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PDF file - 15946K, Representative immunohistochemical staining patterns for CD44 (A) and OTP (B-C), in (A) a large cell neuroendocrine carcinoma with absence of membranous CD44 staining in the tumor cells but presence of CD44 near necrotic areas and in inflammatory cells. (B) SCLC with moderate to strong nuclear OTP reactivity. This patient had no evidence of disease seven years after initial diagnosis, and was still alive 11 years after diagnosis. (C) Small cell lung cancer (SCLC) with faint cytoplasmic OTP staining and absence of nuclear staining. Original magnifications 200x.

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PDF file - 1346K, Kaplan-Meier analysis showing 20-year overall survival rates for a combined group of carcinoids and high-grade neuroendocrine carcinomas. A gradient of OTP immunostaining is shown for the 312 out of 348 cases with available follow-up. The solid lines indicate cases with nuclear staining which may or may not display cytoplasmic immunoreactivity, the dashed lines indicate tumors which have lost nuclear staining but retain cytoplasmic positivity, and the dotted lines indicate negative tumors. The period is indicated in months. Censored cases are indicated by an asterisk.

XLSX file - 185K, Significance levels (P-values) for uni- and multivariate analyses (Cox-regression) of pulmonary neuroendocrine tumor subgroups, indicating the relation of clinical and molecular parameters with disease outcome

Representative immunohistochemical staining patterns for CD44 (A), OTP (B) and RET (C). (A) Normal lung tissue adjacent to a carcinoid tumor showing CD44 staining of the bronchial epithelium BE and seromucous glands G (original magnification 100x). The insets show higher magnifications of a seromucous gland and the bronchial epithelium (original magnification 200x). (B) Normal tissue adjacent to carcinoid tumor tissue with cytoplasmic OTP reactivity of the bronchial epithelium and seromucous glands (original magnification 200x), in agreement with the Human Protein Atlas (http://www.proteinatlas.org/ENSG00000171540). The inset shows cytoplasmic reactivity in macrophages (original magnification 400x). (C) Normal tissue adjacent to a carcinoid tumor showing cytoplasmic RET positivity of the bronchial epithelium and seromucous glands (original magnification 200x), in agreement with the Human Protein Atlas (http://www.proteinatlas.org/ENSG00000165731). The inset shows strong membranous RET staining in normal colon (original magnification 200x).

XLSX file - 17K, Comparison of gene expression levels (quantitative RT-PCR) and protein expression levels (immunohistochemistry) of CD44, OTP and RET in pulmonary neuroendocrine tumours

Purpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histologic classification in typical and atypical carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, that is, orthopedia homeobox (OTP), CD44, and rearranged during transfection (RET).Experimental Design: These genes were analyzed in 56 frozen carcinoids by quantitative real-time PCR (qRT-PCR). RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was conducted on 292 carcinoids.Results: Low mRNA expression levels of CD44 (P = 1.8e−5) and OTP (P = 0.00054), and high levels of RET (P = 0.025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP but not for RET. Within all carcinoids as well as atypical carcinoids, absence of CD44 protein was significantly associated with low 20-year survival (P = 0.00014 and 0.00013, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (P = 5.2−6). Multivariate analyses revealed that age at diagnosis, histopathology, stage, and cytoplasmic OTP immunoreactivity were independent predictors of prognosis.Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories. Clin Cancer Res; 19(8); 2197–207. ©2013 AACR.

BACKGROUND: The definition of objective, clinically applicable evaluation criteria for FISH 1c/7c in laryngeal precursor lesions for the detection of chromosome instability (CI). Copy Number Variations (CNV) for chromosomes 1 and 7 reflect the general ploidy status of premalignant head and neck lesions and can therefore be used as a marker for CI.METHODS: We performed dual-target FISH for chromosomes 1 and 7 centromeres on 4 µm formalin-fixed, paraffin-embedded tissue sections of 87 laryngeal premalignancies to detect CNVs. Thirty-five normal head and neck squamous cell samples were used as a control. First, the chromosome 7:1 ratio (CR) was evaluated per lesion. The normal range of CRs (≥0.84 ≤ 1.16) was based on the mean CR +/- 3 x SD found in the normal population. Second, the percentage of aberrant nuclei, harboring > 2 chromosomes of chromosome 1 and/or 7 (PAN), was established (cut-off value for abnormal PAN ≥ 10%).RESULTS: PAN showed a stronger correlation with malignant progression than CR (resp. OR 5.6, p = 0.001 and OR 3.8, p = 0.009). PAN combined with histopathology resulted in a prognostic model with an area under the ROC curve (AUC) of 0.75 (s.e. 0.061, sensitivity 71%, specificity 70%).CONCLUSIONS: evaluation criteria for FISH 1c/7c based on PAN ≥ 10% provide the best prognostic information on the risk of malignant progression of premalignant laryngeal lesions as compared with criteria based on the CR. FISH 1c/7c detection can be applied in combination with histopathological assessment.

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

Summary 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 immuno-stainings 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 histo-pathological 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 (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16(INK4A) is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16(INK4A) overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)malignant and benign head and neck lesions. P16(INK4A) immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or -11. P16(INK4A) immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPV-negative OPSCC (p < 0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16(INK4A) immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or -11-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16(INK4A) immunostaining is highly variable and cannot be recommended to predict HPV-presence.

Background: The local recurrence rate in oral squamous cell cancer (OSCC) hardly decreases. This is partly due to the presence of (pre) malignant cells in the remaining tissue after resection, that may lead to the development of a new tumor in time. Detection of histologically (pre) malignant cells in the tumor resection margins should predict these patients at risk for recurrence, however this appears to be difficult in routine practice. Purpose of this study was to apply easy-to-use molecular tests for more accurate detection of (pre) malignant cells in histopathologically tumor-free margins, to improve diagnosis of patients at risk.Methods: 42 patients with firstly diagnosed, radically resected primary OSCC with histopathologically confirmed tumor-free resection margins (treated between 1994 and 2003) were included. Inclusion criteria comprised of follow-up >= 5 years, and radical surgery without postoperative treatment. Formalinf-ixed paraffine-embedded tissue sections of 42 tumors, 290 resection margins, and 11 recurrences were subjected to fluorescence in situ hybridization (FISH) to examine chromosome 1 and 7 copy number variations (CNV), and to p53 immunohistochemistry (IHC).Results: 11 out of the 42 patients developed a local recurrence within 5 years. FISH analysis showed that nine of eleven recurrences exhibited CI in at least one of the resection margins (p = 0.008). P53 overexpression and routine histopathologic classification were not correlated with recurrent disease. The presence of CI in the resection margins revealed a significantly worse progression-free survival (log-rank p = 0.012).Conclusions: CI in the resection margins of OSCC can reliably identify patients at risk for developing a local recurrence. (C) 2016 Elsevier Ltd. All rights reserved.

AIMS: Oncogenic human papillomavirus (HPV) type 16 has been strongly associated with tonsillar squamous cell carcinoma (TSCC) and appears to be of prognostic significance. Because HPV+ TSCC also accumulates p16(INK4A), this cyclin-dependent kinase inhibitor has been proposed as a potential biomarker for HPV in clinical diagnosis. The aim of this study was to determine the prevalence of HPV in tumour-free tonsillar tissue and the value of p16(INK4A) overexpression in predicting its presence. METHODS AND RESULTS: p16(INK4A) overexpression was detected by immunohistochemistry in tissue sections of tumour-free tonsils of 262 patients. They were treated for non-oncological reasons (snoring or chronic/recurrent tonsillitis) consisting of tonsillectomy. Genomic DNA isolated from these tissues was subjected to HPV-specific polymerase chain reaction (PCR) analysis. p16(INK4A) immunoreactivity was detected in 28% of samples in both crypt epithelium (49/177) and lymphoid germinal centres (52/187), which correlated with each other (P < 0.0001). No reactivity was observed in superficial squamous cell epithelium. HPV16 and 18 were detected by PCR analysis in 2/195 cases (1%), which, however, were negative on fluorescence in situ hybridization analysis and discrepant on p16(INK4A) immunostaining. CONCLUSIONS: No proof was found for the presence of HPV in tumour-free tonsil tissue, despite increased p16(INK4A) expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16(INK4A) up-regulation.

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 p(16INK4A)-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

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.

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.

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.

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16(INK4A) immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS-as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q) PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16(INK4A) is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16(INK4A) overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)malignant and benign head and neck lesions. P16(INK4A) immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or -11. P16(INK4A) immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPV-negative OPSCC (p0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16(INK4A) immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or -11-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16(INK4A) immunostaining is highly variable and cannot be recommended to predict HPV-presence.

Summary .Second neoplasms after allogenic bone marrow transplantation (BMT) occur in donor cells; however, host origin generally cannot be excluded. Using fluorescence in situ hybridization (FISH) the origin of the malignant population can be proven indisputably. In a female patient with CD30+ large-cell anaplastic non-Hodgkin's lymphoma (LCAL) after BMT with an HLA-identical brother donor, FISH using anti-CD30 immunocytochemistry in combination with anti-Y- and anti-EBV RNA probes was applied. In pathological lymph nodes the majority of cells were of donor type (Y). CD30-positive cells were Y-positive; these cells were also EBV-positive. Using FISH and immunocytochemistry we have demonstrated convincingly that this, possibly EBV-induced, LCAC originated in donor cells.

Oncogenic human papillomavirus (HPV) type 16 has been strongly associated with tonsillar squamous cell carcinoma (TSCC) and appears to be of prognostic significance. Because HPV+ TSCC also accumulates p16(INK4A), this cyclin-dependent kinase inhibitor has been proposed as a potential biomarker for HPV in clinical diagnosis. The aim of this study was to determine the prevalence of HPV in tumour-free tonsillar tissue and the value of p16(INK4A) overexpression in predicting its presence.p16(INK4A) overexpression was detected by immunohistochemistry in tissue sections of tumour-free tonsils of 262 patients. They were treated for non-oncological reasons (snoring or chronic/recurrent tonsillitis) consisting of tonsillectomy. Genomic DNA isolated from these tissues was subjected to HPV-specific polymerase chain reaction (PCR) analysis. p16(INK4A) immunoreactivity was detected in 28% of samples in both crypt epithelium (49/177) and lymphoid germinal centres (52/187), which correlated with each other (P0.0001). No reactivity was observed in superficial squamous cell epithelium. HPV16 and 18 were detected by PCR analysis in 2/195 cases (1%), which, however, were negative on fluorescence in situ hybridization analysis and discrepant on p16(INK4A) immunostaining.No proof was found for the presence of HPV in tumour-free tonsil tissue, despite increased p16(INK4A) expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16(INK4A) up-regulation.

Unknown primary adenocarcinoma (UPA) comprises a group of heterogeneous cancers of great clinical and biological interest. UPA presents as metastatic disease without a detectable primary site after medical workup. Here we investigated whether or not a clonal relationship exists between multiple tumors within individual UPA patients. A molecular resemblance would argue for an early clonal outgrowth of tumor cells from the primary lesion, a mutual feature observed within this group of neoplasms. In 14 patients with UPA multiple tumors, obtained at autopsy, were analyzed by molecular allelotyping and immunohistochemistry. In addition, tumors of 4 patients could be analyzed by comparative genomic hybridization (CGH). Similar genetic and phenotypic profiles were used as indicator for a clonal relationship, whereas different profiles implicate independent tumors. The molecular data indicated that the multiple lesions in the 14 UPA patients, including the primary tumors, are clonally related. In agreement with the theory of tumor progression, some metastatic lesions showed additional genetic alterations besides the characteristics that were shared with the primary tumor. Furthermore, 8 UPA patients had tumors with a high frequency of allelic loss and/or imbalance (FALI; 43-71%), while 6 patients demonstrated a lower FALI (14-29%), suggesting the occurrence of chromosomal instability in the former group. Our data provide molecular evidence for a clonal relationship between multiple metastases and the primary tumor within individual UPA patients, independent of the anatomical origin of the cancer. This finding is in agreement with the suggestion that tumor progression is rapid in UPA patients, limiting the chance of clonal divergence. The identification of 2 groups of UPAs with either a high or low FALI indicates that chromosomal instability is not the only driving force behind early tumor cell dissemination. Thus, other molecular mechanisms must underlie the common biology of these tumors.

Human papillomavirus type 16 (HPV16) plays a role in the development of a subgroup of head and neck squamous cell carcinomas (HNSCC). However, uncertainty exists about the true impact of HPV in this tumor type as conflicting reports have been published with prevalence rates from 0 to 100%. We aimed to find a detection algorithm of a biologically and thus clinically meaningful infection, applicable for high-throughput screening of frozen and formalin-fixed paraffin embedded (FFPE) specimens. By considering detection of HPV E6 oncogene expression in frozen biopsies as gold standard for a meaningful HPV infection, the value of several assays was evaluated on FFPE tumor specimens and sera of 48 HNSCC patients. The following assays were evaluated on FFPE tissue samples: HPV DNA general primer (GP)5+/6+ PCR, viral load analysis, HPV16 DNA FISH detection, HPV16 E6 mRNA RT-PCR, p16 immunostaining, and on corresponding serum samples detection of antibodies against the HPV16 proteins L1, E6 and E7. Comparing single assays on FFPE tissue samples detection of E6 expression by RT-PCR was superior, but application remains at present limited to HPV16 detection. Most suitable algorithm with 100% sensitivity and specificity appeared p16 immunostaining followed by GP5+/6+ PCR on the p16-positive cases. We show that clinically meaningful viral HPV infections can be more reliably measured in FFPE HNSCC samples in a standard and high throughput manner, paving the way for prognostic and experimental vaccination studies, regarding not only HNSCC, but possibly also cancer types with HPV involvement in subgroups such as penile and anal cancer.

Purpose Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates to the level of viral gene transcription and influences the expression of disrupted genes. Material and methods HPV16 integration sites were assessed in 75 patients with HPV16-DNA- and p16 INK 4 A -positive OSCC using Detection of Integrated Papillomavirus Sequences (DIPS)- as well as Amplification of Papillomavirus Oncogene Transcripts (APOT)-PCR. Quantitative RT-PCR assays were carried out to determine the viral E2/E4, E6 and E7 gene expression levels. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCC. Nuclear localization of HPV16 was visualized using Fluorescence In Situ Hybridization (FISH) in 59 OSCC, and corresponding viral copy numbers were assessed by quantitative RT-PCR in 61 tumors. Results We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we neither found significant differences in the mean expression of the viral genes E2/E4, E6 and E7, the nuclear FISH staining patters nor the viral copy numbers per cell. The expression of the HPV-invaded genes also did not significantly differ when comparing the expression in the affected tumor with the expression of that gene in either group of OSCC. Conclusions In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Purpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histological classification in typical and atypical (AC) carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, i.e., OTP, CD44 and RET. Methods: These genes were analyzed in 56 frozen carcinoids by quantitative RT-PCR. RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was performed on 292 carcinoids. Results: Low mRNA expression levels of CD44 (p=1.3e-5) and OTP (p=0.012), and high levels of RET (p=0.0025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP, but not for RET. Within all carcinoids as well as ACs, absence of CD44 protein was significantly associated with low 20-year survival (p=8.6e-5 and p=0.00015, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (p=2.9e-5). Multivariate analysis revealed that CD44 and OTP immunostaining combined with histopathology is the optimal indicator for patient outcome. Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories. Citation Format: Dorian RA Swarts, Mieke Henfling, Leander van Neste, Robert J. van Suylen, Anne-Marie Dingemans, Winand NM Dinjens, Annick Haesevoets, Martina Rudelius, Erik Thunnissen, Marco Volante, Wim van Criekinge, Manon van Engeland, Frans CS Ramaekers, Ernst-Jan M. Speel. Reduced CD44 and OTP gene expression are strong indicators of poor prognosis in pulmonary carcinoids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2013-2380

Objective. To date, there is no consensus on the optimal diagnostic and therapeutic strategy for patients with carcinoma of unknown primary (CUP) of the neck. These tumors are heterogeneous in their clinical and biological characteristics, and a pre-operative prognostic marker is desirable to optimize therapy and improve outcome and survival. Human papillomavirus (HPV) is an etiologic factor in a subgroup of head and neck squamous cell carcinomas and has been identified as a significant prognostic biomarker. We sought to determine if HPV also may add relevant information on the origin and prognosis of these tumors. Material and method. 47 patients (mean age 58.7 years; 40 men, 7 women) presenting with CUP of the neck between 1994 and 2008 in Cologne were examined by standard diagnostic procedures including radiological imaging of the head, neck, thorax and abdomen, positron emission tomography (PET), panendoscopy, curettage of the nasopharynx, bilateral tonsillectomy and blind probes of the base of tongue. All patients were surgically treated with neck dissection of the diseased neck as well as adjuvant chemoradiation. The mean follow up time was 34 months. Formaldehyde-fixed, paraffin-embedded tissue specimens of the 47 metastases were examined retrospectively by means of p16INK4A immunohistochemistry, HPV-specific PCR and fluorescence in situ hybridization. Results were correlated with clinical follow-up data. Results. Oncogenic HPV was present in 12/47 (26%) metastases (10 with HPV 16), which showed also p16INK4A overexpression. In 42% of CUP patients the primary tumor was discovered during follow-up. A significant correlation between HPV positivity and later detection of the primary tumor in the oropharynx was found (p=0.038). Moreover, patients with a p16INK4A- (13/47) or HPV-positive tumor had a more favorable overall 5-year survival rate then p16INK4A- and HPV-negative tumors (69% vs 33%, p=0.05; 65% vs 37%, p=0.093; respectively). Conclusion. HPV is present in a quarter of neck metastases of CUP patients and the presence of oncogenic HPV and p16INK4A expression can serve to locate the primary tumor in the oropharynx. In addition, both biomarkers are indicators of a favorable prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2011-2255

A major dilemma in the management of patients with precursor lesions of the oral mucosa lies in deciding which lesions will progress into carcinoma. Because a strong association between histological differentiation and clinical prognosis is lacking, the aim of this study was to evaluate the value of chromosomal instability (CIN) detected by fluorescence in situ hybridization (FISH) for the identification of oral premalignant lesions at risk for progression. We examined a series of premalignant oral mucosa lesions of 106 patients by means of FISH on paraffin-embedded tissue sections using biotin- and digoxigenin-labeled probes specific for the centromeric regions of chromosomes 1 and 7. CIN was indicated by the presence of chromosome imbalances and/or polyploidization for chromosomes 1 and 7. Results were correlated with histopathological data as well as with clinical follow up data. The 5-year progression-free survival rate was 83%. Outcome of routine histopathology did only predict malignant progression when comparing severe dysplasia with lower stage precursor lesions (hyperplasia, mild en moderate dysplasia) (p In conclusion, tumorigenesis of the oral mucosa is associated with the development of CIN, which can reliably identify lesions at risk for progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5720.

Merkel cell polyomavirus (MCPyV) DNA has been detected by PCR in 75-100% of Merkel cell carcinomas (MCC), an aggressive neuroendocrine skin cancer. MCPyV is a 5.4 kb DNA virus that expresses tumor (T) antigen in tumor tissues. The aim of this study was to analyze which subset of MCC is biologically associated with MCPyV using various viral detection methods. Genomic DNA isolated from paraffin-embedded tissue of 61 MCC was analyzed by MCPyV-specific PCR. Using a 5kb MCPyV DNA probe, the physical status of MCPyV was determined by fluorescence in situ hybridization (FISH) using tyramide signal amplification and ApoTome microscopy. Expression of large T antigen was assessed by immunohistochemistry (IHC) using monoclonal antibody CM2B4 (kindly provided by Y. Chang). A total of 44 of 61 (72%) MCC were positive for MCPyV by PCR. 34 of these 44 cases (77%) were also positive for MCPyV by FISH and 28 (64%) showed strong nuclear large T antigen expression by IHC. In contrast, none of the 17 PCR-negative tumors showed FISH signals and only 1 case showed a weak immunostaining for large T antigen. FISH analysis showed in 16 cases (47%) tumor nuclei with a single punctate signal indicating viral integration. In 8 of these tumors also areas were observed with a granular pattern with >1 nuclear signals varying significantly in size and intensity, indicating the presence of episomal viral DNA and/or RNA. In the remaining 18 cases (53%) an exclusive granular FISH pattern was observed. Our data indicate that a subset of MCC is caused by MCPyV as determined by PCR, FISH and IHC analysis (46% is positive with all 3 detection methods). FISH and IHC showed a strong positive and negative concordance. FISH signal evaluation furthermore suggests viral integration in half of the positive MCC and a non-clonal viral persistence in the remaining positive cases. Further studies have to validate these findings and elucidate the molecular mechanisms underlying MCV-positive and -negative MCC development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5352.