Anton H. N. Hopman

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

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) and p16INK4A‐specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16INK4A accumulation. Only 5 out of 48 HPV‐negative tumors showed p16INK4A immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non‐smoking patients with HPV‐containing TSCC show a remarkably better disease‐specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16INK4A overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV‐positive tumors show a favorable prognosis as compared to those with HPV‐negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non‐smokers differ from those occurring in smokers, the former being related to HPV 16 infection.

A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8.

Besides well‐known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV‐induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single‐strand conformation polymorphism (SSCP) analysis of exons 5–8. Paraffin‐embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16INK4A overexpression in all 10 HPV‐positive tumors. Although FISH is considered to be less sensitive than PCR‐based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV‐positive carcinomas. However, in none of the latter cases could mutations in exons 5–8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors.

Genetic Signatures of HPV-related and Unrelated Oropharyngeal Carcinoma and Their Prognostic Implications

Purpose: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. Experimental Design: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16INK4A immunostaining. The results were correlated with HPV status and clinical data from patients. Results: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P = 0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P = 0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P = 0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P = 0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P = 0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P = 0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P = 0.008; disease-free survival, P = 0.01) and none of these patients had a tumor recurrence. Conclusions: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

Amplification Methods to Increase the Sensitivity of In Situ Hybridization: Play CARD(S)

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.

Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalities.

Cervical intraepithelial neoplasia (CIN I, II, and III) and cases of CIN III associated with micro‐invasive cervical carcinoma (CIN III & mCA) were analysed for evidence of episomal or integrated human papillomavirus (HPV) 16/18 DNA by fluorescence in situ hybridization (FISH). In parallel, numerical aberrations of chromosomes 1, 17, and X were determined in these lesions as indicators of genomic instability. HPV 16/18 DNA was present in 2 of 12 CIN I, 19 of 23 CIN II/III, and 10 of 12 CIN III & mCA. None of the CIN I and only two of the 19 HPV 16/18‐positive solitary CIN II/III showed an integrated HPV pattern. However, all ten cases of HPV‐positive CIN III & mCA showed this pattern. Transition of CIN II/III to CIN III & mCA therefore correlates strongly with viral integration (p < 0.001). Chromosomal aberrations were detected in 23 of 31 HPV 16/18‐positive lesions (14 solitary CIN I–III and nine CIN III & mCA) and 5 of 16 HPV‐negative lesions. Nine of 21 HPV 16/18‐positive solitary CIN I–III showed tetrasomy for all chromosomes tested, while trisomies for a single chromosome were seen in a further five of these HPV‐positive lesions. In eight of ten HPV‐positive CIN III & mCA, predominantly aneusomies and/or polysomies were detected. A significant correlation (p < 0.02) was found between the chromosome copy number and the physical status of HPV, indicating that in its episomal form HPV induces genomic changes such as tetrasomies and single trisomies, while HPV integration correlates with aneusomies and polysomies, predominantly detected in CIN III & mCA. These data indicate that integration of HPV 16/18 DNA is a pivotal step in the transition of CIN to micro‐invasive carcinoma. Copyright

Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer

Recently proposed events associated with the progression of cervical intraepithelial neoplasia (CIN) 2/3 to cervical carcinoma include integration of human papillomavirus (HPV) into the host genome, genomic instability, and an increase in chromosome 3q copy number. In particular, the gene coding for the RNA component of telomerase (TERC) at 3q26 has been implicated as a possible candidate gene. Since it is not known to date how these events are temporally related during cervical carcinogenesis, the aim of the present study was to assess the correlation between TERC gene copy number and the physical status of HPV during progression in cervical neoplasia. Solitary precursor lesions of the uterine cervix (CIN 2/3, n = 17), lesions associated with a micro‐invasive carcinoma (CIN 3&mCA, n = 13), and advanced invasive carcinomas (invCA, n = 7) were analysed by fluorescence in situ hybridization (FISH) to determine the physical status of the virus and TERC gene copy number. The TERC gene was increasingly gained with progression of CIN 2/3 (3 of 17) through CIN 3&mCA (7 of 13) to invCA (5 of 7). In the lesions exhibiting gain of TERC, the virus was predominantly integrated. This was seen in eight of ten diploid lesions, indicating that these events can occur prior to aneuploidization and are strongly associated with the progression of CIN 3 to mCA and invCA (p < 0.001). With progression to carcinoma, a number of these lesions show polyploidization, resulting in aneuploidy and high TERC gene copy numbers. In conclusion, genomic integration of oncogenic HPV and gain of the human telomerase gene TERC appear to be important associated genetic events in the progression of uterine cervical dysplasia to invasive cancer. Copyright

A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.

Sensitive Multicolor Fluorescence In Situ Hybridization Using Catalyzed Reporter Deposition (CARD) Amplification

We describe the simultaneous localization of DNA sequences in cell and chromosome preparations by means of differently fluorochrome-labeled (AMCA, FITC, TRITC) tyramides using the catalyzed reporter deposition (CARD) procedure. For this purpose, repeated as well as single-copy DNA probes were labeled with biotin, digoxigenin, and FITC, hybridized, and visualized with three different cytochemical detection systems based on horseradish peroxidase conjugates. These were sequentially applied to interphase nuclei and metaphase chromosomes at low concentrations to prevent crossreaction and nonspecific background. In situ localized peroxidase activity was visualized by the deposition of fluorochrome-labeled tyramide molecules. To allow specific deposition of a second and a third tyramide conjugate for multiple-target fluorescence in situ hybridization (FISH), remaining peroxidase activity was always completely inactivated by a mild acid treatment before application of the next peroxidase conjugate. The CARD reactions were optimized for maximal signal-to-noise ratio and discrete localization by tuning reaction time, H2O2, and tyramide concentrations. For both repeated and single-copy DNA targets, high FISH signal intensities were obtained, providing improvement of sensitivity over conventional indirect detection systems. In addition, the fluorescence CARD detection system proved to be highly efficient and easy to implement in multiple-labeling studies, such as reported here for FISH.

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

Although there is consensus that HPV integration is common in invasive cervical carcinomas and uncommon or absent in low‐grade uterine cervical intraepithelial neoplasia (CIN I), estimates for HPV integration in CIN II/III range from 5 to 100% using different PCR‐based and in situ hybridization (ISH) approaches. It has been suggested that HPV integration can be identified using ISH by scoring of punctate signals. The increased sensitivity of fluorescence ISH (FISH) methods, allowing the detection of single copies of HPV, complicates the distinction between integrated and episomal HPV. Recently it has been suggested that, in such assays, the signals originating from integrated virus can be hidden in a background of episomal HPV. We therefore compared 2 different FISH protocols for the detection of integrated HPV in a series of CIN II/III lesions: 1) a mild protocol in which episomal HPV and RNA is retained and 2) a harsh protocol that extensively extracts proteins and RNA, and which promotes the partial loss of episomal HPV but not integrated HPV. A series of 28 HPV 16/18 positive CIN II/III lesions (17 solitary lesions and 11 lesions adjacent to microinvasive carcinoma) were studied. A punctate signal pattern was identified in 7 of these lesions with both protocols. Punctate signal was also present in control samples from lesions that are known to be associated with HPV integration (invasive squamous cell carcinoma (n = 3), adenocarcinoma in situ (n = 3), and invasive adenocarcinoma (n = 1). HPV RNA contributed significantly to the intensity of punctate FISH signal, especially when applying the mild protocol, as shown by omitting DNA denaturation, including RNase pretreatment steps and measuring the fluorescence signal intensity. Also, HPV RNA was frequently detected in addition to episomal/integrated HPV DNA in the majority of the other 21 CIN II/III lesions; this resulted in intense granular/diffuse FISH signals throughout the epithelium. However, in 7 of these lesions, the harsh protocol gave a more consistent punctate pattern in cells throughout the full thickness of the epithelium. This supports the hypothesis that the harsh protocol unmasks integrated HPV more efficiently by extracting RNA and episomal HPV. Overall, with this harsh protocol, a clonally expanded population of cells containing punctate HPV signals was found in 5 of 17 (29%) solitary CIN II/III lesions and in 9 of 11 (88%) CIN II/III lesions associated with microinvasive carcinoma. Combining these data with the results from our previous study, with the harsh protocol in 7 of 40 (18%) solitary CIN II/III lesions and 19/21 (90%) CIN II/III lesions associated with microinvasive carcinoma (p < 0.001), this pattern was found. This indicates that, when robustly defined, a punctate HPV pattern in CIN II/III lesions is associated with the presence of an invasive carcinoma.

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.