C. René Leemans

The molecular biology of head and neck cancer

Head and neck squamous cell carcinomas (HNSCCs) are caused by tobacco and alcohol consumption and by infection with high-risk types of human papillomavirus (HPV). Tumours often develop within preneoplastic fields of genetically altered cells. The persistence of these fields after treatment presents a major challenge, because it might lead to local recurrences and second primary tumours that are responsible for a large proportion of deaths. Aberrant signalling pathways have been identified in HNSCCs and inhibition of epidermal growth factor receptor (EGFR) has proved a successful therapeutic strategy. In this Review, we discuss the recent literature on tumour heterogeneity, field cancerization, molecular pathogenesis and the underlying causative cancer genes that can be exploited for novel and personalized treatments of patients with HNSCC.

Decoding tumour phenotype by noninvasive imaging using a quantitative radiomics approach.

Human cancers exhibit strong phenotypic differences that can be visualized noninvasively by medical imaging. Radiomics refers to the comprehensive quantification of tumour phenotypes by applying a large number of quantitative image features. Here we present a radiomic analysis of 440 features quantifying tumour image intensity, shape and texture, which are extracted from computed tomography data of 1,019 patients with lung or head-and-neck cancer. We find that a large number of radiomic features have prognostic power in independent data sets of lung and head-and-neck cancer patients, many of which were not identified as significant before. Radiogenomics analysis reveals that a prognostic radiomic signature, capturing intratumour heterogeneity, is associated with underlying gene-expression patterns. These data suggest that radiomics identifies a general prognostic phenotype existing in both lung and head-and-neck cancer. This may have a clinical impact as imaging is routinely used in clinical practice, providing an unprecedented opportunity to improve decision-support in cancer treatment at low cost.

Second primary tumors and field cancerization in oral and oropharyngeal cancer: Molecular techniques provide new insights and definitions†

Second primary tumors (SPTs) are a significant problem in treating oral and oropharyngeal squamous cell carcinoma and have a negative impact on survival. In most studies the definition of SPT is based on the criteria of Warren and Gates, published in 1932. These criteria, however, are ill‐defined and lead to confusion. Recent molecular studies have shown that a tumor can be surrounded by a mucosal field consisting of genetically altered cells. Furthermore, evidence has been provided that SPTs (defined by classical criteria) can share some or even all genetic markers with the index tumor, indicating that both tumors have arisen from a common cell clone. We propose that these secondary neoplastic lesions should not be considered SPTs, implying that the present concept of SPT needs revision. This review describes a novel classification of the secondary tumors that develop after treatment of a carcinoma in the oral cavity or oropharynx. On the basis of the molecular analysis of the tumors and the genetically altered mucosal field in between, we propose definitions for a “true SPT,” a local recurrence, a “SFT” (second field tumor derived from the same genetically altered mucosal field as the primary tumor), and a metastasis. Considering the etiologic differences of these lesions, we believe that an accurate molecular definition is essential to make headway with the clinical management of oral and oropharyngeal cancer.

Intensity-Modulated Radiotherapy Reduces Radiation-Induced Morbidity and Improves Health-Related Quality of Life: Results of A Nonrandomized Prospective Study Using A Standardized Follow-Up Program

PURPOSE The purpose of this study was to compare intensity-modulated radiation therapy (IMRT) and three-dimensional conventional radiotherapy (3D-CRT) with regard to patient-rated xerostomia, Radiation Therapy Oncology Group (RTOG) acute and late xerostomia and health-related quality of life (HRQoL) among patients with head and neck squamous cell carcinoma (HNSCC). METHODS AND MATERIALS Included were 241 patients with HNSCC treated with bilateral irradiation +/- chemotherapy. Since 2000, all patients treated with HNSCC were included in a program, which prospectively assessed acute and late morbidity according to the RTOG and HRQoL on a routine basis at regular intervals. Before October 2004, all patients were treated with 3D-CRT (N = 150). After clinical implementation in October 2004, 91 patients received IMRT. In this study, the differences regarding RTOG toxicity, xerostomia, and other items of HRQoL were analyzed. RESULTS The use of IMRT resulted in a significant reduction of the mean dose of the parotid glands (27 Gy vs. 43 Gy (p < 0.001). During radiation, Grade 2 RTOG xerostomia was significantly less with IMRT than with 3D-CRT. At 6 months, the prevalence of patient-rated moderate to severe xerostomia and Grade 2 or higher RTOG xerostomia was significantly lower after IMRT versus 3D-CRT. Treatment with IMRT also had a positive effect on several general and head and neck cancer-specific HRQoL dimensions. CONCLUSIONS IMRT results in a significant reduction of patient- and observer-rated xerostomia, as well as other head and neck symptoms, compared with standard 3D-CRT. These differences translate into a significant improvement of the more general dimensions of HRQoL.

The mutational landscape of adenoid cystic carcinoma

Adenoid cystic carcinomas (ACCs) are among the most enigmatic of human malignancies. These aggressive salivary gland cancers frequently recur and metastasize despite definitive treatment, with no known effective chemotherapy regimen. Here we determined the ACC mutational landscape and report the exome or whole-genome sequences of 60 ACC tumor-normal pairs. These analyses identified a low exonic somatic mutation rate (0.31 non-silent events per megabase) and wide mutational diversity. Notably, we found mutations in genes encoding chromatin-state regulators, such as SMARCA2, CREBBP and KDM6A, suggesting that there is aberrant epigenetic regulation in ACC oncogenesis. Mutations in genes central to the DNA damage response and protein kinase A signaling also implicate these processes. We observed MYB-NFIB translocations and somatic mutations in MYB-associated genes, solidifying the role of these aberrations as critical events in ACC. Lastly, we identified recurrent mutations in the FGF-IGF-PI3K pathway (30% of tumors) that might represent new avenues for therapy. Collectively, our observations establish a molecular foundation for understanding and exploring new treatments for ACC.

Improved tumor targeting of anti–epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology

The ∼15-kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti–epidermal growth factor receptor (EGFR) Nanobody (αEGFR-αEGFR) by fusion to a Nanobody unit binding to albumin (αAlb). Biodistributions of αEGFR-αEGFR, αEGFR-αEGFR-αAlb (∼50 kDa), αTNF-αTNF-αAlb (control, binding tumor necrosis factor-α), and the ∼150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with 177Lu to facilitate quantification. Tumor uptake of 177Lu-αEGFR-αEGFR decreased from 5.0 ± 1.4 to 1.1 ± 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of αAlb. Blood levels of αEGFR-αEGFR-αAlb were 21.2 ± 2.5, 11.9 ± 0.6, and 4.0 ± 1.4 and tumor levels were 19.4 ± 5.5, 35.2 ± 7.5, and 28.0 ± 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 ± 3.9, 27.1 ± 7.9, and 25.6 ± 6.1 %ID/g) and significantly higher than for αTNF-αTNF-αAlb. αEGFR-αEGFR-αAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of αEGFR and αAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned. [Mol Cancer Ther 2008;7(8):2288–97]

Multiple Head and Neck Tumors Frequently Originate from a Single Preneoplastic Lesion

The development of second primary tumors has a negative impact on the prognosis of head and neck squamous cell carcinoma. Previously, we detected genetically altered and tumor-related mucosal lesions in the resection margins in 25% of unselected head and neck squamous cell carcinoma patients (Tabor MP, Brakenhoff RH, van Houten VMM, Kummer JA, Snel MHJ, Snijders PJF, Snow GB, Leemans CR, Braakhuis BJM: Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications. Clin Cancer Res 2001, 7: 1523-1532). The aim of this study was to determine whether first and second primary tumors are clonally related and originate from a single genetically altered field. From 10 patients we analyzed the first tumor of the oral cavity or oropharynx, the >3-cm remote second primary tumor, and the mucosa from the tumor-free margins from both resection specimens. We compared TP53 mutations and loss of heterozygosity profiles using 19 microsatellite markers at chromosomes 3p, 9p, 13q, and 17p. In all patients, genetically altered mucosal lesions were detected in at least one resection margin from both first and second primary tumor. Evidence for a common clonal origin of the first tumor, second primary tumor, and the intervening mucosa was found for at least 6 of 10 patients. Our results indicate that a proportion of multiple primary tumors have developed within a single preneoplastic field. Based on different etiology and clinical consequences, we propose that independent second primary tumors should be distinguished from second field tumors, that arise from the same genetically altered field the first tumor has developed from.

Peripheral blood IFN-γ-secreting Vα24+Vβ11+ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load

Natural killer T (NKT) cells are CD1d‐restricted lymphoid cells and are characterized by an invariant T‐cell receptor, which in humans consists of a Vα24 chain paired with a Vβ11 chain. These cells are known for their rapid production of large amounts of cytokines (e.g., IFN‐γ and IL‐4), thereby modulating other cells of the immune system such as T cells, NK cells and dendritic cells. NKT cells have been reported to play important regulatory roles in many immune responses, including antitumor immune responses. Here, we demonstrate an age‐dependent decrease in circulating Vα24+Vβ11+ NKT cell numbers in both healthy controls and cancer patients and demonstrate that in both groups females have higher NKT cell levels compared to males. In a large group of 120 cancer patients, we show that circulating Vα24+Vβ11+ NKT cell numbers are about 50% lower than in age‐ and gender‐matched healthy controls and that this decrease is independent of tumor type or tumor load. This decrease was not restored upon tumor removal by means of surgery or radiotherapy. Even though the percentage of NKT cells that secrete IFN‐γ, as detected by ELISPOT, is normal in cancer patients, the absolute number of circulating IFN‐γ‐secreting NKT cells is reduced. Together, our results suggest that the reduced circulating Vα24+Vβ11+ NKT cell numbers in cancer patients are not affected by tumor load, but might actually reflect a risk factor for tumor development, e.g., by hampering efficient tumor immunosurveillance.

A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus.

Purpose: To assess safety, pharmacokinetics, maximum tolerated dose, and preliminary efficacy of bivatuzumab mertansine. Bivatuzumab is a humanized monoclonal antibody directed against CD44v6, which previously seemed to be safe in phase I radioimmunotherapy trials, whereas the conjugated mertansine is a potent maytansine derivative. Experimental Design: Patients with incurable squamous cell carcinoma of the head and neck or esophagus were eligible. Bivatuzumab was given weekly for 3 consecutive weeks by i.v. infusion. One patient was planned to be treated at each dose tier as long as toxicity did not reach grade 2; otherwise, three patients had to be treated until dose-limiting toxicity occurred. Starting dose was 20 mg/m2 and dose was subsequently escalated in steps of 20 mg/m2. Patients without disease progression and not experiencing dose-limiting toxicity were eligible for repeated courses. Blood serum samples were taken throughout the treatment period to determine the pharmacokinetic properties of bivatuzumab mertansine and to assess the human anti–bivatuzumab mertansine antibody response. Results: Seven patients received a total of 23 weekly doses of bivatuzumab mertansine. One patient at the 100 mg/m2 and one at the 120 mg/m2 level experienced stable disease during treatment phase but also developed grade 1 skin toxicity (desquamation). One of them received a second treatment course. At the highest dose level achieved in this study (140 mg/m2), one patient developed toxic epidermal necrolysis after two infusions and died. Massive apoptosis of skin keratinocytes had occurred, whereas only symptomatic therapy for skin toxicity was available. The risk-benefit assessment of all patients treated in the total phase I program (4 clinical trials, 70 patients) turned out to be negative after consideration of this case of a toxic epidermal necrolysis and the skin-related adverse events observed in the other trials. Therefore, development of the conjugate was discontinued. Interindividual variability in pharmacokinetic variables was low and exposure to BIWI 1 increased proportionally with dose. No anti–bivatuzumab mertansine reactions were observed. Conclusion: The main toxicity of bivatuzumab mertansine was directed against the skin, most probably due to CD44v6 expression in this tissue. The majority of skin reactions was reversible; however, one fatal drug-related adverse event had occurred. Clinical development was discontinued before reaching maximum tolerated dose.

Performance of Immuno–Positron Emission Tomography with Zirconium-89-Labeled Chimeric Monoclonal Antibody U36 in the Detection of Lymph Node Metastases in Head and Neck Cancer Patients

Purpose: Immuno–positron emission tomography (PET), the combination of PET with monoclonal antibodies (mAb), is an attractive option to improve tumor detection and to guide mAb-based therapy. The long-lived positron emitter zirconium-89 (89Zr) has ideal physical characteristics for immuno-PET with intact mAbs but has never been used in a clinical setting. In the present feasibility study, we aimed to evaluate the diagnostic imaging performance of immuno-PET with 89Zr-labeled-chimeric mAb (cmAb) U36 in patients with squamous cell carcinoma of the head and neck (HNSCC), who were at high risk of having neck lymph node metastases. Experimental Design: Twenty HNSCC patients, scheduled to undergo neck dissection with or without resection of the primary tumor, received 75 MBq 89Zr coupled to the anti-CD44v6 cmAb U36 (10 mg). All patients were examined by computed tomography (CT) and/or magnetic resonance imaging (MRI) and immuno-PET before surgery. Six patients also underwent PET with 18F-fluoro-2-deoxy-d-glucose. Immuno-PET scans were acquired up to 144 hours after injection. Diagnostic findings were recorded per neck side (left or right) as well as per lymph node level (six levels per side), and compared with histopathologic outcome. For this purpose, the CT/MRI scores were combined and the best of both scores was used for analysis. Results: Immuno-PET detected all primary tumors (n = 17) as well as lymph node metastases in 18 of 25 positive levels (sensitivity 72%) and in 11 of 15 positive sides (sensitivity 73%). Interpretation of immuno-PET was correct in 112 of 121 operated levels (accuracy 93%) and in 19 of 25 operated sides (accuracy 76%). For CT/MRI, sensitivities of 60% and 73% and accuracies of 90% and 80% were found per level and side, respectively. In the six patients with seven tumor-involved neck levels and sides, immuno-PET and 18F-fluoro-2-deoxy-d-glucose PET gave comparable diagnostic results. Conclusion: In this study, immuno-PET with 89Zr-cmAb U36 performed at least as good as CT/MRI for detection of HNSCC lymph node metastases.