Fredrik J. Bot

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.

P21Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis.

Human papillomavirus is involved in the carcinogenesis of tonsillar squamous cell carcinomas. Here, we investigated the expression and the prognostic value of key cell cycle proteins in the pRb and p53 pathways in both human papillomavirus type 16-positive and -negative tonsillar squamous cell carcinomas. Using immunohistochemistry, 77 tonsillar squamous cell carcinomas with known human papillomavirus type 16 status and clinical outcome were analyzed for expression of Ki67, p16INK4A, cyclin D1, pRb, p14ARF, MDM2, p53, p21Cip1/WAF1, and p27KIP1. Results were correlated with each other and with clinical and demographic patient data. A total of 35% of tonsillar carcinomas harbored integrated human papillomavirus type 16 DNA and p16INK4A overexpression, both being considered essential features for human papillomavirus association. These tumors also showed the overexpression of p14ARF (P<0.0001) and p21Cip1/WAF1 (P=0.001), and downregulation of pRb (P<0.0001) and cyclin D1 (P=0.027) compared with the human papillomavirus-negative cases. Univariate Cox regression analyses revealed a favorable survival rate for non-smokers (P=0.006), as well as for patients with T1-2 tumors (P<0.0001) or tumors showing low expression of cyclin D1 (P=0.028), presence of human papillomavirus and overexpression of p16INK4A (P=0.01), p14ARF (P=0.02) or p21Cip1/WAF1 (P=0.004). In multivariate regression analyses, smoking and tumor size, as well as expression of cyclin D1 and p21Cip1/WAF1, were found to be independent prognostic markers. We conclude that human papillomavirus positivity in tonsillar squamous cell carcinomas strongly correlates with p21Cip1/WAF1 and p14ARF overexpression and downregulation of pRb and cyclin D1. In particular p21Cip1/WAF1 overexpression is an excellent favorable prognosticator in tonsillar squamous cell carcinomas.

Gastric low-grade MALT lymphoma, high-grade MALT lymphoma and diffuse large B cell lymphoma show different frequencies of trisomy.

Gastric MALT lymphoma is a distinct entity related to Helicobacter pylori gastritis. Some studies suggest a role for trisomy 3 in the genesis of these lymphomas, but they mainly focused on low-grade MALT lymphoma. Gastric MALT lymphoma, however, comprises a spectrum from low- to high-grade cases. Furthermore, its exact relation to primary diffuse large B cell lymphoma (DLBCL) of the stomach is not clear. We applied in situ hybridisation (ISH) with centromeric probes on 43 samples of 39 patients with primary gastric lymphoma (13 samples with low-grade MALT lymphoma, 25 with high-grade MALT lymphoma and five with DLBCL) to detect numerical aberrations of 10 chromosomes. ISH was performed immunohistochemically on nuclei isolated from paraffin-embedded resection tissue and on whole paraffin sections using immunofluorescence. In six of 13 low-grade MALT lymphomas trisomy was detected (46%) and mostly involved chromosome 3 (33%). In high-grade MALT lymphomas, trisomies were found in 16 of 25 cases (64%), mainly involving chromosomes 12 and 18. Trisomy 3 was present in only 13% of these cases. Of five DLBCL, only one showed trisomy. Nine of the 16 aberrant high-grade MALT lymphomas (56%) showed trisomy of more than one chromosome per case vs two of six for low-grade cases. In lymphomas with separate low- and high-grade tumour components some trisomies were detected in both components, whereas others occurred only in the high-grade tumour cells. This supports the hypothesis that high-grade MALT lymphomas can develop from a low-grade type and that this progression is accompanied by the acquisition of more genetic aberrations. However, trisomy 3 probably does not play a major role in this progression.

Chromosome Instability as an Indicator of Malignant Progression in Laryngeal Mucosa

PURPOSE Routine histologic examination cannot predict whether premalignant laryngeal lesions will progress toward invasive growth. The acquisition of changes in chromosome constitution has been suggested to be essential for driving tumor progression by enhancing mutagenic mechanisms. The aim of the present study was to determine whether chromosomal changes occur in the subsequent stages of early laryngeal carcinogenesis and, if so, whether these changes can be of prognostic value. MATERIALS AND METHODS Numerical aberrations for chromosomes 1 and 7 were detected in tissue sections from archival material using an improved in situ hybridization protocol. In total, eight benign laryngeal lesions, 37 premalignant laryngeal lesions, and 16 specimens containing histologically normal epithelia adjacent to laryngeal squamous cell carcinomas were studied. Both the histologic and the cytogenetic classifications were correlated with progression to laryngeal cancer. RESULTS No evidence for chromosome alterations was obtained in the control group, nor in histologically normal epithelia adjacent to laryngeal squamous cell carcinomas, nor in all but one hyperplastic lesion (n = 11). In contrast, 14 of 15 dysplastic lesions and nine of 11 carcinomas-in-situ contained numerical chromosomal aberrations. Tetrasomy was present in the majority of the dysplastic lesions. An unstable chromosome content (indicated by the presence of chromosome imbalances and/or polyploidization) in the premalignant lesion strongly predicted its malignant progression. CONCLUSION Our results show that laryngeal tumor development involves chromosome tetraploidization. The further change from a stable to an unstable chromosome constitution is of importance for malignant progression.

Mapping of resection margins of oral cancer for p53 overexpression and chromosome instability to detect residual (pre)malignant cells

Oral squamous cell cancers (OSCCs) have a high local recurrence rate, partly due to problems in the recognition of minimal residual disease. The use of molecular markers is shown to increase the sensitivity of detection of residual malignant cells in tumour margins of OSCC. p53 immunohistochemistry was combined with in situ hybridization for chromosomes 1 and 7 to determine the presence of genetically unstable cells in resection specimens of OSCC containing invasive cancer. An increased frequency of genetically aberrant cells was observed, as detected by p53 overexpression and/or aneusomy, with histological progression of normal mucosa via hyperplasia to dysplasia. Of clinical importance was the finding that 11 of 20 resection margins, all of which were initially diagnosed as being tumour‐free, were found to contain genetically aberrant (pre)malignant cells. In these areas, closer histological examination of the genetically aberrant compartment within these margins often also revealed small dysplastic areas that were missed in the initial diagnosis, showing that this genetic approach can assist in diagnosis. Copyright

Comparison of A and B-type lamin expression in reactive lymph nodes and nodular sclerosing Hodgkin's disease

In order to clarify the differentiation and proliferation status of the Reed–Sternberg and Hodgkin cells we studied A and B‐type lamin expression with specific monoclonal antibodies in nodular sclerosing Hodgkins disease. Its normal counterpart, the reactive lymph node, was also examined for lamin subtype expression.

Chromosome stability in tonsillar squamous cell carcinoma is associated with HPV16 integration and indicates a favorable prognosis

Tonsillar squamous cell carcinoma (TSCC) is frequently associated with human papillomavirus (HPV) and chromosome instability. Data from cellular model systems are, however, controversial concerning a relation between HPV and chromosome instability development. Here we studied this association in 77 primary TSCC with known clinical outcome and cell cycle protein expression profiles. Thirty‐two tumors (42%) showed HPV16‐integration. All 77 cases were analyzed by fluorescence in situ hybridization using chromosome 1‐ and 7‐specific centromere DNA probes to detect chromosome instability, indicated by the presence of chromosome imbalances and/or polyploidization for these chromosomes. In addition, eight HPV‐positive dysplasias, seven of which were adjacent to a carcinoma, were analyzed. Disomy for chromosome 1 and 7 was present in 29 out of 77 TSCC (38%), of which 19 were HPV16‐positive (p = 0.002). Aneusomy was observed in the remaining 48 TSCC, of which 13 were HPV‐positive. Aneusomies correlated significantly with tobacco‐ and alcohol consumption (p = 0.001 and p = 0.016, respectively) and a higher T‐stage (p = 0.018). Both HPV‐positivity and chromosome disomy were significantly associated with a favorable disease‐free survival (p = 0.001 and p = 0.025, respectively). Particularly in the HPV16‐positive group chromosome instability is a very strong indicator for an unfavorable prognosis (p = 0.032). In the dysplasias an identical HPV and chromosome copy number status was identified as in the adjacent tumors. We conclude that HPV‐positive TSCC and their precursor lesions are more often genetically stable than HPV‐negative lesions and that these tumors are associated with a favorable prognosis. Chromosome instability is an indicator for unfavorable prognosis, particularly in the HPV‐positive patient group.

Specific steps in aneuploidization correlate with loss of heterozygosity of 9p21, 17p13 and 18q21 in the progression of pre-malignant laryngeal lesions.

Laryngeal squamous-cell carcinoma is often preceded by pre-malignant lesions. In this study, pre-malignant as well as malignant laryngeal lesions were analyzed using p53 immunohistochemistry and in situ hybridization for chromosomes 1, 7, 9, 17 and 18. Microsatellite analysis was performed on laser-microdissected tissue fragments with the aim of studying loss of heterozygosity (LOH) of 9p21, 17p13 and 18q21. Sequential biopsies were analyzed from a few cases to study genetic progression in more detail. The following genetic progression patterns were observed: (i) histologically normal mucosa and hyperplastic lesions without malignant progression were typically disomic for all chromosomes tested and showed no or only basal cell layer positivity for p53 and no allelic loss; (ii) hyperplastic lesions preceding dysplastic/invasive growth frequently showed trisomy for chromosome 7 and LOH of 9p21 and 17p13, and small foci within these lesions sometimes showed tetraploidization and p53 positivity; (iii) dysplastic lesions were characterized by a tetraploid chromosome content, LOH of 9p21 and 17p13 and p53 positivity; (iv) carcinoma in situ lesions and invasive laryngeal carcinomas showed a more unbalanced chromosome pattern and an additional 18q21 LOH. These results show that different steps in aneuploidization correlate with LOH of 9p21, 17p13 and 18q21 in early laryngeal carcinogenesis. These genomic changes could be of potential use in the diagnosis and prognosis of pre-malignant laryngeal lesions.

Detection of Chromosomal Aberrations in Cytologic Brush Specimens from Head and Neck Squamous Cell Carcinoma

Detection of genetic changes in the mucosa of the upper aerodigestive tract may provide a target for the screening of cytologic specimens to identify premalignant transformation in this region. In this pilot study, the feasibility of the fluorescence in situ hybridization (FISH) technique to detect genetically aberrant cells in brush specimens was evaluated.

Chromosomal abnormalities in Hodgkin's disease are not restricted to Hodgkin/Reed–Sternberg cells

Hodgkin and Reed–Sternberg cells are considered to represent the malignant fraction in Hodgkins disease. Several studies have shown that the Hodgkin and Reed‐Sternberg cells are chromosomally abnormal, but genetic data about the morphologically normal cell population in Hodgkins disease are very limited. This latter cell population has therefore been examined for chromosomal aberrations, using the in situ hybridization (ISH) procedure, making use of DNA probes for chromosomes 1, 7, 8, 9, 11, 12, 15, 17, and 18. Nuclei were isolated from freshly frozen (10 cases) and paraffin‐embedded (16 cases) biopsy samples and 1000 nuclei per case were evaluated. The cases of Hodgkins disease were compared with reactive lymph nodes, which show aberrant chromosome copy numbers in less than 1 per cent of the cells. Using strict scoring criteria, nuclei in the tumour were found to show an abnormal genotype, in the range of 1–12 per cent, with trisomies occurring most frequently. No characteristic numerical chromosome abnormality was observed. ISH on 4 μm thick paraffin sections of six cases of Hodgkins disease revealed numerical aberrations for chromosome 1 in cells which appeared to be morphologically normal. The genomically abnormal nuclei did not differ in morphology or size from the nuclei of morphologically normal cells, but differed considerably in size when compared with the nuclei of Hodgkin/Reed–Sternberg cells after the ISH procedure. Three of these six cases revealed a population of apparently normal cells with an aberrant copy number which differed notably from the fraction observed in reactive lymph nodes. It is concluded, therefore, that a subset of morphologically normal cells, next to the Hodgkin/Reed–Sternberg cells, are chromosomally aberrant and may participate in the malignant cell fraction of Hodgkins disease.