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HPV infection and EGFR activation/alteration in HIV-infected East African patients with conjunctival carcinoma.

Yu JJ, Fu P, Pink JJ, Dawson D, Wasman J, Orem J, Mwanda WO, Zhu H, Liang X, Guo Y, Petros WP, Mitsuyasu RT, Wabinga H, Remick SC - PLoS ONE (2010)

Bottom Line: HPV 18 was found in 61% of samples, with HPV 16 double-genotype in 6 patients (16%).A SNP in 10 patients and one missense mutation were found within EGFR tyrosine kinase domain.Agents that target the EGFR pathway may have potential therapeutic benefit.

View Article: PubMed Central - PubMed

Affiliation: Mary Babb Randolph Cancer Center and Molecular Medicine Core Facility, School of Medicine, West Virginia University, Morgantown, West Virginia, United States of America. jyu@hsc.wvu.edu

ABSTRACT

Background: There has been substantial growth in the numbers of patients with conjunctival squamous cell carcinoma infected with HIV in East Africa. The natural history of the conjunctival squamous cell carcinoma appears to be unique in this region of the world, but the etiologic mechanism unclear and therapeutic options limited. This research was carried out to determine if conjunctival squamous cell carcinoma harbors human papillomavirus DNA and is associated with activation of the EGFR signaling pathway. Positive findings would identify etiologic causes and provide clinical guidance to improve treatment.

Methods/findings: Expression of p-MAPK/MAPK, p-Akt/Akt and p-EGFR/EGFR in cell nuclei and cytoplasm of 38 FFPE specimens were assessed by immunohistochemistry; HPV genotype was detected by qPCR assay; EGFR mutation was assessed by DNA sequencing analysis; and EGFR mRNA expression was measured using relative qPCR. Statistical analyses included two-sided Fisher exact test or chi-square test, Spearman correlation coefficient and ANOVA. HPV 18 was found in 61% of samples, with HPV 16 double-genotype in 6 patients (16%). Immunohistochemistry and qPCR data suggest that activation and expression of the EGFR signaling pathway is related to disease progression of conjunctival cancer. The associations between cytoplasmic p-MAPK, cytoplasmic p-Akt and tumor invasiveness were significant (p = 0.05 or 0.028). Nuclear p-EGFR appeared only in invasive tumors. A significant positive association between EGFR expression and disease invasiveness was observed (p = 0.01). A SNP in 10 patients and one missense mutation were found within EGFR tyrosine kinase domain. Statistical analysis indicates that patients with measurable EGFR expression more likely harbor EGFR mutations, compared to those with negative EGFR expression (35.3% vs. 0%).

Conclusions/significance: We conclude that HPV types 16/18 infection is frequent in East African patients with AIDS-associated squamous cell carcinoma of the conjunctiva. EGFR activation/alteration may contribute to and sustain the high prevalence of this cancer. Our findings hint that adoption of HPV vaccination strategies may impact the incidence of conjunctival carcinoma. Agents that target the EGFR pathway may have potential therapeutic benefit.

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Prevalence of high-risk HPV genotypes in conjunctival squamous cell carcinoma by real-time RQ PCR assay.Tumor DNA extraction was performed using Pico Pure DNA Extraction kit. The viral DNA of 4 HPV genotypes were determined using a TaqMan-based real-time quantitative PCR analysis. Type-specific primers and probes for HPV types 16, 18, 52 and 59 were selected to target genome segments of the E6/E7 region and synthesized by Applied Biosystems. RNase-P as endogenous control for each sample (in duplicate) was applied in this assay. Amplification results from the endogenous control were used to normalize the amplification results from the target HPV types. Finally, Ct, ΔCt, ΔΔCt, RQ and gene expression plots were generated by the ABI 7500 Fast System SDS Software (Version 1.4).
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pone-0010477-g001: Prevalence of high-risk HPV genotypes in conjunctival squamous cell carcinoma by real-time RQ PCR assay.Tumor DNA extraction was performed using Pico Pure DNA Extraction kit. The viral DNA of 4 HPV genotypes were determined using a TaqMan-based real-time quantitative PCR analysis. Type-specific primers and probes for HPV types 16, 18, 52 and 59 were selected to target genome segments of the E6/E7 region and synthesized by Applied Biosystems. RNase-P as endogenous control for each sample (in duplicate) was applied in this assay. Amplification results from the endogenous control were used to normalize the amplification results from the target HPV types. Finally, Ct, ΔCt, ΔΔCt, RQ and gene expression plots were generated by the ABI 7500 Fast System SDS Software (Version 1.4).

Mentions: Viral DNA of high-risk HPV genotypes 16 and 18 was detected in surveyed specimens by absolute- and relative-quantitation real-time PCR assays. HPV 18, the most common oncogenic strain, was found in 23 of 38 (61%) conjunctival cancer patients. Three in situ diseases and 3 invasive tumors of the 23 HPV 18 positive patients also showed coincident HPV 16 infection (i.e., double genotypes of HPV 16 and 18). High-risk HPV types 52 and 59 were not characterized in these samples (Table 1 & Fig. 1). This technique appears to be a sensitive, reproducible and reliable method to determine HPV genotypes in FFPE samples of conjunctival squamous cell carcinoma.


HPV infection and EGFR activation/alteration in HIV-infected East African patients with conjunctival carcinoma.

Yu JJ, Fu P, Pink JJ, Dawson D, Wasman J, Orem J, Mwanda WO, Zhu H, Liang X, Guo Y, Petros WP, Mitsuyasu RT, Wabinga H, Remick SC - PLoS ONE (2010)

Prevalence of high-risk HPV genotypes in conjunctival squamous cell carcinoma by real-time RQ PCR assay.Tumor DNA extraction was performed using Pico Pure DNA Extraction kit. The viral DNA of 4 HPV genotypes were determined using a TaqMan-based real-time quantitative PCR analysis. Type-specific primers and probes for HPV types 16, 18, 52 and 59 were selected to target genome segments of the E6/E7 region and synthesized by Applied Biosystems. RNase-P as endogenous control for each sample (in duplicate) was applied in this assay. Amplification results from the endogenous control were used to normalize the amplification results from the target HPV types. Finally, Ct, ΔCt, ΔΔCt, RQ and gene expression plots were generated by the ABI 7500 Fast System SDS Software (Version 1.4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871792&req=5

pone-0010477-g001: Prevalence of high-risk HPV genotypes in conjunctival squamous cell carcinoma by real-time RQ PCR assay.Tumor DNA extraction was performed using Pico Pure DNA Extraction kit. The viral DNA of 4 HPV genotypes were determined using a TaqMan-based real-time quantitative PCR analysis. Type-specific primers and probes for HPV types 16, 18, 52 and 59 were selected to target genome segments of the E6/E7 region and synthesized by Applied Biosystems. RNase-P as endogenous control for each sample (in duplicate) was applied in this assay. Amplification results from the endogenous control were used to normalize the amplification results from the target HPV types. Finally, Ct, ΔCt, ΔΔCt, RQ and gene expression plots were generated by the ABI 7500 Fast System SDS Software (Version 1.4).
Mentions: Viral DNA of high-risk HPV genotypes 16 and 18 was detected in surveyed specimens by absolute- and relative-quantitation real-time PCR assays. HPV 18, the most common oncogenic strain, was found in 23 of 38 (61%) conjunctival cancer patients. Three in situ diseases and 3 invasive tumors of the 23 HPV 18 positive patients also showed coincident HPV 16 infection (i.e., double genotypes of HPV 16 and 18). High-risk HPV types 52 and 59 were not characterized in these samples (Table 1 & Fig. 1). This technique appears to be a sensitive, reproducible and reliable method to determine HPV genotypes in FFPE samples of conjunctival squamous cell carcinoma.

Bottom Line: HPV 18 was found in 61% of samples, with HPV 16 double-genotype in 6 patients (16%).A SNP in 10 patients and one missense mutation were found within EGFR tyrosine kinase domain.Agents that target the EGFR pathway may have potential therapeutic benefit.

View Article: PubMed Central - PubMed

Affiliation: Mary Babb Randolph Cancer Center and Molecular Medicine Core Facility, School of Medicine, West Virginia University, Morgantown, West Virginia, United States of America. jyu@hsc.wvu.edu

ABSTRACT

Background: There has been substantial growth in the numbers of patients with conjunctival squamous cell carcinoma infected with HIV in East Africa. The natural history of the conjunctival squamous cell carcinoma appears to be unique in this region of the world, but the etiologic mechanism unclear and therapeutic options limited. This research was carried out to determine if conjunctival squamous cell carcinoma harbors human papillomavirus DNA and is associated with activation of the EGFR signaling pathway. Positive findings would identify etiologic causes and provide clinical guidance to improve treatment.

Methods/findings: Expression of p-MAPK/MAPK, p-Akt/Akt and p-EGFR/EGFR in cell nuclei and cytoplasm of 38 FFPE specimens were assessed by immunohistochemistry; HPV genotype was detected by qPCR assay; EGFR mutation was assessed by DNA sequencing analysis; and EGFR mRNA expression was measured using relative qPCR. Statistical analyses included two-sided Fisher exact test or chi-square test, Spearman correlation coefficient and ANOVA. HPV 18 was found in 61% of samples, with HPV 16 double-genotype in 6 patients (16%). Immunohistochemistry and qPCR data suggest that activation and expression of the EGFR signaling pathway is related to disease progression of conjunctival cancer. The associations between cytoplasmic p-MAPK, cytoplasmic p-Akt and tumor invasiveness were significant (p = 0.05 or 0.028). Nuclear p-EGFR appeared only in invasive tumors. A significant positive association between EGFR expression and disease invasiveness was observed (p = 0.01). A SNP in 10 patients and one missense mutation were found within EGFR tyrosine kinase domain. Statistical analysis indicates that patients with measurable EGFR expression more likely harbor EGFR mutations, compared to those with negative EGFR expression (35.3% vs. 0%).

Conclusions/significance: We conclude that HPV types 16/18 infection is frequent in East African patients with AIDS-associated squamous cell carcinoma of the conjunctiva. EGFR activation/alteration may contribute to and sustain the high prevalence of this cancer. Our findings hint that adoption of HPV vaccination strategies may impact the incidence of conjunctival carcinoma. Agents that target the EGFR pathway may have potential therapeutic benefit.

Show MeSH
Related in: MedlinePlus