Annals of Clinical Microbiology and Antimicrobials Emphysematous cystitis: An unusual disease of the Genito-Urinary system suspected on imaging

Ravi K Bobba
Department of Internal Medicine Veterans Affairs Medical Center
University of Virginia
Roanoke-Salem Program
VirginiaUSA

Edward L Arsura
Department of Internal Medicine Veterans Affairs Medical Center
University of Virginia
Roanoke-Salem Program
VirginiaUSA

Pawanjit S Sarna email@example.com
Department of Internal Medicine Veterans Affairs Medical Center
University of Virginia
Roanoke-Salem Program
VirginiaUSA

Amar K Sawh
Department of Internal Medicine Veterans Affairs Medical Center
University of Virginia
Roanoke-Salem Program
VirginiaUSA

Annals of Clinical Microbiology and Antimicrobials Emphysematous cystitis: An unusual disease of the Genito-Urinary system suspected on imaging
10.1186/1476-0711-3-20BioMed Central Page 1 of 4 (page number not for citation purposes) Case report * Corresponding author hematuriaemphysematous cystitis
Emphysematous cystitis is a rare disease entity caused by gas fermenting bacterial and fungal pathogens. Clinical symptoms are nonspecific and diagnostic clues often arise from the unanticipated imaging findings. We report a case of 52-year-old male who presented with fever, dysuria and gross hematuria who was found to have emphysematous cystitis.

Introduction

Emphysematous cystitis is an uncommon, but severe manifestation of infection of the urinary bladder produced by gas forming organisms. The presentation may be atypical and contrary to the degree of inflammation, patients may present with subtle clinical findings. A high index of suspicion, especially in susceptible populations, is needed. We report a case of middle-aged, poorly controlled, diabetic male who presented with dysuria, fever and hematuria and was found to have Escherichia coli emphysematous cystitis that resolved with antibiotic treatment.

Case Report

A 52-year-old male presented with fever, chills and blood in the urine. Four days prior to admission, he noted increased urinary frequency, urgency, occasional incontinence and burning sensation during micturition. His past medical history is significant for coronary artery disease, hyperlipidemia, hypertension and insulin dependent diabetes mellitus (most recent HgbA1c of 11.7 two weeks prior). He had 66-pack year history of smoking. Medications on admission included, aspirin, atenolol, cyclobenzaprine, gabapentin, gemfibrozil, glipizide, NPH insulin, hydrochlorthiazide/triamterene, nifedipine, nitroglycerin patch, omeprazole, and sertraline.

On physical examination, his temperature was 100.9°F, pulse 88, BP 123/71 mm/hg. Cardiac and pulmonary examinations were unremarkable. His abdomen was soft, nontender with positive bowel sounds and no organomegaly detected. The rectal examination was normal with a non-tender, smooth prostate. Neurological exam was exam was unremarkable.

Laboratory evaluation revealed white cell count 17,400 cells/mm 3 , hemoglobin 15.1 gm/dl, sodium 128 mmol/l, potassium 4.1 mmol/l, chloride 91 mmol/l, bicarbonate 24 mmol/L, glucose 273 mg/dl and an anion gap of 13. Urine analysis revealed red, cloudy urine with pH 5.0, nitrite positive and numerous white and red blood cells on microscopy. A pelvic X-ray (figure 1) showed circumferential air in the bladder wall. CT scan of the pelvis (figure 2) revealed gas in the bladder lumen and wall extending to the right ureter. He was empirically treated with gentamicin and piperacillin/tazobactam. He responded with defervescence and a decline in his white cell count. Urine cultures grew Escherichia coli and based on the sensitivities he was discharged home on ciprofloxacin.

Discussion

Emphysematous cystitis is a rare entity characterized by pockets of gas in and around the bladder wall produced by bacterial or fungal fermentation [1,2]. Patients may complain of irritative symptoms, abdominal discomfort or pneumaturia. A history of pneumaturia is highly suggestive, but is rarely offered by the patient. As occurred in our case and in a number of cases in the literature, the clinical features were inconclusive or actually unhelpful [3][4][5][6]. The radiographic findings provided the first and only diagnostic clue. The disease is often associated with female sex, immunocompromised state, diabetes mellitus, previous recurrent urinary tract infections, urinary stasis, neurogenic bladder and in transplant recipients [7]. Therefore, in susceptible patients, with the above risk factors along with signs and symptoms of urinary tract infection, the index of suspicion for this entity should be high. The most common organism is E. coli [2], but other organisms reported to produce emphysematous cystitis include Enterobacter aerogenes, Klebsiella pneumonia, Proteus mirabilis, Staphylococcus aureus, streptococci, Clostridium perfringens [8], and Candida albicans [9]. The mechanism by which gas appears in the wall of the bladder may involve either X-ray of pelvis showing gas in the urinary bladder wall (Arrow) Diagnostic entities associated with gas in the genitourinary tract include emphysematous pyelonephritis, emphysematous pyelitis, and gas-forming renal abcess. Patients with emphysematous cystitis are not as acutely ill as those with pyelonephritis or pyelitis. Abdomino-pelvic CT scan can further delineate the extent of disease. It is important to differentiate emphysematous cystitis from emphysematous pyelonephritis, in which gas involves the renal parenchyma, since the latter has an increased mortality and generally requires nephrectomy. In contrast surgical intervention is rarely needed in emphysematous cystitis except when an anatomical abnormality like an obstruction or stone is present [10].

The source of this gas within the urinary tract is from infection, trauma, vesico-enteric fistulas from radiation therapy, rectal carcinoma, diverticular disease or Crohn's disease and iatrogenic causes, such as diagnostic or surgical instrumentation. History, physical exam and imaging are the best modalities to differentiate the above etiologic causes. Fistulous tracts, abscess, can be excluded on CT scan.

Emphysematous cystitis requires aggressive treatment with parenteral antibiotics and bladder drainage [11].

CT scan of the pelvis revealing gas in the bladder and the bladder wall Delayed diagnosis may lead to unfavorable outcomes including overwhelming infection, extension to ureters and renal parenchyma, bladder rupture and death. Improved outcomes may be achieved by early recognition of the infection, by clinical and radiological assessment, and by appropriate antibiotic therapy.

Conclusion

Emphysematous cystitis most often is not diagnosed by routine or systematic approach. It is a rare entity, detected on imaging, and the physician should be cautious, tailor the diagnostic approach to individual patients based on the suspicion, available clinical and radiological data, and consider emphysematous cystitis in the differential diagnosis of hematuria in a patient with known risk factors.

Figure 1 X(
1-ray of pelvis showing gas in the urinary bladder wall (Arrow) page number not for citation purposes) transluminal dissection of gas or true infection of the bladder wall with pathogens.

Figure 2 (
2CT scan of the pelvis revealing gas in the bladder and the bladder wall.Publish with Bio Med Central and every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours -you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Annals of Clinical Microbiology and Antimicrobials 2004, page number not for citation purposes)
(page number not for citation purposes)

Emphysematous cystitis: a review of the spectrum of disease. H J Quint, G W Drach, W D Rappaport, C J Hoffmann, J Urol. 147Quint HJ, Drach GW, Rappaport WD, Hoffmann CJ: Emphysema- tous cystitis: a review of the spectrum of disease. J Urol 1992, 147:134-137.

Cystitis emphysematosa: 19 cases with intraluminal and interstitial collections of gas. H Bailey, Am J Roentgenol Radium Ther Nucl Med. 86Bailey H: Cystitis emphysematosa: 19 cases with intraluminal and interstitial collections of gas. Am J Roentgenol Radium Ther Nucl Med 1961, 86:850-862.

An unusual presentation of emphysematous cystitis. J Weddle, B Brunton, D R Rittenhouse, Am J Emerg Med. 16Weddle J, Brunton B, Rittenhouse DR: An unusual presentation of emphysematous cystitis. Am J Emerg Med 1998, 16:664-6.

Emphysematous cystitis presenting as painless gross hematuria. J Davidson, C V Pollack, J Emerg Med. 13Davidson J, Pollack CV: Emphysematous cystitis presenting as painless gross hematuria. J Emerg Med 1995, 13:317-20.

. T: Emphysematous Knutson, Cystitis, Scand J Urol Nephrol. 37Knutson T: Emphysematous cystitis. Scand J Urol Nephrol 2003, 37:361-3.

Images in clinical medicine. Emphysematous cystitis. S Asada, T Kawasaki, N Engl J Med. 17349258Asada S, Kawasaki T: Images in clinical medicine. Emphysema- tous cystitis. N Engl J Med 2003, 17(349 (3)):258.

Emphysematous cystitis and pyelitis in a diabetic renal transplant recipient. E Akalin, C Hyde, G Schmitt, J Kaufman, R J Hamburger, Transplantation. 62Akalin E, Hyde C, Schmitt G, Kaufman J, Hamburger RJ: Emphyse- matous cystitis and pyelitis in a diabetic renal transplant recipient. Transplantation 1996, 62:1024-1026.

Emphysematous cystitis due to Clostridium perfringens. T E West, H P Holley, A D Lauer, JAMA. 246West TE, Holley HP, Lauer AD: Emphysematous cystitis due to Clostridium perfringens. JAMA 1981, 246:363-364.

Emphysematous prostatitis and cystitis secondary to Candida albicans. D P Bartkowski, J R Lanesky, J Urol. 1395Bartkowski DP, Lanesky JR: Emphysematous prostatitis and cys- titis secondary to Candida albicans. J Urol 1988, 139(5):1063-5.

Emphysematous cystitis: a complication of urinary tract infection occurring predominantly in diabetic women. F Ankel, A B Wolfson, J S Stapczynski, Ann Emerg Med. 19Ankel F, Wolfson AB, Stapczynski JS: Emphysematous cystitis: a complication of urinary tract infection occurring predomi- nantly in diabetic women. Ann Emerg Med 1990, 19:404-6.

Emphysematous cystitis. R Yasumoto, M Asakawa, N Nishisaka, Br J Urol. 636644Yasumoto R, Asakawa M, Nishisaka N: Emphysematous cystitis. Br J Urol 1989, 63(6):644.


BMC Medical Genomics Genomic profiling identifies common HPV-associated chromosomal alterations in squamous cell carcinomas of cervix and head and neck

Saskia M Wilting email@example.com
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

Serge J Smeets
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

Department of Otolaryngology/Head-Neck Surgery
VU University Medical Center
AmsterdamThe Netherlands

Peter Jf Snijders
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

Wessel N Van Wieringen email@example.com
Department of Epidemiology and Biostatistics
VU University Medical Center
AmsterdamThe Netherlands

Mark A Van De Wiel
Department of Epidemiology and Biostatistics
VU University Medical Center
AmsterdamThe Netherlands

Gerrit A Meijer
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

Bauke Ylstra firstname.lastname@example.com
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

C René Leemans
Department of Otolaryngology/Head-Neck Surgery
VU University Medical Center
AmsterdamThe Netherlands

Chris Jlm Meijer
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

Ruud H Brakenhoff
Department of Otolaryngology/Head-Neck Surgery
VU University Medical Center
AmsterdamThe Netherlands

J M Boudewijn
Braakhuis
Department of Otolaryngology/Head-Neck Surgery
VU University Medical Center
AmsterdamThe Netherlands

Renske Dm
Department of Pathology
VU University Medical Center
AmsterdamThe Netherlands

Steenbergen
BMC Medical Genomics Genomic profiling identifies common HPV-associated chromosomal alterations in squamous cell carcinomas of cervix and head and neck
10.1186/1755-8794-2-32BioMed Central Page 1 of 11 (page number not for citation purposes) Research article
Background: It is well known that a persistent infection with high-risk human papillomavirus (hrHPV) is causally involved in the development of squamous cell carcinomas of the uterine cervix (CxSCCs) and a subset of SCCs of the head and neck (HNSCCs). The latter differ from hrHPVnegative HNSCCs at the clinical and molecular level.Methods:To determine whether hrHPV-associated SCCs arising from different organs have specific chromosomal alterations in common, we compared genome-wide chromosomal profiles of 10 CxSCCs (all hrHPV-positive) with 12 hrHPV-positive HNSCCs and 30 hrHPV-negative HNSCCs. Potential organ-specific alterations and alterations shared by SCCs in general were investigated as well.Results: Unsupervised hierarchical clustering resulted in one mainly hrHPV-positive and one mainly hrHPV-negative cluster. Interestingly, loss at 13q and gain at 20q were frequent in HPVpositive carcinomas of both origins, but uncommon in hrHPV-negative HNSCCs, indicating that these alterations are associated with hrHPV-mediated carcinogenesis. Within the group of hrHPVpositive carcinomas, HNSCCs more frequently showed gains of multiple regions at 8q whereas CxSCCs more often showed loss at 17p. Finally, gains at 3q24-29 and losses at 11q22.3-25 were frequent (>50%) in all sample groups.Conclusion:In this study hrHPV-specific, organ-specific, and pan-SCC chromosomal alterations were identified. The existence of hrHPV-specific alterations in SCCs of different anatomical origin, suggests that these alterations are crucial for hrHPV-mediated carcinogenesis.

Background

In the pathogenesis of uterine cervical cancer the necessary and causal involvement of high-risk types of the human papillomavirus (hrHPV) is widely accepted and supported by strong epidemiological and molecular evidence [1]. HrHPV is present in virtually all cervical carcinomas and the viral oncogenes E6 and E7 are consistently expressed in cervical cancers and precancers. Deregulated expression of these oncogenes in the basal, dividing cells of the epithelium interferes with cell cycle control due to their ability to induce degradation of the tumour suppressor proteins p53 and pRb, respectively. This results in uncontrolled cell proliferation and accumulation of specific (epi)genetic changes in the host cell genome, driving progression to a malignant phenotype [2,3]. In a previous study we have used array-based comparative genomic hybridisation (array CGH) to determine frequent chromosomal alterations in cervical cancer, which included gains at 1q, 3q and 20q and losses at 8q, 10q, 11q, and 13q [4]. The necessity of these and other additional (epi)genetic alterations in the carcinogenic process is illustrated by the fact that their frequency increases with increasing severity of cervical disease.

On the other hand, head and neck squamous cell carcinoma (HNSCC) is known to be mainly caused by wellestablished life-style related habits, such as tobacco and excessive alcohol consumption. However, besides the influence of these life-style carcinogens, high risk human papillomavirus (hrHPV) is present in 15-35% of HNSCCs and has been suggested to be a separate aetiological factor in head-and-neck carcinogenesis [5][6][7][8]. Several studies have shown that hrHPV-positive HNSCCs are associated with a better clinical outcome [7][8][9]. Moreover, molecular differences were found between hrHPV-positive and hrHPV-negative HNSCCs, supporting the idea of two separate carcinogenic pathways to HNSCC, one determined by life-style carcinogens and the other by hrHPV [5,6,[10][11][12][13].

In a previous study, using array CGH, we identified a number of chromosomal alterations specific for hrHPVnegative HNSCCs that were absent in hrHPV-positive HNSCCs, including loss at 3p, 5q, and 9p, and amplifications at 11q [13]. The hrHPV-positive HNSCCs were characterised by a lower level of chromosomal alterations, none of which were hrHPV-specific. To investigate potential organ-independent, hrHPV-associated chromosomal alterations, genomic profiles of cervical SCCs (CxSCCs), hrHPV-positive and hrHPV-negative HNSCCs were compared using sophisticated clustering and statistical approaches [4,13]. In addition, we also investigated the presence of organ-specific alterations and alterations shared by all SCCs included in this study.

Methods

CGH microarrays

We used chromosomal profiles of 30 hrHPV-negative HNSCCs, 12 hrHPV-positive HNSCCs and 10 CxSCCs all of which were previously described (Table 1) [4,13,14]. To avoid possible confounding of our results, we excluded a subset of hrHPV-negative HNSCCs described in Smeets et al, which showed little to no alterations and should therefore be considered a separate group [14].

CGH BAC microarrays produced at the Microarray facility of the VU Medical Center were used. These arrays included the 1 Mb resolution Sanger BAC clone set and a subset of clones from the Children's Hospital Oakland Research Institute (CHORI). Spots were quantified using ImaGene 5.6.1 software (BioDiscovery Ltd, Marina del Rey, CA, USA) with default settings for the flagging of bad quality spots.

The entire dataset described here is available from the Gene Expression Omnibus (GEO, http:// www.ncbi.nlm.nih.gov/projects/geo/) through series accession numbers GSE6473 (CxSCC) and GSE12020 (HNSCC).

This study followed the ethical guidelines of the Institutional Review Board of the VU University Medical Center and informed consent was obtained from all patients included.

Array CGH analysis

Calling of gains and losses BAC clones were positioned along the genome according to the May 2004 freeze. After exclusion of clones with one or more flagged spots, the average of the triplicate spots was calculated for each BAC clone. Log 2 ratios were normalised per spotted sub-array by subtraction of the median value of all BAC clones spotted within that subarray. Segmentation and subsequent calling of gained, amplified and lost regions was done using CGHCall, an automated calling algorithm. Segments with a probability score of ≥ 0.5 were considered gained, amplified or lost [15].

Reduction of dataset into chromosomal regions

We used the CGHregions algorithm to reduce our dataset to chromosomal regions, accepting maximally 0.1% information loss (Threshold = 0.001). It was shown by Van de Wiel et al that the use of regions instead of single BAC clones improved the effectiveness of subsequent statistical analyses and facilitated interpretation of the results [16].

Clustering analysis

The samples were clustered by means of a modified version of WECCA [17]. WECCA is a hierarchical clustering method tailor-made for called aCGH data. The modified version accommodates the use of call probabilities instead of calls. The use of the call probabilities in the unsupervised analysis will give a more subtle picture of the similarities and differences between the samples. The modified version of WECCA defines the distance between two features as the symmetric Kullback-Leibler divergence. The distance between the call probability profiles of two samples is then defined as the average of these divergences over all features. In the construction of the dendrogram we used Ward's linkage as it yields compact and well-separated clusters.

Statistical analysis

The association between clustering results and HPV status was determined by chi-square testing. The average total number of altered regions was compared between HPVpositive and HPV-negative carcinomas using the non-parametric Mann Whitney test. Two-sided p-values below 0.05 were considered statistically significant. Alteration patterns between HPV-positive (HNSCCs and CxSCCs) and HPV-negative tumours as well as between HNSCCs and CxSCCs were compared using a binomial differential proportion test. The test procedure includes a permutation-based false discovery rate (FDR) correction for multiple testing, needed to discriminate real differences from chance effects [18]. An FDR below 0.10 was considered statistically significant.

Gene ontology analysis

To interpret the biological significance of the genes that are located at altered chromosomal regions of interest, a gene ontology analysis was performed using Ingenuity Pathways Analysis (Ingenuity Systems ® , Redwood City, USA). Biological/molecular functions were considered to be significantly overrepresented if they contained more than 1 gene and the Benjamini-Hochberg corrected pvalue was p < 0.10 [19].

Results

hrHPV-positive carcinomas cluster together

To obtain an overview of the similarities between samples, unsupervised hierarchical clustering was performed. This method enabled us to determine in an unbiased manner whether chromosomal profiles of hrHPV-positive HNSCCs were more closely related to hrHPV-negative HNSCCs or hrHPV-positive CxSCCs.

As is shown in Figure 1, two clusters emerged. Cluster 1 contained 24 samples, 18 of which were hrHPV-positive Result from the unsupervised hierarchical clustering analysis Figure 1 Result from the unsupervised hierarchical clustering analysis. Cluster 1 contains in majority hrHPV-positive carcinomas, as is indicated by the black boxes in the legend underneath the heatmap (p < 0.0001).

(75%) and 6 were hrHPV-negative. Cluster 2 contained 28 samples of which 4 samples were hrHPV-positive and 24 were hrHPV-negative (86%). This association between cluster assignment and hrHPV status was statistically significant (p < 0.0001). The hrHPV-positive cluster 1 included both samples of cervical (n = 8) and head and neck origin (n = 10), indicating similarities between the chromosomal profiles of hrHPV-induced carcinomas of different anatomical origins. Within cluster 1, however, 7 out of 8 cervical samples formed a separate sub-cluster. This suggests that organ-specific alterations exist as well in hrHPV-positive HNSCCs and CxSCCs.

hrHPV-associated loss at chromosome 13q and gain at 20q

To assess the differences between all three sample groups, the frequency of gains (including amplifications) and losses was analysed for all chromosomal regions ( Figure  2). In general, hrHPV-negative carcinomas showed significantly more altered regions than hrHPV-positive carcinomas (p = 0.022).

To determine hrHPV-specific chromosomal alterations, the frequency of alterations was compared between hrHPV-positive (12 HNSCCs and 10 CxSCCs) and hrHPV-negative tumours (30 HNSCCs) for all chromosomal regions. Regions showing a significant difference (FDR < 0.10) in this comparison are shown in Table 2.

Interestingly, loss of 13q21.1-21.33 and gain of 20p12.1-q13.33 were significantly more frequent in HPV-positive SCCs compared to hrHPV-negative HNSCCs (66.7% and 58.5% in hrHPV-positive SCCs compared to 33% and 24% in hrHPV-negative SCCs, respectively). As was also shown in our previous study, loss of regions at 3p and 5q, and gains/amplifications of a small region at 11q (CCND1 locus) were specific for hrHPV-negative HNSCCs [13]. A region at 8p showed loss in hrHPV-negative HNSCCs, gain in hrHPV-positive HNSCCs and no alteration in CxSCCs. Gain at chromosome 8q was more frequent in hrHPV-negative HNSCCs, which was due to absence of this alteration in CxSCCs.

Our unsupervised classification results indicated that, within our hrHPV-positive cluster, CxSCCs formed a separate group. To identify potential organ-specific alterations, we therefore also compared the frequency of alterations for all regions between hrHPV-positive HNSCCs (n = 12) and CxSCCs (n = 10) ( Table 3). Gains at 3q and losses at 17p were significantly more frequent in CxSCCs than HNSCCs (FDR < 0.10). On the other hand, HNSCCs showed frequent gains at chromosome 8q and losses at 11q. It is important to note that even though gain at 3q and loss at 11q were significantly different between hrHPV-positive HNSCCs and CxSCCs, these alterations were frequent in all sample groups (>50%). Significant differences for these regions can mainly be explained by the fact that the size of the exact altered region differed between groups. Therefore, the smallest regions of overlap between all samples, namely 3q24-29 and 11q22.3-25, may represent general alterations in carcinomas derived from squamous epithelium.

All results described above are summarised in a Venn diagram, showing a general overview of the frequently altered chromosomal arms specific to or common between the (sub) groups ( Figure 3).

Biological functions of genes located within hrHPV-specific chromosomal alterations

As described above gain at 20p12.1-q13. 33  Within the SRO at chromosome 20, two genes reside, i.e. NCOA6 and RBM39, which showed elevated expression in hrHPV16 E7 expressing cells in vitro [20]. Other cancerrelated genes located within this SRO include PIGU, E2F1, and DNMT3B. The SRO on chromosome 13 encompasses the PCDH17 gene and a cluster of five identical loci all of which are predicted to encode proline-rich proteins that contain several dopamine D4 receptor signatures.

Subsequent gene ontology analysis of all genes located in these SROs identified a number of significantly overrepresented GO biological/molecular functions, including cell cycle, cell-to-cell signalling and interaction, cellular growth and proliferation, and a number of cellular maintenance functions (i.e. DNA replication, recombination and repair, gene expression, cellular function and maintenance, cellular assembly and organisation, cellular compromise, cell death, cell morphology, cellular development, nucleic acid metabolism, and lipid metabolism).

Discussion

In the present study we compared genome-wide chromosomal profiles of hrHPV-negative HNSCCs, hrHPV-positive HNSCCs and hrHPV-positive CxSCCs, to determine whether hrHPV-associated carcinomas of different origins have similar chromosomal signatures. In addition, potential organ-specific alterations were determined within the total group of hrHPV-positive SCCs. mosomal profiles of hrHPV-induced carcinomas. Subsequent supervised statistical analysis identified a number of hrHPV-associated chromosomal alterations, including gains at 20p13-q13.33 and losses at 13q21.1-21.33, which were frequent (>50%) in hrHPV-positive carcinomas, but were only rarely observed in hrHPV-negative carcinomas. In contrast, lost regions at 3p and 5q as well as amplifications at 11q13.3 (CCND1 locus) were frequent in hrHPV-negative carcinomas, but not in hrHPV-positive carcinomas. In our previous study comparing only hrHPV-positive and hrHPV-negative HNSCCs, the same regions were identified as well as being specific for hrHPVnegative HNSCCs [13].

In the present study we found that gains at 20q and losses at 13q are specific for hrHPV-positive carcinomas of dif-    ferent anatomical origins. Our previous study, including only hrHPV-positive and hrHPV-negative HNSCCs, did not identify these alterations as specific for hrHPV-positive HNSCCs. However, in the present study a larger number of hrHPV-negative hrHNSCCs and HPV-positive SCCs was included. Furthermore, a sophisticated, objective calling method was presently used to determine gained and lost regions [15]. Interestingly, in a number of studies describing hrHPV E6 and/or E7 mediated immortalisation of keratinocytes of multiple anatomical origins, gains of chromosome 20q and losses of chromosome 13q were consistently observed [20][21][22][23]. Low-level gains of chromosome 20q are suggested to be caused by E7 expression and consequent inactivation of the pRb pathway in epithelial cells [20,21,24]. Deletion of part of the long arm of chromosome 13 is frequently found in a number of hrHPV-associated tumours, including cervical, anal and head and neck carcinomas [4,13,25]. Sabbir et al showed that loss of 13q in HNSCCs was associated with the presence of hrHPV, which is in agreement with our findings [26].

In a recent study Pyeon et al showed that the gene expression patterns of hrHPV-positive HNSCCs and CxSCCs dif-fered yet shared many changes compared to hrHPVnegative HNSCCs [10]. Our study shows that the same holds true on a chromosomal level. Interestingly, 28% of the genes Pyeon et al found to be differentially expressed between hrHPV-positive and hrHPV-negative carcinomas is located within the chromosomal regions identified in this study and showed expression changes concordant with the chromosomal alterations. Of these genes, 39% was located at chromosome 1p, 25% at 5q, and 14% at 3p. The other genes were located at 11q, 18q and 21q. Only one gene, SYCP2, was located within the hrHPV-specific chromosomal alterations found in this study (20q), but was not located within our SRO. To the best of our knowledge none of the genes are known to directly interact with hrHPVE6 and/or E7 [27,28]. Pathway analysis of all genes overlapping with our findings identified cell cycle/proliferation as most overrepresented biological function, which is in concordance with the observations made by Pyeon et al [10].

Pathway analysis of all genes located within the hrHPVassociated SROs at chromosome 20q and 13q found in this study, again underlined the importance of cell cycle (replication and proliferation) related genes in hrHPVmediated carcinogenesis. This may be related to the continuous E7-regulated E2F1 activation and is accompanied by changes in overall cellular maintenance systems, such as nucleic acid metabolism, as was also found in HPV16 E7 expressing epithelial cells in vitro [20]. Two genes, NCOA6 and RBM39, overlapped between this in vitro study and our results, warranting further investigation of their role in hrHPV-mediated transformation. NCOA6 encodes a transcriptional coactivator interacting with basal transcription factors, histone acetyltransferases, and methyltransferases. RBM39 encodes an RNA binding protein and possible splicing factor and may act as a transcriptional coactivator for the AP-1 transcription activator complex and estrogen receptors. Other cancer-related genes at 20q include E2F1, which is specifically targeted by hrHPV-mediated degradation of pRb, PIGU, which may play a role in cell cycle control and was identified as an oncogene in bladder cancer [29], and DNMT3B, a de novo DNA methyl transferase. We previously showed that DNMT3B is amplified in the cervical cancer cell line SiHa and found a correlation between increased DNMT3B gene copy numbers and elevated mRNA expression in 78% of CxSCCs [4]. The increased DNMT3B levels are most likely related to the high frequency of tumour suppressor gene promoter hypermethylation events during hrHPV-mediated carcinogenesis [30][31][32]. The SRO at chromosome 13q includes one known gene, PCDH17, a member of the protocadherin family, which is a subfamily of the cadherin superfamily. At present no reports are available describing interactions between hrHPV and protocadherins. How-Summary of common and specific chromosomal alterations in the different sample groups ever hrHPV presence has been related to decreased E-cadherin and subsequent impaired immune response [33][34][35].

Next to the hrHPV-related common events, organ-specific alterations for CxSCCs and HNSCCs were identified as well. CxSCCs more frequently showed loss at 17p, while HNSCCs were characterised by frequent gains at chromosome 8q, harbouring the oncogene c-Myc [36].

Gains at 3q and losses at 11q were found frequently in all SCCs included in this study, suggesting their involvement in carcinogenesis of squamous epithelial cells in general. Interestingly, we previously showed that gains of 3q were highly frequent in CxSCCs but not in adenocarcinomas of the same organ, further emphasising that this alteration may be specific for squamous epithelium [4].

Conclusion

Together with results from previous studies, our findings support a causal role for hrHPV in the development of a subset of HNSCCs. Consequently, hrHPV-positive and hrHPV-negative HNSCCs should be regarded as different disease entities requiring different diagnostic and therapeutic approaches. The fact that hrHPV-associated SCCs of different organs have chromosomal alterations in common, suggests that these alterations are crucial for hrHPVinduced carcinogenesis. Diagnostic and/or therapeutic targets based on these alterations may therefore be relevant to hrHPV-associated SCCs of all anatomical origins.

Abbreviations

hrHPV: high-risk human papillomavirus; CxSCC: squamous cell carcinoma of the uterine cervix; HNSCC: squamous cell carcinoma of the head and neck; SCC: squamous cell carcinoma; CGH: comparative genomic hybridisation; BAC: bacterial artificial chromosome; Mb: megabase; FDR: false discovery rate; SRO: smallest region of overlap; GO: gene ontology.

and loss at 13q21.1-21.33 were significantly more frequent in hrHPV-positive SCCs compared to hrHPV-negative ones. In fact, a 4.5 megabase (Mb) region on chromosome 20 (20q11.21-q11.23), and a 2 Mb region on chromosome 13 (13q21.1) formed the smallest regions of overlap (SRO) at these respective loci when all hrHPV-positive carcinomas were considered (Figure 4A and 4B). The SRO at chromosome 20q contains 78 genes and the one at chromosome 13q contains 6 genes [see Additional file 1].

Unsupervised hierarchical clustering resulted in a separate hrHPV-positive cluster, indicating similarities in the chro-Frequency plots for all 3 sample groupsFigure 2 Frequency plots for all 3 sample groups. The frequency of gains (positive axis) and losses (negative axis) are shown for A. hrHPV-negative HNSCCs, B. hrHPV-positive HNSCCs and C. CxSCCs for chromosome 1-22.

specific alterations are printed in bold. Alterations specific for hrHPV-negative HNSCCs are italicised. Mb; Megabases, FDR; False Discovery Rate, amp; amplification

Alterations specific for HNSCCs are printed in bold, alterations specific for CxSCCs are italicised. Mb;Megabases, FDR; False Discovery Rate, amp; amplification

Figure 3
3Summary of common and specific chromosomal alterations in the different sample groups. Chromosome arms showing frequent alterations (>50%) in one or more sample groups (hrHPV-positive HNSCCs; hrHPV-negative HNSCCs; CxSCCs) are shown in a Venn diagram. ↑ indicates gain; ↓ indicates loss Genomic coordinates of A. losses at chromosome 13 and B. gains at chromosome 20 are shown for all hrHPV-positive carci-nomas Figure 4 Genomic coordinates of A. losses at chromosome 13 and B. gains at chromosome 20 are shown for all hrHPVpositive carcinomas. Chromosomal alterations in CxSCCs are shown by dashed lines and alterations in hrHPV-positive HNSCCs by solid lines. In C. the smallest regions of overlap (SROs) between hrHPV-positive carcinomas at chromosome 13 and 20 are summarised.

Table 1 :
1Summary of clinical data of carcinomas included in this study.Sample ID
origin
hrHPV (+/-) age (yrs) sex

CxSCC2
cervix
+
39
female
CxSCC4
cervix
+
62
female
CxSCC12
cervix
+
44
female
CxSCC15
cervix
+
47
female
CxSCC27
cervix
+
49
female
CxSCC28
cervix
+
48
female
CxSCC32
cervix
+
37
female
CxSCC36
cervix
+
72
female
CxSCC38
cervix
+
51
female
CxSCC39
cervix
+
40
female

HNSCC9741 oropharynx
-
53
female
HNSCC9762 oral cavity
-
76
female
HNSCC9773 oral cavity
-
49
male
HNSCC9830 oral cavity
-
42
male
HNSCC9848 oropharynx
-
59
male
HNSCC9892 oral cavity
-
67
male
HNSCC9897 oral cavity
-
72
male
HNSCC9902 oral cavity
-
53
female
HNSCC9942 oral cavity
-
55
female
HNSCC9952 oral cavity
-
61
female
HNSCC9956 oral cavity
-
38
male
HNSCC2014 oral cavity
-
68
female
HNSCC2034 oral cavity
-
45
female
HNSCC9738 oral cavity
-
60
male
HNSCC9745 oropharynx
-
52
male
HNSCC9750 oral cavity
-
65
male
HNSCC9812 oral cavity
-
65
female
HNSCC9827 oropharynx
-
57
male
HNSCC9829 oral cavity
-
74
female
HNSCC9831 oral cavity
-
55
male
HNSCC9841 oropharynx
-
55
female
HNSCC9847 oropharynx
-
57
male
HNSCC9880 oral cavity
-
71
female
HNSCC9907 oral cavity
-
46
male
HNSCC9914 oropharynx
-
78
male
HNSCC9926 oropharynx
-
60
male
HNSCC9957 oral cavity
-
51
male
HNSCC9970 oral cavity
-
49
female
HNSCC9981 oropharynx
-
55
female
HNSCC9987 oral cavity
-
79
male

HNSCC9881 oral cavity
+
51
male
HNSCC9729 oropharynx
+
52
male
HNSCC9838 oropharynx
+
60
male
HNSCC9860 oropharynx
+
67
female
HNSCC9901 oropharynx
+
57
female
HNSCC9948 oropharynx
+
70
male
HNSCC9951 oropharynx
+
46
male
HNSCC9808 oral cavity
+
46
male
HNSCC9859 oral cavity
+
40
male
HNSCC9924 oral cavity
+
41
female
HNSCC9947 oral cavity
+
72
male
HNSCC2015 oral cavity
+
65
male

Table 2 :
2Significantly different chromosomal alterations between hrHPV+ and hrHPV-carcinomas.HPV+ (HNSCC; CxSCC)
HPV-

Table 3 :
3Significantly different chromosomal alterations between hrHPV-positive HNSCCs and CxSCCs.HNSCC+
CxSCC

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AcknowledgementsWe would like to thank the Mapping Core and Map Finishing groups of the Wellcome Trust Sanger Institute for initial clone supply and verification. This work was partly supported by the Centre for Medical Systems Biology (CMSB) in the framework of the Netherlands Genomic Initiative (NGI).Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSMW and SJS performed array experiments and data analysis and drafted the manuscript. RDMS and BJMB participated in the design of the study and the drafting of the manuscript. WNvW and MAvdW contributed to the statistical analyses. BY and GAM were involved in the development of the micorarray platform and provided the facilities for the microarray experiments. PJFS, CRL, CJLMM and RHB contributed to the conception of the study and critically revised the manuscript. All authors read and approved the final manuscript.chromosomal alterations at chromosomes 13q and 20qAdditional materialAdditional file 1BAC clones and genes included in the SROs of the hrHPV-specificPre-publication historyThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/2/32/prepub
Papillomaviruses and cancer: from basic studies to clinical application. H Zur Hausen, Nat Rev Cancer. 2zur Hausen H: Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002, 2:342-350.

HPV-mediated transformation of the anogenital tract. R D Steenbergen, J De Wilde, S M Wilting, A A Brink, P J Snijders, C J Meijer, J Clin Virol. 321SupplSteenbergen RD, de Wilde J, Wilting SM, Brink AA, Snijders PJ, Meijer CJ: HPV-mediated transformation of the anogenital tract. J Clin Virol 2005, 32(Suppl 1):S25-S33.

Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. H Zur Hausen, J Natl Cancer Inst. 92zur Hausen H: Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Can- cer Inst 2000, 92:690-698.

Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix. S M Wilting, P J Snijders, G A Meijer, B Ylstra, Ijssel Pr Van Den, A M Snijders, D G Albertson, J Coffa, J P Schouten, M A Wiel, Van De, J Pathol. 209Wilting SM, Snijders PJ, Meijer GA, Ylstra B, Ijssel PR van den, Snijders AM, Albertson DG, Coffa J, Schouten JP, Wiel MA van de, et al.: Increased gene copy numbers at chromosome 20q are fre- quent in both squamous cell carcinomas and adenocarcino- mas of the cervix. J Pathol 2006, 209:220-230.

Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. B J Braakhuis, P J Snijders, W J Keune, C J Meijer, H J Ruijter-Schippers, C R Leemans, R H Brakenhoff, J Natl Cancer Inst. 96Braakhuis BJ, Snijders PJ, Keune WJ, Meijer CJ, Ruijter-Schippers HJ, Leemans CR, Brakenhoff RH: Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst 2004, 96:998-1006.

Comparative genomic hybridization analysis of tonsillar cancer reveals a different pattern of genomic imbalances in human papillomavirus-positive and -negative tumors. L Dahlgren, H Mellin, D Wangsa, K Heselmeyer-Haddad, L Bjornestal, J Lindholm, E Munck-Wikland, G Auer, T Ried, T Dalianis, Int J Cancer. 107Dahlgren L, Mellin H, Wangsa D, Heselmeyer-Haddad K, Bjornestal L, Lindholm J, Munck-Wikland E, Auer G, Ried T, Dalianis T: Com- parative genomic hybridization analysis of tonsillar cancer reveals a different pattern of genomic imbalances in human papillomavirus-positive and -negative tumors. Int J Cancer 2003, 107:244-249.

Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. M L Gillison, W M Koch, R B Capone, M Spafford, W H Westra, L Wu, M L Zahurak, R W Daniel, M Viglione, D E Symer, J Natl Cancer Inst. 92Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, Zahurak ML, Daniel RW, Viglione M, Symer DE, et al.: Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000, 92:709-720.

Human papillomavirus and head and neck cancer: epidemiology and molecular biology. R G Mckaig, R S Baric, A F Olshan, Head Neck. 20McKaig RG, Baric RS, Olshan AF: Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head Neck 1998, 20:250-265.

Human papillomaviruses in head and neck cancer: 8 year-survival-analysis of 73 patients. M Hoffmann, T Gorogh, S Gottschlich, C Lohrey, W Rittgen, P Ambrosch, E Schwarz, T Kahn, Cancer Lett. 218Hoffmann M, Gorogh T, Gottschlich S, Lohrey C, Rittgen W, Ambro- sch P, Schwarz E, Kahn T: Human papillomaviruses in head and neck cancer: 8 year-survival-analysis of 73 patients. Cancer Lett 2005, 218:199-206.

Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. D Pyeon, M A Newton, P F Lambert, J A Den Boon, S Sengupta, C J Marsit, C D Woodworth, J P Connor, T H Haugen, E M Smith, Cancer Res. 67Pyeon D, Newton MA, Lambert PF, den Boon JA, Sengupta S, Marsit CJ, Woodworth CD, Connor JP, Haugen TH, Smith EM, et al.: Fun- damental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. Cancer Res 2007, 67:4605-4619.

Gene expression profiles in HPV-infected head and neck cancer. N F Schlecht, R D Burk, Adrien L Dunne, A Kawachi, N Sarta, C Chen, Q Brandwein-Gensler, M Prystowsky, M B Childs, G , J Pathol. 213Schlecht NF, Burk RD, Adrien L, Dunne A, Kawachi N, Sarta C, Chen Q, Brandwein-Gensler M, Prystowsky MB, Childs G, et al.: Gene expression profiles in HPV-infected head and neck cancer. J Pathol 2007, 213:283-293.

Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma. R J Slebos, Y Yi, K Ely, J Carter, A Evjen, X Zhang, Y Shyr, B M Murphy, A J Cmelak, B B Burkey, Clin Cancer Res. 12Slebos RJ, Yi Y, Ely K, Carter J, Evjen A, Zhang X, Shyr Y, Murphy BM, Cmelak AJ, Burkey BB, et al.: Gene expression differences asso- ciated with human papillomavirus status in head and neck squamous cell carcinoma. Clin Cancer Res 2006, 12:701-709.

Genome-wide DNA copy number alterations in head and neck squamous cell carcinomas with or without oncogene-expressing human papillomavirus. S J Smeets, B J Braakhuis, S Abbas, P J Snijders, B Ylstra, M A Wiel, Van De, G A Meijer, C R Leemans, R H Brakenhoff, Oncogene. 25Smeets SJ, Braakhuis BJ, Abbas S, Snijders PJ, Ylstra B, Wiel MA van de, Meijer GA, Leemans CR, Brakenhoff RH: Genome-wide DNA copy number alterations in head and neck squamous cell car- cinomas with or without oncogene-expressing human papil- lomavirus. Oncogene 2006, 25:2558-2564.

Genetic classification of oral and oropharyngeal carcinomas identifies subgroups with a different prognosis. S J Smeets, R H Brakenhoff, B Ylstra, W N Van Wieringen, Wiel Ma Van De, C R Leemans, B J Braakhuis, Cell Oncol. in pressSmeets SJ, Brakenhoff RH, Ylstra B, van Wieringen WN, Wiel MA van de, Leemans CR, Braakhuis BJ: Genetic classification of oral and oropharyngeal carcinomas identifies subgroups with a differ- ent prognosis. Cell Oncol 2009 in press.

CGHcall: calling aberrations for array CGH tumor profiles. Wiel Ma Van De, K I Kim, S J Vosse, W N Van Wieringen, S M Wilting, B Ylstra, Bioinformatics. 23Wiel MA van de, Kim KI, Vosse SJ, van Wieringen WN, Wilting SM, Ylstra B: CGHcall: calling aberrations for array CGH tumor profiles. Bioinformatics 2007, 23:892-894.

CGHRegions: Dimension Reduction for Array CGH Data with Minimal Information Loss. Wiel Ma Van De, W N Van Wieringen, Cancer Informatics. 2Wiel MA van de, van Wieringen WN: CGHRegions: Dimension Reduction for Array CGH Data with Minimal Information Loss. Cancer Informatics 2007, 2:55-63.

Weighted clustering of called array CGH data. W N Van Wieringen, Wiel Ma Van De, B Ylstra, Biostatistics. 9van Wieringen WN, Wiel MA van de, Ylstra B: Weighted cluster- ing of called array CGH data. Biostatistics 2008, 9:484-500.

Generation of patterns from gene expression data by assigning confidence to differentially expressed genes. E Manduchi, G R Grant, S E Mckenzie, G C Overton, S Surrey, C J StoeckertJr, Bioinformatics. 16Manduchi E, Grant GR, McKenzie SE, Overton GC, Surrey S, Stoeck- ert CJ Jr: Generation of patterns from gene expression data by assigning confidence to differentially expressed genes. Bio- informatics 2000, 16:685-698.

Controlling the false discovery rate in behavior genetics research. Y Benjamini, D Drai, G Elmer, N Kafkafi, I Golani, Behav Brain Res. 125Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I: Controlling the false discovery rate in behavior genetics research. Behav Brain Res 2001, 125:279-284.

Amplification of the chromosome 20q region is associated with expression of HPV-16 E7 in human airway and anogenital epithelial cells. A J Klingelhutz, Q Qian, S L Phillips, F A Gourronc, B W Darbro, S R Patil, Virology. 340Klingelhutz AJ, Qian Q, Phillips SL, Gourronc FA, Darbro BW, Patil SR: Amplification of the chromosome 20q region is associ- ated with expression of HPV-16 E7 in human airway and ano- genital epithelial cells. Virology 2005, 340:237-244.

Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification. Y Jin, H Zhang, S W Tsao, Jin C Lv, M Strombeck, B Wiegant, J Wan, T S Yuen, P W Kwong, Y L , 20Jin Y, Zhang H, Tsao SW, Jin C, Lv M, Strombeck B, Wiegant J, Wan TS, Yuen PW, Kwong YL: Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20.

. Gynecol Oncol. 92Gynecol Oncol 2004, 92:183-191.

20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells. E Savelieva, C D Belair, M A Newton, S Devries, J W Gray, F Waldman, C A Reznikoff, Oncogene. 14Savelieva E, Belair CD, Newton MA, DeVries S, Gray JW, Waldman F, Reznikoff CA: 20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithe- lial cells. Oncogene 1997, 14:551-560.

Non-random allelic losses at 3p, 11p and 13q during HPV-mediated immortalization and concomitant loss of terminal differentiation of human keratinocytes. R D Steenbergen, M A Hermsen, J M Walboomers, G A Meijer, J P Baak, C J Meijer, P J Snijders, Int J Cancer. 76Steenbergen RD, Hermsen MA, Walboomers JM, Meijer GA, Baak JP, Meijer CJ, Snijders PJ: Non-random allelic losses at 3p, 11p and 13q during HPV-mediated immortalization and concomi- tant loss of terminal differentiation of human keratinocytes. Int J Cancer 1998, 76:412-417.

Dominant genetic alterations in immortalization: role for 20q gain. S Cuthill, P Agarwal, S Sarkar, E Savelieva, C A Reznikoff, Genes Chromosomes Cancer. 26Cuthill S, Agarwal P, Sarkar S, Savelieva E, Reznikoff CA: Dominant genetic alterations in immortalization: role for 20q gain. Genes Chromosomes Cancer 1999, 26:304-311.

Ried T: A recurrent pattern of chromosomal aberrations and immunophenotypic appearance defines anal squamous cell carcinomas. K Heselmeyer, M S Du, H Blegen, B Friberg, C Svensson, E Schrock, T Veldman, K Shah, G Auer, Br J Cancer. 76Heselmeyer K, du MS, Blegen H, Friberg B, Svensson C, Schrock E, Veldman T, Shah K, Auer G, Ried T: A recurrent pattern of chro- mosomal aberrations and immunophenotypic appearance defines anal squamous cell carcinomas. Br J Cancer 1997, 76:1271-1278.

Deletion mapping of chromosome 13q in head and neck squamous cell carcinoma in Indian patients: correlation with prognosis of the tumour. M G Sabbir, A Roy, S Mandal, A Dam, S Roychoudhury, C K Panda, Int J Exp Pathol. 87Sabbir MG, Roy A, Mandal S, Dam A, Roychoudhury S, Panda CK: Deletion mapping of chromosome 13q in head and neck squamous cell carcinoma in Indian patients: correlation with prognosis of the tumour. Int J Exp Pathol 2006, 87:151-161.

The human papillomavirus E7 oncoprotein. M E Laughlin-Drubin, K Munger, Virology. 384Laughlin-Drubin ME, Munger K: The human papillomavirus E7 oncoprotein. Virology 2009, 384:335-344.

Papillomavirus E6 and E7 proteins and their cellular targets. T M Wise-Draper, S I Wells, Front Biosci. 13Wise-Draper TM, Wells SI: Papillomavirus E6 and E7 proteins and their cellular targets. Front Biosci 2008, 13:1003-1017.

CDC91L1 (PIG-U) is a newly discovered oncogene in human bladder cancer. Z Guo, J F Linn, G Wu, S L Anzick, C F Eisenberger, S Halachmi, Y Cohen, A Fomenkov, M O Hoque, K Okami, Nat Med. 10Guo Z, Linn JF, Wu G, Anzick SL, Eisenberger CF, Halachmi S, Cohen Y, Fomenkov A, Hoque MO, Okami K, et al.: CDC91L1 (PIG-U) is a newly discovered oncogene in human bladder cancer. Nat Med 2004, 10:374-381.

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis. F E Henken, S M Wilting, R M Overmeer, J G Van Rietschoten, A O Nygren, A Errami, J P Schouten, C J Meijer, P J Snijders, R D Steenbergen, Br J Cancer. 97Henken FE, Wilting SM, Overmeer RM, van Rietschoten JG, Nygren AO, Errami A, Schouten JP, Meijer CJ, Snijders PJ, Steenbergen RD: Sequential gene promoter methylation during HPV-induced cervical carcinogenesis. Br J Cancer 2007, 97:1457-1464.

Utility of methylation markers in cervical cancer early detection: appraisal of the state-of-the-science. N Wentzensen, M E Sherman, M Schiffman, S S Wang, Gynecol Oncol. 112Wentzensen N, Sherman ME, Schiffman M, Wang SS: Utility of methylation markers in cervical cancer early detection: appraisal of the state-of-the-science. Gynecol Oncol 2009, 112:293-299.

Human papillomavirus and molecular considerations for cancer risk. M A Whiteside, E M Siegel, E R Unger, Cancer. 113Whiteside MA, Siegel EM, Unger ER: Human papillomavirus and molecular considerations for cancer risk. Cancer 2008, 113:2981-2994.

High-risk HPV/ ErbB-2 interaction on E-cadherin/catenin regulation in human carcinogenesis. Al Moustafa, A E Kassab, A Darnel, A Yasmeen, A , Curr Pharm Des. 14Al Moustafa AE, Kassab A, Darnel A, Yasmeen A: High-risk HPV/ ErbB-2 interaction on E-cadherin/catenin regulation in human carcinogenesis. Curr Pharm Des 2008, 14:2159-2172.

Silencing of E7 oncogene restores functional Ecadherin expression in human papillomavirus 16-transformed keratinocytes. J H Caberg, P M Hubert, D Y Begon, M F Herfs, P J Roncarati, J J Boniver, P O Delvenne, Carcinogenesis. 29Caberg JH, Hubert PM, Begon DY, Herfs MF, Roncarati PJ, Boniver JJ, Delvenne PO: Silencing of E7 oncogene restores functional E- cadherin expression in human papillomavirus 16-trans- formed keratinocytes. Carcinogenesis 2008, 29:1441-1447.

E-cadherin-dependent adhesion of dendritic and Langerhans cells to keratinocytes is defective in cervical human papillomavirus-associated (pre)neoplastic lesions. P Hubert, J H Caberg, C Gilles, L Bousarghin, E Franzen-Detrooz, J Boniver, P Delvenne, J Pathol. 206Hubert P, Caberg JH, Gilles C, Bousarghin L, Franzen-Detrooz E, Boniver J, Delvenne P: E-cadherin-dependent adhesion of den- dritic and Langerhans cells to keratinocytes is defective in cervical human papillomavirus-associated (pre)neoplastic lesions. J Pathol 2005, 206:346-355.

Oncogenes related to head and neck cancer. W J Issing, T P Wustrow, W J Heppt, Anticancer Res. 13Issing WJ, Wustrow TP, Heppt WJ: Oncogenes related to head and neck cancer. Anticancer Res 1993, 13:2541-2551.