HPV <i>in situ</i> hybridization: Impact of different protocols on the detection of integrated HPV

Hopman, Anton H. N.; Kamps, M.; Smedts, Frank; Speel, Ernst‐Jan M.; Herrington, C. Simon; Ramaekers, Frans C.S.

doi:10.1002/ijc.20862

Cited by 72 publications

(71 citation statements)

References 44 publications

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“…In addition to the PCR-based approach, it has been suggested that in situ hybridization can also detect the viral status, viral DNA load, and even transcript levels. 30 As shown in Figure 2E, the HPV-16 E6 in situ hybridization demonstrates not only a mixed granular and diffuse staining pattern but also diverse signal intensity from zero to high levels in the tumor. Because HPV RNA can also be detected, the diffuse staining may represent episomal forms or active transcripts.…”

Section: Association Of Viral Status With Cell Cycle Proteinmentioning

confidence: 85%

Use of combined molecular biomarkers for prediction of clinical outcomes in locally advanced tonsillar cancers treated with chemoradiotherapy alone

Chung¹,

Lee²,

Horng

et al. 2008

Head & Neck

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Background. Environmental exposures to tobacco, alcohol, human papillomavirus (HPV) and/or Epstein-Barr virus (EBV), all of which can perturb multiple cell cycle proteins or tumor suppressors, have been implicated in the pathogenesis of different subsets of head and neck cancers. The aim of this study was to investigate to which extent the virus infection by itself, and/or the altered cell cycle proteins, contributes to prognosis in locally advanced tonsillar squamous cell carcinomas (TSCCs) treated with concurrent chemoradiotherapy (CCRT) alone.Methods. Serial tumor tissue arrays from archival samples were tested for the presence of HPV genome integration or EBV episome by means of DNA sequencing, realtime polymerase chain reaction (PCR), and in situ hybridization. Alterations of cell cycle proteins (p53, pRb, and p21) were evaluated by immunohistochemical staining. The association of viral presence with altered cell cycle proteins was correlated to clinical outcomes.Results. Of the 46 patients with the same T2N2bM0 stage IVA among consecutive patients with TSCC, 23 (50%) had integrated HPV DNA and only 1 (2%) had EBV episome. The HPV types detected were almost all HPV-16. A reduced expression pattern of p53, pRb, and p21 was noted in HPVpositive tumors, and the incremental number of alterations in the 3 proteins was significantly associated with HPV-negative tumors. The presence or absence of HPV together with the number of altered expression of the 3 cell cycle markers resulted in further identification of 4 biologically and clinically distinct subgroups with different outcomes after CCRT.Conclusions. Use of combined biomarkers of oncogenic HPV and tumor suppressors of p53, pRb, and p21 in advanced TSCC provides prognostic molecular classification superior to the TNM stage system and identifies low-risk patients for organ preservation by CCRT alone and high-risk patients who might benefit from planned tonsillectomy and neck dissection before

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Section: Association Of Viral Status With Cell Cycle Proteinmentioning

confidence: 85%

Use of combined molecular biomarkers for prediction of clinical outcomes in locally advanced tonsillar cancers treated with chemoradiotherapy alone

Chung¹,

Lee²,

Horng

et al. 2008

Head & Neck

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“…Hopman et al (2005) have shown by in situ hybridization that 88% of CIN2/3 associated with microinvasive carcinoma contained integrated viral DNA. In contrast, the characteristic punctuate signal Figure 3 Relative copy numbers of E6all transcripts (white boxes) and spliced E6*I transcripts (hatched boxes) in CIN and CxCa samples in relation to the physical state of the HPV genome (e, episomes; i, integrates; e þ i, episomes þ integrates).…”

Section: Discussionmentioning

confidence: 99%

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts

et al. 2007

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Virus integration into the host genome is a characteristic step during cervical carcinogenesis. Experimental data provide evidence that integration could result in increased levels of oncogene (E6/E7) transcripts. This is the first study in which the level of viral transcripts is correlated to the physical state of the viral genome in cervical intraepithelial neoplasia (CIN) and cervical carcinomas (CxCa). Using the APOT-assay integrate-derived transcripts only were detected in 3/28 (11%) CIN and in 28/55 (51%) carcinomas, respectively. The remaining biopsies contained either episome-derived transcripts only or both mRNA species. SybrGreen real time reverse transcriptase-PCR assays were used to quantify viral gene expression for (i) all transcripts initiated from p97, (ii) full-length E6, (iii) E6*I and (iv) E5 transcripts. E6/E7 transcript levels showed a broad distribution but similar median values irrespective of histopathological grading and physical state of the viral genome. Biopsies with integrate-derived transcripts only generally lacked E5-specific mRNA. Our data do not support the hypothesis that HPV integration invariably results in high levels of oncogene transcripts. Instead, constitutive expression of oncogene transcripts rather than the level of expression appears to be decisive for transformation and the maintenance of the malignant phenotype.

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“…Data concerning the physical status of HPV in TSCC are so far limited and confusing, ranging from virus being present only in contribute to the observed FISH signal, 46 but that the protocol used in this study is optimal for unmasking integrated HPV DNA. Preliminary data on 2 mucosal dysplasia and 4 carcinoma in situ lesions identified adjacent to the 33 TSCC specimens also showed punctate FISH signals indicative for integrated HPV 16.…”

Section: Hpv 16 Is Integrated In Tsccmentioning

confidence: 99%

Marked differences in survival rate between smokers and nonsmokers with HPV 16‐associated tonsillar carcinomas

Hafkamp

Manni

Haesevoets

et al. 2008

Intl Journal of Cancer

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305

292

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Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV-DNA and the use of p16INK4A overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridization (FISH) Head and neck squamous cell carcinomas (HNSCC) account for 4% of all malignancies in the Western world, for up to 50% of all malignancies in Southeast Asian countries and for 6.5% of all annual cancer cases worldwide.1 HNSCC is associated with severe disease-and treatment-related morbidity and because treatment has not improved greatly in recent years, the 5-year survival rate remains 50%. HNSCC develop in various anatomical defined regions, including the oral cavity, larynx and pharynx. These organ-specific tumors each show specific clinical presentations and outcome, and are treated by different strategies. 2,3 The median age at presentation is 60 years and approximately two-third of patients are male. Well-known risk factors in the etiology of HNSCC are cigarette smoking combined with alcohol consumption in Western countries, or with betel quid chewing in Asia. A history of tobacco use is present in 90% of patients who develop oral cavity cancers. 2,3Despite these evident associations, the exact mechanisms by which these factors cause tumor initiation and progression are not fully understood. Furthermore, the fact that most tobacco and alcohol users do not develop HNSCC and that in recent years more often individuals without a history of these traditional risk factors have been witnessed, 4 underlines the complexity of HNSCC pathogenesis and a role for additional factors in the disease process.Increasing evidence suggests that human oncogenic papillomaviruses (HPVs), known to cause uterine cervical and other anogenital cancers, may also be of importance in the pathogenesis of HNSCC. 5 The strongest association has been found for oropharyngeal carcinomas, especially tonsillar carcinomas.6-11 Sero-positive patients for HPV 16 or with a history of HPV-related anogenital cancer also show increased risk rates of developing oropharyngeal cancer.12,13 The prevalence of HPV-exhibiting HNSCC, however, varies broadly amongst several studies (2-76%) due to differences in the population, combination of histological subsites, type and number of specimens analyzed, and detection methods used. 7,14 Thus, besides determining the presence of HPV DNA it has been suggested to better define the biological association of oncogenic HPV with these tumors, e.g., by means of assessing the viral copy number per cell, the viral oncoprotein E6/E7 expression levels, perturbation of pRb-dependent cell cycle control, or the physical status of the virus (episomal or integrated). 15 In this way, several reports have shown that HPV 16 is predomi...

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HPV in situ hybridization: Impact of different protocols on the detection of integrated HPV

Cited by 72 publications

References 44 publications

Use of combined molecular biomarkers for prediction of clinical outcomes in locally advanced tonsillar cancers treated with chemoradiotherapy alone

Use of combined molecular biomarkers for prediction of clinical outcomes in locally advanced tonsillar cancers treated with chemoradiotherapy alone

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts

Marked differences in survival rate between smokers and nonsmokers with HPV 16‐associated tonsillar carcinomas

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