cancer 80:506–510, 1999.

Expression of p16INK4A in Pap smears containing atypical glandular cells from the uterine cervix.

OBJECTIVE To verify one of the diagnostic dilemmas concerning atypical glandular cells (AGC) by immunocytochemical detection of p16INK4A (p16) applied to routine Pap smears with correlation of follow-up biopsies for improvement of cytologic diagnoses. STUDY DESIGN The study included 36 Pap smears in AGC diagnostic categories, all of which were correlated histologically. The cytologic diagnoses of AGC were further classified according to the 2001 Bethesda System. All Pap smears were decolorized and immunostained with the primary anti-p16 antibody, clone E6H4. Immunoreactivity for p16 was correlated with histologic sections in a semiblind fashion. RESULTS Of the 36 smears containing AGC, 22 (61%) were reclassified as general AGC and 14 (39%) as AGC--favor neoplasia. Follow-up biopsies revealed that 15 (42%) cervixes had no obvious abnormalities and that 21 (58%) cases had different cervical lesions. More than half the cases (19/36, 53%) of follow-up biopsies concerning AGC-containing smears represented significant lesions. There was a much higher proportion of significant lesions (13/14, 93%) in AGC--favor neoplasia than those (6/22, 27%) in general AGC cases. Fifteen of 36 (36%) AGC-containing cases were actually squamous abnormalities on follow-up biopsies. p16 Immunocytochemical stain was reactive in 22 (61%) of 36 smears, either weakly/sporadically (2 cases, 6%) or strongly positively (20 cases, 55%). Conversely, 14 (39%) of the smears were negative for p16 and displayed predominantly reactive changes. However, there was 1 case of high grade squamous intraepithelial lesion showing negative immunostaining for p16. From the view-point of clinical significance, this analysis was highly sensitive (sensitivity, 95%) and specific (specificity, 88%) and had favorable positive (90%) and negative (94%) predictive values. CONCLUSION On the basis of both morphologic and immunostaining patterns, there was a clear association between strong p16 immunostaining of atypical cells in smears and the presence of significant lesions in the cervix except in 1 patient. Similarly, there was a clear association between lack of p16 expression and absence of cervical lesions. p16 Immunocytochemical stain can be applied successfully to conventional Pap smears and may serve as a useful biomarker in diagnoses of AGC-containing smears. This may offer a more objective parameter to help clarify this ambiguous area of gynecologic cytopathology.

HPV‐associated cervical cancers show frequent allelic loss at 3p14 but no apparent aberration of FHIT mRNA

The genetic aberration involved in the loss of heterozygosity (LOH) at 3p14 has recently been attributed to the disruption of the FHIT gene in many cancers. This study analyzed HPV DNA and allelic status of 5 microsatellite markers spaning 3p13‐3p25 in 57 cases of cervical cancer. With no homozygous deletion found in any case, a 39% overall frequency of LOH was noted. The presence of tumorigenic HPV DNA (91%) did not correlate with the allelic loss at any marker, including THRB (3p22‐24) and D3S1228 (3p14) which were found with high LOH rates of 43% (12/28) and 37% (11/30), respectively. Further analysis of FHIT mRNA in 29 cancers by reverse transcription (RT)‐PCR showed a full‐length transcript in all cases. However, additional minor transcripts were occasionally observed in cancer tissues (9/29) as well as in normal tissues (12/31) by nested PCR of the RT products. Sequence analysis of these transcripts showed exclusive internal exon deletions, suggesting a source of minor splicing variants. No apparent mutation of the mRNA sequences was found in 8 transcripts examined, except for a silent polymorphism and a site of alternative splicing. The results suggest that, although frequently reported to be abrogated in several cancers, the mRNA of FHIT remains intact in cervical cancer. Other genes closely linked to FHIT may be responsible for frequent LOH at 3p14 observed in cervical cancer. Int. J. Cancer 75:199–204, 1998.

Seldi-tof MS Profiling of Plasma Proteins in Ovarian Cancer

OBJECTIVE Proteomic profiling of plasma or serum is a technique to identify new biomarkers in disease. The objective of this study was to identify new plasma biomarkers in ovarian cancer patients using mass spectrometry protein profiling and artificial intelligence. METHODS A total of 65 plasma samples obtained from women with ovarian cancer (n = 35) and age-matched disease-free controls (n = 30) were applied to anion exchange protein chips for protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS SELDI-TOF MS was highly reproducible in detecting ovarian tumor-specific protein profiles. One protein peak (relative molecular mass, Mr, 11,537 Da) was identified in plasma from women with ovarian cancer but not in controls. Two peaks, Mr 5,147 and 8,780 Da, were present in the plasma of controls but not of women with ovarian cancer. After a training analysis, classification analysis generated by univariant or linear combination split was performed to reach a discriminant protein signature pattern. After cross validation, a sensitivity of 84% and specificity of 89% for all studied cases and controls was reached. CONCLUSION This study clearly demonstrates that the combined technology of SELDI-TOF MS and artificial intelligence is effective in distinguishing protein expression between normal and ovarian cancer plasma. The identified protein peaks may be candidate proteins for early detection of ovarian cancer or evaluation of therapeutic response.