Annick Haesevoets

A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen.

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.

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 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.

A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8.

Besides well‐known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV‐induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single‐strand conformation polymorphism (SSCP) analysis of exons 5–8. Paraffin‐embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16INK4A overexpression in all 10 HPV‐positive tumors. Although FISH is considered to be less sensitive than PCR‐based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV‐positive carcinomas. However, in none of the latter cases could mutations in exons 5–8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors.

p16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Klingenberg B, Hafkamp H C, Haesevoets A, Manni J J, Slootweg P J, Weissenborn S J, Klussmann J P & Speel E‐J M
(2010) Histopathology 56, 957–967
p16INK4Aoverexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression

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 p16INK4A 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.

P16(INK4A) immunostaining is a strong indicator for high-risk-HPV-associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low-risk-HPV-infection in head and neck papillomas and laryngeal dysplasias

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16INK4A 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 p16INK4A overexpression can accurately predict both high‐risk and low‐risk‐HPV‐presence in (pre)malignant and benign head and neck lesions. P16INK4A 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. P16INK4A 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 p16INK4A immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16INK4A 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, p16INK4A immunostaining is highly variable and cannot be recommended to predict HPV‐presence.

CD44 and OTP Are Strong Prognostic Markers for Pulmonary Carcinoids

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.

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

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

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 Hodgkins 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 Hodgkins 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. Copyright

Large-cell anaplastic non-Hodgkin's lymphoma originating in donor cells after allogenic bone marrow transplantation

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‐Hodgkins 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.