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 p16(INK4A) 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 p16(INK4A)-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 p16(INK4A) accumulation. Only 5 out of 48 HPV-negative tumors showed p16(INK4A) 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 p16(INK4A) 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.

[Kikuchi's histiocytic necrotising lymphadenitis: a benign disorder--not to be confused with malignant lymphoma]. / Histiocytaire necrotiserende lymfadenitis van Kikuchi: een benigne aandoening--niet te verwarren met maligne lymfoom.

A 26-year-old woman who later turned out to have the rarely seen histiocytic necrotising lymphadenitis of Kikuchi was twice diagnosed incorrectly with malignant T-cell lymphoma. She was treated with standard chemotherapy, whereas Kikuchi's disease has a self-limiting course. Fear for recurrent lymphoma greatly affected the patient's life until the proper diagnosis was ultimately made. This occurred after the patient herself had seen in her dossier that the diagnosis 'Kikuchi's histiocytic lymphadenitis' had been proposed by two pathologists of the consulted regional lymphoma board in the past, but had been rejected by the board after external consultation.

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.

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.

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.

False-positive cytology in diagnostic laparoscopy due to ectopic pancreas.

BACKGROUND: Report on a case of incorrect diagnosis after laparoscopy and peritoneal fluid sampling. METHODS: Case description and literature review. RESULTS: Diagnostic laparoscopy is a frequently used tool. In a patient with chronic abdominal pain, a diagnostic laparoscopy was performed, and a peritoneal fluid sample was taken. Cytology of the aspirated peritoneal fluid revealed an adenocarcinoma. At laparotomy, ectopic pancreas was found as the source of the false-positive cytology. CONCLUSION: In the diagnosis of adenocarcinomas from peritoneal fluid aspirates without an obvious clinical location (tumor), ectopic pancreatic tissue should be considered in the differential diagnosis.

Expression of CD10, CD75 and CD43 in MALT lymphoma and their usefulness in discriminating MALT lymphoma from follicular lymphoma and chronic gastritis.

AIMS: While it may be difficult to discriminate between chronic gastritis, MALT lymphoma and a gastric location of a nodal lymphoma using histology of small endoscopic biopsies alone, additional markers like CD10, CD75 and CD43 and proliferative activity may be of value. We assessed the expression of these antigens in MALT lymphoma and their usefulness in discriminating MALT lymphoma from follicular lymphoma and from gastritis. METHODS AND RESULTS: Tissue samples of 38 patients with gastric MALT lymphoma were immunohistochemically stained for expression of CD10, CD75 and/or CD43. Proliferation index was scored using MIB-1 staining. Ten cases of nodal follicle centre B-cell lymphomas (n = 11) and 18 cases of high-grade MALT lymphoma (n = 22) showed moderate to high CD75 expression (25-100% positive cells). All tested low-grade MALT lymphomas (n = 9) and chronic gastritis (n = 6) were negative (0-25%) for CD75. All MALT lymphomas (n = 25) were negative for CD10. High-grade lymphomas show significantly higher proliferation indices (67% vs. 16%) than low-grade lymphomas. Only four of 26 MALT lymphomas were slightly positive for CD43. All gastritis biopsies (n = 4) were negative for CD43. CONCLUSIONS: These data suggest that combining both CD10 and CD75 may be useful in discriminating between low-grade MALT, high-grade MALT lymphoma and extranodal location of follicular lymphoma. However, CD43 expression cannot in the majority of cases be used to distinguish between low-grade MALT lymphoma and gastritis.

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.

Morphologically normal, CD30-negative B-lymphocytes with chromosome aberrations in classical Hodgkin's disease: the progenitor cell of the malignant clone?

A recent study observed that numerical chromosome abnormalities in Hodgkin's disease (HD) are detected not only in morphologically abnormal Hodgkin/Reed-Sternberg cells, but also in a fraction of morphologically normal cells. However, the phenotypic constitution of these genetically abnormal, morphologically normal cells and their relationship to the malignant Hodgkin/Reed-Sternberg cells could not be established in the earlier cases studied, because of the low frequency of these cells. The present study investigated two cases of classical Hodgkin's disease containing a relatively large population of such apparently normal cells with aberrant chromosome copy numbers. The phenotype and their position within the developmental route of the malignant compartment were examined by a combined in situ hybridization and immunocytochemistry approach. Numerical abnormalities for chromosome 1 in one case and for chromosomes X, Y, and 1 in the other case were observed not only in CD30-positive Hodgkin/Reed-Sternberg cells, but also in CD30-negative, morphologically normal cells. It was shown that these genetically aberrant cells expressed the B-cell antigen CD19, thus confirming their B-cell nature. These studies indicate a relationship between the genome aberrations in these genetically abnormal, morphologically normal B-cells and the Hodgkin/Reed-Sternberg cells, suggesting that they are progenitor cells of the malignant cell fraction.

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 Hodgkin's 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 Hodgkin's 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 Hodgkin's 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 microns thick paraffin sections of six cases of Hodgkin's 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 Hodgkin's disease.

Double-target fluorescence in situ hybridization distinguishes multiple genetically aberrant clones in head and neck squamous cell carcinoma.

Genomic heterogeneity has been observed in several solid tumor types. To investigate this phenomenon in head and neck squamous cell carcinoma (HNSCC), we analyzed macroscopically distinct tissue samples of 12 resected tumors by a combination of fluorescence in situ hybridization (FISH) and DNA flow cytometry. Using a panel of centromeric DNA probes, numerical chromosomal aberrations were detected in 10 tumors, 9 of which showed a single DNA aneuploid peak. Imbalances in chromosomal copy numbers resulted in unique patterns of chromosomal aberrations for each tumor case. Two types of tumors could be distinguished, i.e., tumors (n = 5) containing a single aneusomic clone and tumors (n = 5) with multiple aneusomic clones. The center of this latter group of tumors was shown to be genetically more heterogeneous than the tumor margin. In conclusion, this study showed that 1) the pattern of chromosomal aberrations varies greatly between different HNSCC, 2) a major clone with a specific pattern of chromosomal aberrations has spread throughout most HNSCC, and 3) a subgroup of HNSCCs contains additional clones with a different pattern of chromosomal aberrations. Based on these results, HNSCC can be divided into a genetically more homogeneous and a genetically more heterogeneous group.

Cell turnover parameters in small and large cell varieties of primary intestinal non-Hodgkin's lymphoma.

BACKGROUND: In contrast to primary gastric lymphoma, primary intestinal lymphoma is an uncommon condition with a poorer outcome, perhaps due to differences in its pathogenesis. In this study, the authors analyzed the roles of proliferation and apoptosis in the pathogenesis of intestinal lymphoma. METHODS: Fifty-one cases of intestinal non-Hodgkin's lymphoma (NHL) (10 small B-cell mucosa-associated lymphoid tissue [MALT] NHLs, 12 large B-cell MALT NHLs, 18 large B-cell NHLs, 2 small T-cell NHLs, 7 large T-cell NHLs, and 2 mantle cell NHLs) were studied for the immunohistochemical expression of MIB-1 and the TUNEL assay as well as the expression of bcl-2 and p53, both of which are regulatory gene products involved in apoptosis. RESULTS: The median proliferation index (PI) was 37.3%, and the median apoptotic index (AI) was 1.10%. The respective values of PI and AI were 5.8% and 0.06% in small B-cell MALT lymphoma, 52.8% and 0.24% in large B-cell MALT lymphoma, 58.85% and 1.36% in large B-cell lymphoma, 30.9% and 1.93% in mantle cell lymphoma, 18.13% and 1.25% in small T-cell lymphoma, and 43.4% and 1.93% in large T-cell lymphoma. In an analysis of B-cell NHL only (with mantle cell NHL excluded), proliferative and apoptotic indices were positively correlated (correlation coefficient=0.563, P < 0.001). Furthermore, high bcl-2 expression was inversely correlated with both PI and AI. Expression of p53 was observed in 8 cases (1 small cell lymphoma and 7 large cell lymphomas). CONCLUSIONS: Small cell lymphomas had low AI and PI values, whereas large cell lymphomas had high AI and PI values. Apoptosis and proliferation were positively correlated, and higher expression of bcl-2 was associated with lower rates of apoptosis.

Molecular assessment of clonality leads to the identification of a new germ line TP53 mutation associated with malignant cystosarcoma phyllodes and soft tissue sarcoma.

Cystosarcoma phyllodes (CSP) is a rare breast neoplasm composed of stromal and epithelial elements. It usually runs a benign course but it may metastasize. In a 31-year-old patient with recurring CSP, a mesenchymal tumor in the leg developed. The question arose whether the latter tumor could be a metastasis from the CSP, which would have major treatment consequences. The problem was addressed using molecular methods, i.e., comparison of the pattern of polymorphic repeat markers on chromosome 17p as well as single strand conformation polymorphism analysis and sequencing of exons 5 to 8 of the TP53 gene in both tumor and normal tissue. An identical pattern of loss of heterozygosity in both breast tumors was demonstrated, but a different pattern was shown in the tumor in the leg. This led to the conclusion that the latter tumor had to be a new primary tumor. A mutation in codon 162 of the TP53 gene was found in the tumor tissue as well as in the normal tissue of this patient. This germ line mutation leads to the replacement of isoleucine by asparagine and most likely has functional consequences. In all four examined tumors of this patient, the normal TP53 allele was lost. This is strong evidence that this germ line TP53 mutation causes the genesis of these two rare primary mesenchymal tumors in this young patient. The current study exemplifies the power of molecular diagnostic methods in investigating the specific clinical problem of clonal relation between two separate tumors. The germ line mutation found in codon 162 of the TP53 gene and the association with cystosarcoma phyllodes have not been described previously.

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

AIMS: 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 Hodgkin's disease. Its normal counterpart, the reactive lymph node, was also examined for lamin subtype expression. METHODS AND RESULTS: The CD20 positive centrocytes and centroblasts of the follicle centre in the reactive lymph nodes expressed lamin B1, but were not or only very weakly positive for lamin B2 or A-type lamin antibodies. Mantle zone lymphocytes displayed lamins B1 and B2, but were negative for A-type lamins. Furthermore, CD3- and CD20-positive lymphocytes in the medulla and paracortex lacked A-type lamins, but were positive for both B-type lamins. Finally, the proliferation marker Ki67 was mainly detected in the centroblasts, but also in a fraction of the A-type lamin negative cells in the paracortex and medulla. In Hodgkin's disease, all cells expressed lamins B1 and B2, whereas A-type lamins were primarily observed in CD30-positive Reed-Sternberg and Hodgkin cells. About 20% of the Reed-Sternberg and Hodgkin cells expressed Ki67, with co-expression of lamin A in most of these cells. CONCLUSIONS: Ki67 and A-type lamin staining were in general mutually exclusive in lymph nodes, indicating that A-type lamin positive cells are not proliferative. This suggests also that the A-type lamin expression in Reed-Sternberg and Hodgkin cells is correlated with a relatively mature phenotype of these malignant cells. However, some of these differentiated malignant cells still have a capacity to proliferate as indicated by Ki67 positivity. Our observation that lamin B2 expression in the follicle centre cells of the reactive lymph node is low or absent indicates that this lamin subtype is not always expressed in nucleated cells, which is in clear contrast to the results obtained in previous studies in other diseases and in normal tissues. Absence of lamin B2 expression may be associated with the follicle centre stage of B-cells.

Proliferation and apoptosis in primary gastric B-cell non-Hodgkin's lymphoma.

To elucidate the role of cell turnover in primary gastric B-cell non-Hodgkin's lymphomas we studied tissue samples of 72 patients-26 small cell (25 MALT lymphomas) and 46 large cell (31 MALT lymphomas). Proliferation indices and apoptotic indices were measured by Mib-1 expression and terminal deoxynucleotidyltransferase (TdT)-mediated nick end labelling, respectively. Furthermore, expression of the apoptosis related gene-products bcl-2 and p53 was studied. Large cell lymphomas showed significantly higher proliferation indices (59.1% vs. 15.4%) and apoptotic indices (3.2% vs.0.7%) than small cell lymphomas. Proliferation and apoptotic indices were positively correlated (r = 0.371, P = 0.03). Expression of the bcl-2 protein was significantly higher in the small cell lymphomas. Furthermore, cases with a high bcl-2 expression showed both a significantly decreased proliferation (P < 0.0001) and apoptotic index (P = 0.0096) compared to bcl-2 negative cases. Expression of the mutant p53 protein was present in 8.0% of small cell and in 18.2% of large cell lymphomas. p53 positive cases showed significantly higher proliferation indices, but showed no correlation with apoptotic index. These data suggest that impaired apoptosis by bcl-2 may be more prominent than proliferation in the genesis of small cell lymphomas, whereas a high cell turnover characterizes large cell primary gastric lymphomas.

Specific detection of Helicobacter pylori and non-Helicobacter pylori flora in small- and large-cell primary gastric B-cell non-Hodgkin's lymphoma.

BACKGROUND: Primary gastric non-Hodgkin's lymphomas possibly develop in response to local infection by Helicobacter pylori (H. pylori). We investigated the presence of H. pylori and non-H. pylori flora histologically in small- and large-cell primary gastric lymphoma using a specific staining method. MATERIALS AND METHODS: Specimens of 52 cases of primary gastric lymphoma (17 small cell, 35 large cell) were stained with modified Giemsa (MG) and immunohistochemically using a polyclonal antibody against H. pylori (IHC). RESULTS: Thirty-two cases (61.5%) (small cell 76% versus large cell 53%, P > 0.05) showed immunoreactivity for H. pylori in the mucosa surrounding the tumor. Remarkably, there was localization of H. pylori in the neck of the gastric glands in 3 cases. Non-H. pylori flora was seen in 35 cases (76.3%) (small cell 53% versus large cell 74%, P > 0.05). In 20 cases, this non-H. pylori flora was mixed with H. pylori. Five cases showed no bacterial flora at all. CONCLUSIONS: (1) Using immunohistochemistry, the prevalence of gastric lymphoma cases with H. pylori (61.5%) approximates that of H. pylori in the normal population. (2) No statistical difference was found between the occurrence of H. pylori and non-H. pylori bacterial flora in small- versus large-cell lymphoma. (3) Our results suggest that H. pylori may not be the only etiologic factor in primary gastric lymphoma.

Detection of chromosomal aberrations in cytologic brush specimens from head and neck squamous cell carcinoma.

BACKGROUND: 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. METHODS: Brush specimens taken from the tumors of 20 patients with head and neck squamous cell carcinoma (HNSCC) and from the normal mucosa of 8 control patients were analyzed by FISH using DNA probes for the chromosomes 1 and 7. The FISH results were compared with DNA flow cytometry and FISH results of the solid tumor specimens. RESULTS: The results of this study showed that 15 of the 20 tumor brush specimens contained numeric chromosomal aberrations in at least 5% of the cells collected. Chromosomal aberrations were detected in all brush specimens taken from tumors that were DNA aneuploid and showed aneusomy. The presence of these aberrations correlated well with the classification "suspicious for malignancy," which was based on Papanicolaou stained slides of the same specimens. In the control group the percentage of chromosomally aberrant cells did not exceed 2%; in addition, no suspiciously malignant cells were observed in this group. CONCLUSIONS: This study reveals that the FISH technique can be applied diagnostically to brush specimens of HNSCC. The presence of chromosomal aberrations in > 5% of the cells in these specimens can be considered as a marker for malignancy.