Ernst-Jan M. Speel

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.

Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV‐DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV‐DNA and expression of p16 and EGFR were analyzed. The 5‐year disease‐free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty‐eight percent of the cases contained oncogenic HPV‐DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV‐DNA (p < 0.001). Univariate analysis of the 5‐year DFS revealed a significantly better outcome for patients with p16‐positive tumors (84% vs. 49%, p = 0.009). EGFR‐negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5‐year DFS of 93% for p16+/EGFR− tumors vs. 39% for p16−/EGFR+ tumors (p = 0.003) and to a 5‐year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5‐fold increased risk for relapse in patients with p16‐negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV‐positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

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.

Genetic Signatures of HPV-related and Unrelated Oropharyngeal Carcinoma and Their Prognostic Implications

Purpose: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. Experimental Design: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16INK4A immunostaining. The results were correlated with HPV status and clinical data from patients. Results: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P = 0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P = 0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P = 0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P = 0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P = 0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P = 0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P = 0.008; disease-free survival, P = 0.01) and none of these patients had a tumor recurrence. Conclusions: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

Molecular and cellular biology of neuroendocrine lung tumors: evidence for separate biological entities.

Pulmonary neuroendocrine tumors (NETs) are traditionally described as comprising a spectrum of neoplasms, ranging from low grade typical carcinoids (TCs) via the intermediate grade atypical carcinoids (ACs) to the highly malignant small cell lung cancers (SCLCs) and large cell neuroendocrine carcinomas (LCNECs). Recent data, however, suggests that two categories can be distinguished on basis of molecular and clinical data, i.e. the high grade neuroendocrine (NE) carcinomas and the carcinoid tumors. Bronchial carcinoids and SCLCs may originate from the same pulmonary NE precursor cells, but a precursor lesion has only been observed in association with carcinoids, termed diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. The occurrence of mixed tumors exclusively comprising high grade NE carcinomas also supports a different carcinogenesis for these two groups. Histopathologically, high grade NE lung tumors are characterized by high mitotic and proliferative indices, while carcinoids are defined by maximally 10 mitoses per 2mm(2) (10 high-power fields) and rarely have Ki67-proliferative indices over 10%. High grade NE carcinomas are chemosensitive tumors, although they usually relapse. Surgery is often not an option due to extensive disease at presentation and early metastasis, especially in SCLC. Conversely, carcinoids are often insensitive to chemo- and radiation therapy, but cure can usually be achieved by surgery. A meta-analysis of comparative genomic hybridization studies performed for this review, as well as gene expression profiling data indicates separate clustering of carcinoids and carcinomas. Chromosomal aberrations are much more frequent in carcinomas, except for deletion of 11q, which is involved in the whole spectrum of NE lung tumors. Deletions of chromosome 3p are rare in carcinoids but are a hallmark of the high grade pulmonary NE carcinomas. On the contrary, mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are restricted to carcinoid tumors. Many of the differences between carcinoids and high grade lung NETs can be ascribed to tobacco consumption, which is strongly linked to the occurrence of high grade NE carcinomas. Smoking causes p53 mutations, very frequently present in SCLCs and LCNECs, but rarely in carcinoids. It further results in other early genetic events in SCLCs and LCNECs, such as 3p and 17p deletions. Smoking induces downregulation of E-cadherin and associated epithelial to mesenchymal transition. Also, high grade lung NETs display higher frequencies of aberrations of the Rb pathway, and of the intrinsic and extrinsic apoptotic routes. Carcinoid biology on the other hand is not depending on cigarette smoke intake but rather characterized by aberrations of other specific genetic events, probably including Menin or its targets and interaction partners. This results in a gradual evolution, most likely from proliferating pulmonary NE cells via hyperplasia and tumorlets towards classical carcinoid tumors. We conclude that carcinoids and high grade NE lung carcinomas are separate biological entities and do not comprise one spectrum of pulmonary NETs. This implies the need to reconsider both diagnostic as well as therapeutic approaches for these different groups of malignancies.

Prevalence and Clinical Outcomes for Patients With ALK-Positive Resected Stage I to III Adenocarcinoma: Results From the European Thoracic Oncology Platform Lungscape Project

PURPOSE The prevalence of anaplastic lymphoma kinase (ALK) gene fusion (ALK positivity) in early-stage non-small-cell lung cancer (NSCLC) varies by population examined and detection method used. The Lungscape ALK project was designed to address the prevalence and prognostic impact of ALK positivity in resected lung adenocarcinoma in a primarily European population. METHODS Analysis of ALK status was performed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) in tissue sections of 1,281 patients with adenocarcinoma in the European Thoracic Oncology Platform Lungscape iBiobank. Positive patients were matched with negative patients in a 1:2 ratio, both for IHC and for FISH testing. Testing was performed in 16 participating centers, using the same protocol after passing external quality assessment. RESULTS Positive ALK IHC staining was present in 80 patients (prevalence of 6.2%; 95% CI, 4.9% to 7.6%). Of these, 28 patients were ALK FISH positive, corresponding to a lower bound for the prevalence of FISH positivity of 2.2%. FISH specificity was 100%, and FISH sensitivity was 35.0% (95% CI, 24.7% to 46.5%), with a sensitivity value of 81.3% (95% CI, 63.6% to 92.8%) for IHC 2+/3+ patients. The hazard of death for FISH-positive patients was lower than for IHC-negative patients (P = .022). Multivariable models, adjusted for patient, tumor, and treatment characteristics, and matched cohort analysis confirmed that ALK FISH positivity is a predictor for better overall survival (OS). CONCLUSION In this large cohort of surgically resected lung adenocarcinomas, the prevalence of ALK positivity was 6.2% using IHC and at least 2.2% using FISH. A screening strategy based on IHC or H-score could be envisaged. ALK positivity (by either IHC or FISH) was related to better OS.

Human papillomavirus reduces the prognostic value of nodal involvement in tonsillar squamous cell carcinomas.

Assessment of the prognostic value of nodal status in relation to human papillomavirus (HPV) status and the various treatment modalities in tonsillar squamous cell carcinomas (TSCC).

HPV in situ hybridization: impact of different protocols on the detection of integrated HPV.

Although there is consensus that HPV integration is common in invasive cervical carcinomas and uncommon or absent in low‐grade uterine cervical intraepithelial neoplasia (CIN I), estimates for HPV integration in CIN II/III range from 5 to 100% using different PCR‐based and in situ hybridization (ISH) approaches. It has been suggested that HPV integration can be identified using ISH by scoring of punctate signals. The increased sensitivity of fluorescence ISH (FISH) methods, allowing the detection of single copies of HPV, complicates the distinction between integrated and episomal HPV. Recently it has been suggested that, in such assays, the signals originating from integrated virus can be hidden in a background of episomal HPV. We therefore compared 2 different FISH protocols for the detection of integrated HPV in a series of CIN II/III lesions: 1) a mild protocol in which episomal HPV and RNA is retained and 2) a harsh protocol that extensively extracts proteins and RNA, and which promotes the partial loss of episomal HPV but not integrated HPV. A series of 28 HPV 16/18 positive CIN II/III lesions (17 solitary lesions and 11 lesions adjacent to microinvasive carcinoma) were studied. A punctate signal pattern was identified in 7 of these lesions with both protocols. Punctate signal was also present in control samples from lesions that are known to be associated with HPV integration (invasive squamous cell carcinoma (n = 3), adenocarcinoma in situ (n = 3), and invasive adenocarcinoma (n = 1). HPV RNA contributed significantly to the intensity of punctate FISH signal, especially when applying the mild protocol, as shown by omitting DNA denaturation, including RNase pretreatment steps and measuring the fluorescence signal intensity. Also, HPV RNA was frequently detected in addition to episomal/integrated HPV DNA in the majority of the other 21 CIN II/III lesions; this resulted in intense granular/diffuse FISH signals throughout the epithelium. However, in 7 of these lesions, the harsh protocol gave a more consistent punctate pattern in cells throughout the full thickness of the epithelium. This supports the hypothesis that the harsh protocol unmasks integrated HPV more efficiently by extracting RNA and episomal HPV. Overall, with this harsh protocol, a clonally expanded population of cells containing punctate HPV signals was found in 5 of 17 (29%) solitary CIN II/III lesions and in 9 of 11 (88%) CIN II/III lesions associated with microinvasive carcinoma. Combining these data with the results from our previous study, with the harsh protocol in 7 of 40 (18%) solitary CIN II/III lesions and 19/21 (90%) CIN II/III lesions associated with microinvasive carcinoma (p < 0.001), this pattern was found. This indicates that, when robustly defined, a punctate HPV pattern in CIN II/III lesions is associated with the presence of an invasive carcinoma.

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

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

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