Marie-Louise F. van Velthuysen

Incidence and survival of neuroendocrine tumours in the Netherlands according to histological grade: experience of two decades of cancer registry.

PURPOSE Epidemiological studies on neuroendocrine tumours (NETs) generally show a major increase in incidence. To investigate this increase, epidemiological data from the Netherlands were evaluated according to histological grade. METHODS All 47,800 patients with NET (diagnosed 1990-2010) from the population-based Netherlands Cancer Registry were stratified according to the latest World Health Organization (WHO) classification for the digestive system: well-differentiated NET grade 1 and 2 (G1NET and G2NET), and poorly differentiated (grade 3) neuroendocrine carcinoma, subdivided into large cell (G3-LCNEC) and small cell (G3-SCNEC). RESULTS The age-standardised incidence rate (excluding G3-SCNEC) increased from 2.1/100,000 in 1990 to 4.9/100,000 in 2010. The incidence of G1NET increased from 2.0 to 3.0; there was a large increase in G2NET from 0.01 in 1990 to 0.2 in 2010, and of the G3-LCNEC from 0.01 to 1.8, respectively. In G3-SCNEC incidence in men decreased from 21.3 to 10.1, whereas in women it increased from 4.5 to 7.7. The 5-year survival improved in G1NET, particularly for metastatic disease, from 30% in 1990-2000 to 47% in 2001-2010. CONCLUSION The increasing incidence of NET (without G3-SCNEC) was mainly due to the increase of G3-LCNEC. This increase is related to improved diagnostic procedures and to shifting in pathology from other entities (such as undifferentiated carcinoma) to NET. Improved survival was seen in all sites and stages, especially in patients with metastatic G1NET. Because of its influence on incidence and survival, we recommend to present epidemiological studies on NET according to histological classification.

Pulmonary squamous cell carcinoma following head and neck squamous cell carcinoma: metastasis or second primary?

Purpose: To distinguish a metastasis from a second primary tumor in patients with a history of head and neck squamous cell carcinoma and subsequent pulmonary squamous cell carcinoma. Experimental Design: For 44 patients with a primary squamous cell carcinoma of the head and neck followed by a squamous cell carcinoma of the lung, clinical data, histology, and analysis of loss of heterozygosity (LOH) were used to differentiate metastases from second primary tumors. Results: Clinical evaluation suggested 38 patients with metastases and 6 with second primaries. We developed a novel interpretation strategy based on biological insight and on our observation that multiple LOH on different chromosome arms are not independent. LOH analysis indicated metastatic disease in 19 cases and second primary squamous cell carcinoma in 24 cases. In one case, LOH analysis was inconclusive. For 25 patients, LOH supported the clinical scoring, and in 18 cases, it did not. These 18 discordant cases were all considered to be second primary tumors by LOH analysis. Conclusions: A considerable number of squamous cell lung lesions (50% in this study), clinically interpreted as metastases, are suggested to be second primaries by LOH analysis. For these patients, a surgical approach with curative intent may be justified.

Amplicon Mapping and Expression Profiling Identify the Fas-Associated Death Domain Gene as a New Driver in the 11q13.3 Amplicon in Laryngeal/Pharyngeal Cancer

Purpose: Amplification of the 11q13 region is a frequent event in human cancer. The highest incidence (36%) is found in head and neck squamous cell carcinomas. Recently, we reported that the amplicon size in 30 laryngeal and pharyngeal carcinomas with 11q13 amplification is determined by unique genomic structures, resulting in the amplification of a set of genes rather than a single gene. Experimental Design: To investigate which gene(s) drive the 11q13 amplicon, we determined the smallest region of overlap with amplification and the expression levels of all genes within this amplicon. Results: Using array-based comparative genomic hybridization analysis, we detected a region of ∼1.7 Mb containing 13 amplified genes in more than 25 of the 29 carcinomas. Quantitative reverse transcription-PCR revealed that overexpression of 8 potential driver genes including, cyclin D1, cortactin, and Fas-associated death domain (FADD), correlated significantly with DNA amplification. FADD protein levels correlated well with DNA amplification, implicating that FADD is also a candidate driver gene in the 11q13 amplicon. Analysis of 167 laryngeal carcinomas showed that increased expression of FADD (P = 0.007) and Ser194 phosphorylated FADD (P = 0.011) were associated with a worse disease-specific survival. FADD was recently reported to be involved in cell cycle regulation, and cancer cells expressing high levels of the Ser194 phosphorylated isoform of FADD proved to be more sensitive to Taxol-induced cell cycle arrest. Conclusion: Because of the frequent amplification of the 11q13 region and concomitant overexpression of FADD in head and neck squamous cell carcinomas, we hypothesize that FADD is a marker to select patients that might benefit from Taxol-based chemoradiotherapy.

Choice of tumour markers in patients with neuroendocrine tumours is dependent on the histological grade. A marker study of Chromogranin A, Neuron specific enolase, Progastrin-releasing peptide and cytokeratin fragments

BACKGROUND Chromogranin A (CgA) is the most important tumour marker for well-differentiated neuroendocrine tumours (NET) and neuron specific enolase (NSE) for poorly differentiated neuroendocrine carcinoma (NEC). This study investigated whether the markers progastrin-releasing peptide (proGRP) and cytokeratin fragments (CKfr) CK8, CK18 and CK19 (MonoTotal) can be of additional value to the histological classification and help predict survival in these patients. METHODS CgA, NSE, proGRP and CKfr were measured in 242 patients with grade 1 NET (G1NET), 38 with grade 2 NET (G2NET), 42 with large cell NEC (LCNEC), 251 with small cell NEC (SCNEC) and in 282 healthy persons. Results were compared with tumour characteristics and survival by means of Receiver Operating Characteristics (ROC) curves and Cox regression analyses. RESULTS The largest area under the ROC curve was for CgA (0.86, 0.91 and 0.90, respectively) when comparing patients with G1NET, G2NET and LCNEC with healthy persons. ProGRP showed the highest sensitivity (73%) at 95% specificity in patients with SCNEC. In a multivariate survival analysis, only CKfr was associated with survival (P<0.0001) for patients with well-differentiated NET (G1NET and G2NET). For patients with poorly differentiated NEC, both CKfr and NSE were associated with survival (P<0.0001 and P=0.003, respectively). CONCLUSION Within all histological groups a combination of tumour markers proved to be more informative as diagnostic and prognostic marker than each marker alone. In patients with well-differentiated NET and LCNEC we recommend the use of CgA and CKfr, whilst in patients with SCNEC, proGRP and CKfr are preferred.

Molecular markers predict outcome in squamous cell carcinoma of the head and neck after concomitant cisplatin-based chemoradiation.

Not all patients with squamous cell carcinomas of the head and neck (HNSCC) benefit from concurrent cisplatin‐based chemoradiation, but reliable predictive markers for outcome after chemoradiation are scarce. We have investigated potential prognostic biomarkers for outcome in a large group of patients. Ninety‐one tumor biopsies taken from consecutive HNSCC patients were evaluated for protein expression on a tissue microarray. Using immunohistochemistry, 18 biomarkers, involved in various cellular pathways were investigated. Univariable and multivariable proportional hazard analyses were performed to investigate associations between each individual marker and outcome. In addition, the global test was used to test all variables simultaneously and selected combinations of markers for an overall association with local control. Univariable analysis showed statistically significant increased relative risks of RB, P16 and MRP2 for local control and MDR1 and HIF‐1α for overall survival. MRP2, MDR1 and P16 levels were positively associated with outcome whereas RB and HIF‐1α had a negative relationship. Using Goemans global testing no combination of markers was identified that was associated with local control. Grouping the markers according to their function revealed an association between a combination of 3 markers (P16, P21 and P27) and outcome (p = 0.05) was found. In the multivariable analysis, MRP2 and RB remained significant independent predictive markers for local control. This study describes the prognostic value of biomarkers for the outcome in patients uniformly treated with concurrent chemoradiation. MRP2 and RB were found to be associated with outcome in patients treated with concurrent chemoradiation.

Interobserver variability of laryngeal mucosal premalignant lesions: a histopathological evaluation

The objective of this study is to measure interobserver variability in the classification of laryngeal mucosal premalignant lesions by reassessing the histopathology of previously diagnosed cases and to determine the possible therapeutic consequences of disagreement among observers. Histopathological assessment of 110 laryngeal mucosal premalignant lesions was done by three pathologists. Each slide had to be classified according to the World Health Organization, Squamous Intraepithelial Neoplasia, and the Ljubljana Squamous Intraepithelial Lesions systems. After the independent assessment, a joint meeting took place. To assess the relation between histopathological grading and subsequent clinical management, we created a two- and a three-grade system besides one comprising all options. For all analyses, the SAS/STAT statistical software was used. The highest unweighted κ-values concerning the all-options system are observed for the Squamous Intraepithelial Neoplasia classification (0.28, 95% confidence interval 0.23–0.33), followed by the World Health Organization and Ljubljana classifications. For the two-grade system the Ljubljana classification shows the highest unweighted κ-values (0.50, 95%, 0.39–0.61), followed by the World Health Organization and Squamous Intraepithelial Neoplasia classifications. For the three-grade system, the unweighted κ-values are similar. The implementation of weighted κ-values led to higher scores within all three classification systems, although these did not exceed 0.55 (moderate agreement). Given the high level of consensus, simultaneous pathological assessment may be said to provide added value in comparison with independent assessment. In the current study, no clear tendency is observed in favor of any one classification system. The proposed three-grade system could be an improved histopathological tool because it is easier to correlate with clinical decision making and because it yields better unweighted κ-values and proportions of concordance than the all-options system. Furthermore, clinical management could benefit from assessment by more than one pathologist in suspected cases of dysplasia or carcinoma.

Validation of tissue array technology in head and neck squamous cell carcinoma.

The recent development of tissue array technology has potentiated large‐scale retrospective cohort studies using archival formalin‐fixed, paraffin‐embedded tissues. This study evaluates the potential for using archival head and neck cancer tissue in such arrays.

HPV-negative squamous cell carcinoma of the anal canal is unresponsive to standard treatment and frequently carries disruptive mutations in TP53

Background:Human papillomavirus (HPV), p16 expression, and TP53 mutations are known prognostic factors in head and neck squamous cell carcinoma, but their role in squamous cell carcinoma of the anal canal (SCCAC) is less well established. The objective of this study was to determine the prognostic significance of tumour HPV status, p16 and p53 expression, and mutations in TP53 in patients with SCCAC receiving (chemo)radiotherapy.Methods:Human papillomavirus DNA was determined using an INNO-LiPA-based assay in tumour tissue of 107 patients with locally advanced SCCAC. Patients were treated with radiotherapy, with or without concurrent chemotherapy consisting of a fluoropyrimidine and mitomycin C. Expression of p16 and p53 was determined using immunohistochemistry. Exons 2–11 of TP53 in tumour tissue were sequenced.Results:DNA of high-risk HPV types was detected in 93 out of 107 tumours (87%), all of which overexpressed p16 (HPV+/p16+). Of 14 HPV-negative (HPV−) tumours (13%), 10 (9%) were p16-negative (HPV−/p16−) and 4 (4%) overexpressed p16 (HPV−/p16+). Patients with HPV−/p16− disease had inferior 3-year locoregional control (LRC) (15%) compared with patients with HPV+/p16+ tumours (82%, P<0.001) and HPV−/p16+ tumours (75%, P=0.078). Similarly, 3-year overall survival (OS) was 35% (HPV−/p16−) vs 87% (HPV+/p16+, P<0.001) and 75% (HPV−/p16+, P=0.219). Disruptive mutations in TP53 were found in 80% of HPV−/p16− tumours vs 6% of HPV+/p16+ tumours (P<0.001). In multivariate analysis, HPV−/p16− status was an independent predictor of inferior LRC and OS.Conclusions:HPV− tumours are frequently TP53 mutated. HPV−/p16− status is a strong predictor for reduced LRC and OS, and alternative treatment strategies for patients with HPV−/p16− disease need to be explored.

Specific genomic aberrations in primary colorectal cancer are associated with liver metastases

BackgroundAccurate staging of colorectal cancer (CRC) with clinicopathological parameters is important for predicting prognosis and guiding treatment but provides no information about organ site of metastases. Patterns of genomic aberrations in primary colorectal tumors may reveal a chromosomal signature for organ specific metastases.MethodsArray Comparative Genomic Hybridization (aCGH) was employed to asses DNA copy number changes in primary colorectal tumors of three distinctive patient groups. This included formalin-fixed, paraffin-embedded tissue of patients who developed liver metastases (LM; n = 36), metastases (PM; n = 37) and a group that remained metastases-free (M0; n = 25).A novel statistical method for identifying recurrent copy number changes, KC-SMART, was used to find specific locations of genomic aberrations specific for various groups. We created a classifier for organ specific metastases based on the aCGH data using Prediction Analysis for Microarrays (PAM).ResultsSpecifically in the tumors of primary CRC patients who subsequently developed liver metastasis, KC-SMART analysis identified genomic aberrations on chromosome 20q. LM-PAM, a shrunken centroids classifier for liver metastases occurrence, was able to distinguish the LM group from the other groups (M0&PM) with 80% accuracy (78% sensitivity and 86% specificity). The classification is predominantly based on chromosome 20q aberrations.ConclusionLiver specific CRC metastases may be predicted with a high accuracy based on specific genomic aberrations in the primary CRC tumor. The ability to predict the site of metastases is important for improvement of personalized patient management.

Ultrasound-guided aspiration cytology for the assessment of the clinically N0 neck: Factors influencing its accuracy†

Ultrasound‐guided fine‐needle aspiration cytology (US‐FNAC) can be used to diminish the risk of missing occult metastases and for early detection during follow‐up.