Ernst J. M. Speel

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.

Role of human papillomavirus in the development of head and neck squamous cell carcinomas

Objective To review the literature on the role of oncogenic human papillomaviruses (HPVs) in the carcinogenesis of the head and neck mucosa. Material and Methods Molecular and epidemiological studies concerning the high-risk HPV types and their role in carcinogenesis in the head and neck region were screened. Results Different studies revealed that: (i) 15–25% of head and neck squamous cell carcinomas (HNSCCs) are clonally associated with high risk HPV types (type 16); (ii) the oropharynx and particularly the tonsils are the most susceptible sites; (iii) patients with HPV-positive tumours present with more advanced stages of disease, are relatively younger, do not have extravagant tobacco and alcohol intake and seem to have a better survival; (iv) HPV-positive tumours are characterized by poor differentiation grade and a basaloid appearance; and (v) HPV-positive tumours exhibit integrated HPV DNA, wild-type p53, pRb downregulation and overexpression of p16INK4A. Conclusion Taken together, these data support the view that HPV-harbouring HNSCC can be considered a discrete tumour entity with, moreover, a favourable prognosis. Screening of patients, especially those with tonsillar cancers, for the presence of HPV may help to further optimize treatment protocols and to provide more accurate prognostic information.

Quantitative real-time PCR does not show selective targeting of p14(ARF) but concomitant inactivation of both p16(INK4A) and p14(ARF) in 105 human primary gliomas.

In many human cancers, the INK4A locus is frequently mutated by homozygous deletions. By alternative splicing this locus encodes two non-related tumor suppressor genes, p16INK4A and p14ARF (p19ARF in mice), which regulate cell cycle and cell survival in the retinoblastoma protein (pRb) and p53 pathways, respectively. In mice, the role of p16INK4A as the critical tumor suppressor gene at the INK4A locus was challenged when it was found that p19ARF only knock-out mice developed tumors, including gliomas. We have analysed the genetic status of the INK4A locus in 105 primary gliomas using both microsatellite mapping (MSM) and quantitative real-time PCR (QRT–PCR). Comparison of the results of the two methods revealed agreement in 67% of the tumors examined. In discordant cases, fluorescence in situ hybridization (FISH) analysis was always found to support QRT–PCR classification. Direct assessment of p14ARF exon 1β, p16INK4A exon 1α and exon 2 by QRT–PCR revealed 43 (41%) homozygous and eight (7%) hemizygous deletions at the INK4A locus. In 49 (47%) gliomas, both alleles were retained. In addition, QRT–PCR, but not MSM, detected hyperploidy in five (5%) tumors. Deletion of p14ARF was always associated with co-deletion of p16INK4A and increased in frequency upon progression from low to high grade gliomas. Shorter survival was associated with homozygous deletions of INK4A in the subgroup of glioblastoma patients older than 50 years of age (P=0.025, Anova test single factor, α=0.05).

Identification of chromosome 9 alterations and p53 accumulation in isolated carcinoma in situ of the urinary bladder versus carcinoma in situ associated with carcinoma.

Carcinoma in situ (CIS) of the urinary bladder is a flat, aggressive lesion and may be the most common precursor of invasive bladder cancer. Although chromosome 9 alterations are among the earliest and most prevalent genetic alterations in bladder cancer, discrepancy exists about the frequency of chromosome 9 losses in CIS. We analyzed 22 patients with CIS of the bladder (15 patients with isolated CIS, 7 patients combined with synchronous pTa or pT1 carcinomas) for gains and losses of chromosome (peri)centromere loci 1q12, 7p11-q11, 9p11-q12, and 9p21 harboring the INK4A/ARF locus (p16(INK4A)/p14(ARF)) and INK4B (p15(INK4B)) by multiple-target fluorescence in situ hybridization, and for p53 protein accumulation by immunohistochemistry. In 15 of 20 (75%) CIS lesions analyzed p53 overexpression was detected, whereas aneusomy for chromosomes 1 and 7 was identified in 20 of 22 (91%) CIS. In 13 of 22 (60%) CIS cases analyzed, 12 of which were not associated with a synchronous pTa or pT1 carcinoma, no numerical losses for chromosome 9 (p11-q12 and 9p21) were detected as compared with chromosomes 1 and 7. Furthermore 6 of 12 (50%) patients showed a metachronous invasive carcinoma within 2 years. In the remaining nine biopsies CIS lesions (40%) were recognized that showed losses of chromosome 9p11-q12 and 9p21, six of these were associated with a synchronous pTa or pT1 carcinoma. Three of these carcinomas were pTa and exhibited loss of 9q12 as well as a homozygous deletion of 9p21. The others were invasive carcinomas in which CIS lesions were also recognized that showed no numerical loss of chromosome 9, but did show an accumulation of p53. In conclusion our data demonstrate that predominantly isolated CIS lesions contained cells with no specific loss of chromosome 9, as opposed to CIS lesions with synchronous carcinomas that showed evidence of chromosome 9 loss. Furthermore our data strengthen the proposition that p53 mutations (p53 overexpression) precede loss of chromosomes 9 and 9p21 in CIS as precursor for invasive bladder cancer, as opposed to noninvasive carcinomas where chromosome 9 (9p11-q12) losses are early and frequently combined with homozygous deletions of 9p21.

Human papillomavirus and cancer of the oropharynx. Molecular interaction and clinical implications

One-third of the cases of oropharyngeal squamous cell carcinoma (OSCC) contain oncogenic human papillomavirus (HR-HPV). Epidemiologic and molecular evidence underlines the causal role of HR-HPV in these tumors, which can be defined as HPV-related OSCC. These tumors differ from chemical/toxin-induced OSCC in several biological aspects, including specific molecular and genetic alterations. This leads to a characteristic clinical profile of HPV-related OSCC. Sexual risk factors play a role; however, the knowledge about natural infection and the rate of persistence of HR-HPV in the oropharynx is marginal. It is shown that the distinct biological behavior of the HPV-related subset of oropharyngeal tumors results in a more favorable prognosis. This might be the result of a better response to chemotherapy and radiotherapy. However, further studies are needed to show whether it will be possible to reliably select patients for individualized therapy depended on the HPV status of their tumors. Therefore, we think it will be mandatory to consider and stratify HPV status in the design of prospective clinical trials in the future.ZusammenfassungEin Drittel aller Oropharynxkarzinome (OSCC) enthalten onkogene humane Papillomviren (HR-HPV). Epidemiologische und molekulare Untersuchungen belegen, dass HR-HPV bei diesen Tumoren ursächlich sind; sie können als HPV-assoziierte OSCC bezeichnen werden. Sexuelle Risikofaktoren scheinen von Bedeutung zu sein. Wenig ist bekannt über die natürliche Infektion und Persistenz von HR-HPV im Oropharynx. Die HPV-assoziierten OSCC unterscheiden sich von denen durch die klassischen Noxen verursachten OSCC grundlegend. Dies betrifft das Expressionsmuster verschiedener Zellzyklusproteine sowie genetische Veränderungen. Das unterschiedliche biologische Verhalten zeigt sich klinisch durch eine günstigere Prognose der HPV-assoziierten OSCC. Die Ursachen sind noch unklar, eine höhere Empfindlichkeit für Strahlen- und Chemotherapie ist aber denkbar. Für Studien bei OSCC ist in Zukunft eine Stratifizierung nach dem HPV-Status sinnvoll. Ob zukünftig die Patienten nach Bestimmung ihres HPV-Status einer entsprechenden maßgeschneiderten Therapie zuzuführen sind, muss in Therapiestudien geklärt werden.AbstractOne-third of the cases of oropharyngeal squamous cell carcinoma (OSCC) contain oncogenic human papillomavirus (HR-HPV). Epidemiologic and molecular evidence underlines the causal role of HR-HPV in these tumors, which can be defined as HPV-related OSCC. These tumors differ from chemical/toxin-induced OSCC in several biological aspects, including specific molecular and genetic alterations. This leads to a characteristic clinical profile of HPV-related OSCC. Sexual risk factors play a role; however, the knowledge about natural infection and the rate of persistence of HR-HPV in the oropharynx is marginal. It is shown that the distinct biological behavior of the HPV-related subset of oropharyngeal tumors results in a more favorable prognosis. This might be the result of a better response to chemotherapy and radiotherapy. However, further studies are needed to show whether it will be possible to reliably select patients for individualized therapy depended on the HPV status of their tumors. Therefore, we think it will be mandatory to consider and stratify HPV status in the design of prospective clinical trials in the future.

Humane Papillomviren und Oropharynxkarzinome

One-third of the cases of oropharyngeal squamous cell carcinoma (OSCC) contain oncogenic human papillomavirus (HR-HPV). Epidemiologic and molecular evidence underlines the causal role of HR-HPV in these tumors, which can be defined as HPV-related OSCC. These tumors differ from chemical/toxin-induced OSCC in several biological aspects, including specific molecular and genetic alterations. This leads to a characteristic clinical profile of HPV-related OSCC. Sexual risk factors play a role; however, the knowledge about natural infection and the rate of persistence of HR-HPV in the oropharynx is marginal. It is shown that the distinct biological behavior of the HPV-related subset of oropharyngeal tumors results in a more favorable prognosis. This might be the result of a better response to chemotherapy and radiotherapy. However, further studies are needed to show whether it will be possible to reliably select patients for individualized therapy depended on the HPV status of their tumors. Therefore, we think it will be mandatory to consider and stratify HPV status in the design of prospective clinical trials in the future.ZusammenfassungEin Drittel aller Oropharynxkarzinome (OSCC) enthalten onkogene humane Papillomviren (HR-HPV). Epidemiologische und molekulare Untersuchungen belegen, dass HR-HPV bei diesen Tumoren ursächlich sind; sie können als HPV-assoziierte OSCC bezeichnen werden. Sexuelle Risikofaktoren scheinen von Bedeutung zu sein. Wenig ist bekannt über die natürliche Infektion und Persistenz von HR-HPV im Oropharynx. Die HPV-assoziierten OSCC unterscheiden sich von denen durch die klassischen Noxen verursachten OSCC grundlegend. Dies betrifft das Expressionsmuster verschiedener Zellzyklusproteine sowie genetische Veränderungen. Das unterschiedliche biologische Verhalten zeigt sich klinisch durch eine günstigere Prognose der HPV-assoziierten OSCC. Die Ursachen sind noch unklar, eine höhere Empfindlichkeit für Strahlen- und Chemotherapie ist aber denkbar. Für Studien bei OSCC ist in Zukunft eine Stratifizierung nach dem HPV-Status sinnvoll. Ob zukünftig die Patienten nach Bestimmung ihres HPV-Status einer entsprechenden maßgeschneiderten Therapie zuzuführen sind, muss in Therapiestudien geklärt werden.AbstractOne-third of the cases of oropharyngeal squamous cell carcinoma (OSCC) contain oncogenic human papillomavirus (HR-HPV). Epidemiologic and molecular evidence underlines the causal role of HR-HPV in these tumors, which can be defined as HPV-related OSCC. These tumors differ from chemical/toxin-induced OSCC in several biological aspects, including specific molecular and genetic alterations. This leads to a characteristic clinical profile of HPV-related OSCC. Sexual risk factors play a role; however, the knowledge about natural infection and the rate of persistence of HR-HPV in the oropharynx is marginal. It is shown that the distinct biological behavior of the HPV-related subset of oropharyngeal tumors results in a more favorable prognosis. This might be the result of a better response to chemotherapy and radiotherapy. However, further studies are needed to show whether it will be possible to reliably select patients for individualized therapy depended on the HPV status of their tumors. Therefore, we think it will be mandatory to consider and stratify HPV status in the design of prospective clinical trials in the future.

Gain of chromosome 8q is a frequent finding in pleuropulmonary blastoma.

Pleuropulmonary blastomas are rare malignant intrathoracic tumors of early childhood. They appear as a pulmonary- and/or pleural-based mass and their pathogenesis and relationship to other pediatric solid tumors is not well understood. In this study, paraffin-embedded material of five cases of pleuropulmonary blastoma was analyzed for genetic alterations by comparative genomic hybridization and five genetic loci by fluorescence in situ hybridization. Comparative genomic hybridization identified aberrations in all pleuropulmonary blastomas, including four amplifications in three tumors at chromosomes 5q33–34, 11q22.2–ter, 15q25–ter, and 19q11–13.2. The most frequent DNA gains involved 8q11–22.2 (four cases) and 20q (two cases), whereas the most common losses included 9p21–24 (two cases) and 11p14 (three cases). Chromosome 8 gains were confirmed by fluorescent in situ hybridization, resulting in the detection of up to five copies of chromosome 8 centromeres per nucleus. In the two surviving patients, chromosome 8 gains were the only genetic abnormality, suggesting that this might be an early event in pleuropulmonary blastoma carcinogenesis. The identification of new genetic alterations as well as the confirmation of previously reported ones (especially 8q gains) in pleuropulmonary blastoma should help to improve our understanding of both the molecular mechanisms underlying the tumorigenesis of pleuropulmonary blastoma and the relationship of pleuropulmonary blastoma with other pediatric tumors.

Limited additive value of the Ki-67 proliferative index on patient survival in World Health Organization-classified pulmonary carcinoids

Currently pulmonary carcinoids are separated into typical and atypical based on mitotic count and presence of necrosis, according to the World Health Organization. At variance with gastroenteropancreatic neuroendocrine tumours, which are graded based on mitotic count and Ki‐67 proliferative index, the use of Ki‐67 for grading pulmonary carcinoids is still under debate.

10q25.3 (DMBT1) copy number changes in astrocytoma grades II and IV.

In the literature, it has been suggested that loss of the 10q25‐26 region, including the DMBT1 gene (10q25.3), is correlated with initiation and/or malignant progression of astrocytomas, although the results of the studies on the loss of heterozygosity that led to this assumption are not unequivocal. For this reason, using double‐target fluorescence in situ hybridization, we compared copy number changes of 10q25.3 to those of the pericentromeric region (10q12) in 10 cases each of astrocytoma grades II and IV. The same specimens were analyzed for copy number changes of chromosome 1, as a marker for polyploidy, and chromosome 7, which is often gained in astrocytomas of all grades. Our results show that selective loss of the 10q25.3 region was present in 2 of 10 specimens in both astrocytoma grade II and grade IV, occurring only in tumors with polysomy for 10q12. Furthermore, astrocytoma grade II often showed polyploidy for chromosomes 1, 7, and 10 (8 of 10 specimens). In addition, astrocytoma grade IV frequently exhibited losses of chromosome 10 in a high percentage of nuclei. Although based on a small number of cases, the results clearly show that loss of the 10q25.3 region is uncommon in astrocytoma grade II and mostly coincident with loss of chromosome 10 in grade IV tumors. These data indicate that selective loss of the 10q25.3 region, including the DMBT1 gene, is not an initiating event in the genesis of astrocytoma grade II.

Prevalence of high-risk human papillomavirus infection and cancer gene mutations in nonmalignant tonsils

OBJECTIVESnTo analyze the prevalence of high-risk HPV (human papillomavirus) and genetic alterations in nonmalignant tonsils.nnnMETHODSnWe collected benign fresh tonsillar tissue specimens from 477 patients undergoing tonsillectomy because of chronic tonsillitis or tonsillar hypertrophy in 2012 (Group A, n=237) and in 2015 (Group B, n=240). Luminex xMAP technique served to detect E6/E7 DNA from 16 different high-risk HPV types. Tonsillar DNA and peripheral blood leukocyte DNA from the infected individuals were analyzed using Nimblegen SeqCap EZ Comprehensive Cancer Design panel. The panel targets 578 different genes that are relevant in carcinogenesis. HPV negative tonsillar specimens from age- and gender matched individuals were used as controls. All specimens harboring high-risk HPV were analyzed using fluorescence in situ hybridization (FISH).nnnRESULTSnFive of 477 (1.0%) patients tested positive for the following HPV types: HPV16 (two cases), HPV52 (one case), HPV66 (one case), HPV52 and HPV68 (coinfection, one case). FISH analyses showed that the appearance of HPV in specimens infected with HPV 16 was episomal. Benign tonsils infected with high-risk HPV harbored mutations in EP300, NF1, PIK3CA, and RB1 which are considered relevant in the development of HPV-associated head and neck squamous cell carcinoma (SCC).nnnCONCLUSIONSnThe prevalence of high-risk HPV in nonmalignant tonsils is low. High-risk HPV positive tonsils harbored mutations in genes that are commonly altered in HPV-associated head and neck SCC. The role of these mutations in tonsillar carcinogenesis is an interesting target for future research.