HistopathologyVolume 64, Issue 4 p. 602-604 Correspondence Virtual microscopy is a valid alternative for the diagnostic assessment of laryngeal premalignancies Bart Sturm, Bart Sturm Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this authorStijn J H M Fleskens, Stijn J H M Fleskens Department of Otorhinolaryngology and Head and Neck Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this authorFredrik J Bot, Fredrik J Bot Department of Pathology, Maastricht University Medical Center, Maastricht, The NetherlandsSearch for more papers by this authorMarie-Louise van Velthuysen, Marie-Louise van Velthuysen Department of Pathology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, The NetherlandsSearch for more papers by this authorErnst-Jan Speel, Ernst-Jan Speel Department of Pathology, Maastricht University Medical Center, Maastricht, The NetherlandsSearch for more papers by this authorPiet J Slootweg, Piet J Slootweg Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this authorJeroen A W M van der Laak, Jeroen A W M van der Laak Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this author Bart Sturm, Bart Sturm Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this authorStijn J H M Fleskens, Stijn J H M Fleskens Department of Otorhinolaryngology and Head and Neck Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this authorFredrik J Bot, Fredrik J Bot Department of Pathology, Maastricht University Medical Center, Maastricht, The NetherlandsSearch for more papers by this authorMarie-Louise van Velthuysen, Marie-Louise van Velthuysen Department of Pathology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, The NetherlandsSearch for more papers by this authorErnst-Jan Speel, Ernst-Jan Speel Department of Pathology, Maastricht University Medical Center, Maastricht, The NetherlandsSearch for more papers by this authorPiet J Slootweg, Piet J Slootweg Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this authorJeroen A W M van der Laak, Jeroen A W M van der Laak Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The NetherlandsSearch for more papers by this author First published: 20 September 2013 https://doi.org/10.1111/his.12289Citations: 3Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume64, Issue4March 2014Pages 602-604 RelatedInformation

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.

The histopathology of premalignant laryngeal lesions does not provide reliable information on the risk of malignant transformation, hence we examined new molecular markers which can easily be implemented in clinical practice. Dual-target fluorescence in situ hybridisation (FISH) for chromosome 1 and 7 centromeres was performed on tissue sections of laryngeal premalignancies in 69 patients. Chromosome instability was indicated by numerical imbalances and/or polysomy for chromosomes 1 and 7. Additionally, immunostainings for p53, Cyclin D1 and (p)FADD expression were evaluated. Malignant progression was recorded. Eighteen patients with carcinoma in situ (CIS) were treated after diagnosis and excluded from follow-up. Chromosome instability was strongly associated with a high risk of malignant transformation, especially in lower grade lesions (hyperplasia, mild and moderate dysplasia; odds ratio = 8.4, p = 0.004). Patients with lesions containing chromosome instability showed a significantly worse 5-year progression-free survival than those with premalignancies without chromosome instability (p = 0.002). Neither histopathology nor the protein markers predicted progression in univariate analysis, although histopathological diagnosis, p53 and FADD contributed positively to chromosome instability in multivariate analysis. Chromosome instability is associated with malignant progression of laryngeal premalignancies, especially in lower grade lesions. These results may contribute to better risk counselling, provided that they can be validated in a larger patient set.

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.

The Journal of PathologyVolume 215, Issue 3 p. 347-348 Letter to the Editor Chromosome instability in resection margins predicts recurrence of oral squamous cell carcinoma VE Bergshoeff, Corresponding Author VE Bergshoeff Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsDepartment of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorAHN Hopman, AHN Hopman Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorIR Zwijnenberg, IR Zwijnenberg Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The Netherlands Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorFCS Ramaekers, FCS Ramaekers Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorFJ Bot, FJ Bot Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorB Kremer, B Kremer Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorJJ Manni, JJ Manni Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorE-JM Speel, E-JM Speel Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The Netherlands Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this author VE Bergshoeff, Corresponding Author VE Bergshoeff Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsDepartment of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorAHN Hopman, AHN Hopman Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorIR Zwijnenberg, IR Zwijnenberg Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The Netherlands Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorFCS Ramaekers, FCS Ramaekers Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorFJ Bot, FJ Bot Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorB Kremer, B Kremer Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorJJ Manni, JJ Manni Department of Otorhinolaryngology and Head and Neck Surgery, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this authorE-JM Speel, E-JM Speel Department of Molecular Cell Biology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The Netherlands Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The NetherlandsSearch for more papers by this author First published: 17 April 2008 https://doi.org/10.1002/path.2349Citations: 16Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume215, Issue3July 2008Pages 347-348 RelatedInformation

Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV‐DNA and the use of p16INK4A overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16‐specific fluorescence in situ hybridization (FISH) and p16INK4A‐specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16INK4A accumulation. Only 5 out of 48 HPV‐negative tumors showed p16INK4A immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non‐smoking patients with HPV‐containing TSCC show a remarkably better disease‐specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16INK4A overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV‐positive tumors show a favorable prognosis as compared to those with HPV‐negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non‐smokers differ from those occurring in smokers, the former being related to HPV 16 infection. © 2008 Wiley‐Liss, Inc.

BACKGROUNDA pleural effusion in children is usually caused by infectious diseases; malignant effusion is very uncommon. In a case of a malignant effusion in a child, a pleura-based metastasis of a neoplasm with a typically high prevalence in childhood has to be considered. Examples are neuroblastoma, nephroblastoma, Wilms' tumor, hepatoblastoma, malignant germ cell tumor and rhabdomyosarcoma.CASEA 4-year old boy presented with a unilateral pleural effusion. Cytologic examination of the effusion revealed malignant small round cells admixed with very large cells with atypical nuclei. In formalin-fixed cell blocks prepared for immunocytochemistry the cells expressed desmin and myf-4. The cytologic diagnosis was consistent with metastatic rhabdomyosarcoma. Subsequent computed tomography revealed a mass above the diaphragm, which was biopsied. Histologic examination of a needle biopsy specimen confirmed the diagnosis of rhabdomyosarcoma. Molecular examination revealed a PAX3-FKHR fusion transcript specific to the alveolar type of rhabdomyosarcoma.CONCLUSIONThis case illustrates the usefulness of immunocytochemistry on cell block preparations in diagnosing difficult cases of effusion cytology.

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 p16 INK4A 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. © 2003 Wiley‐Liss, Inc.

Gastric non-Hodgkin's lymphomas can be divided histologically into mucosa-associated lymphoid tissue (MALT) lymphoma (ML) and diffuse large cell lymphoma (DLCL) with or without evidence of preceding/accompanying ML (DLCL + ML). We studied the incidence of the most frequent structural chromosomal aberration in ML, t(11;18)(q21;q21), and numerical aberrations of seven chromosomes in 36 ML, 39 DLCL + ML and ten gastric DLCL cases, by dual-colour interphase fluorescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). t(11;18)(q21;q21) was exclusively detected in ML (FISH 22%; RT-PCR 24%), being completely absent in DLCL + ML and DLCL. No other translocations involving 11q21 or 18q21 and other partner chromosomes were detected by FISH. In lymphomas harbouring t(11;18)(q21;q21), this translocation was the sole genetic abnormality. In contrast, 45% of the t(11;18)(q21;q21)-negative ML showed trisomies, especially of chromosome 3 and 18. In DLCL + ML with separate small and large cell components, trisomies were either detected in both components or occurred exclusively in large tumour cells. Our results suggest that ML can be divided in lymphomas characterized by the t(11;18)(q21;q21), which are unlikely to transform into high-grade tumours, and t(11;18)(q21;q21)-negative ML that may develop into DLCL + ML after the acquisition of additional genetic aberrations.

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.

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.

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.

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.