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Objectives: The prognostic advantage of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) resulted in the initiation of treatment de-intensification studies. Two randomized controlled trials (RCTs) reported inferior survival of HPV-positive OPSCC treated with radiotherapy plus cetuximab compared to standard of care radiotherapy plus cisplatin. In this study we investigated whether the important role of cisplatin in the treatment of HPV-positive OPSCCs would also emerge from causal inference analyses of real-world data., Material and Methods: A retrospective cohort of 263 advanced-stage OPSCC-patients from 5 European clinics was studied, treated with radiotherapy (RT) alone or cisplatin-based chemoradiotherapy (CRT) based on standard clinical indications. Causal inference was applied to adjust for treatment assignment, thereby simulating a randomized setting. Average treatment effect of concurrent cisplatin on overall survival (OS) probability was estimated using Bayesian Additive Regression Trees (BART) and Bayesian logistic regression., Results: Significantly better survival probabilities were found for HPV-positive OPSCC treated with CRT compared to RT alone (3-year OS probability 0.961 versus 0.798, p = 0.008)., Conclusion: This study using causal inference of retrospective patient data confirms the important role of cisplatin in the treatment of HPV-positive OPSCC. Causal inference analyses of real-world data complements the evidence from the published RCTs., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Numerous clinical and histopathological characteristics have been associated with malignant transformation (MT) of oral leukoplakia (OL), including classic and differentiated epithelial dysplasia, but MT predictions remain suboptimal. The objective of this study was to determine the annual MT rate of OL and to identify clinicopathological risk factors associated with MT., Patients and Methods: 170 patients with OL were included in this retrospective cohort study, 117 females and 53 males. Follow-up ranged from 12 to 219 months (median 54). The analyzed variables included age, gender, smoking habits, clinical presentation, subsite, size and treatment. In a subgroup of 140 patients, histopathological diagnoses were reviewed with regard to the presence of dysplasia, discerning both classic dysplasia and differentiated dysplasia., Results: MT occurred in 23% of the patients, resulting in an annual MT rate of 4.9% (95% CI: 3.5 - 6.6) which remained consistent. High-risk subsite (tongue and floor of mouth) was the only clinical predictor for MT (Hazard Ratio = 2.7, 95% CI: 1.3 - 5.5, p = 0.007). In 140 patients, classic dysplasia (Hazard Ratio = 7.2, 95% CI: 1.6 - 33.1, p = 0.012) and differentiated dysplasia (Hazard Ratio = 6.6, 95% CI: 1.2 - 25.4, p = 0.026) were predictors for MT. Binary grading between dysplasia and no dysplasia was significant for predicting MT (Hazard Ratio = 6.4, 95% CI: 1.5 - 27.5, p = 0.013)., Conclusion: Since annual MT rate of OL remains stable during follow-up, regular long-term or even life-long follow-up is advocated. Specific oral subsites and epithelial dysplasia are predictors for MT of OL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Tumor-specific genetic aberrations in cell-free DNA (cfDNA) from plasma are promising biomarkers for diagnosis of recurrent head and neck squamous cell carcinoma (HNSCC). However, the sensitivity when using somatic mutations only in cfDNA is suboptimal. Here, we combined detection of copy number aberrations (CNAs), human papillomavirus (HPV) DNA and somatic mutations in a single sequencing workflow., Methods: Pretreatment plasmas of 40 patients and 20 non-cancer controls were used for analysis. Plasma DNA underwent low-coverage whole genome sequencing (lcWGS) to detect both CNAs and HPV-DNA, and deep sequencing to detect mutations in 12 frequently altered cancer driver genes in HNSCC using the same sequencing library. A specific analysis pipeline line was developed for data mining. The corresponding tumors were analyzed using slightly adapted protocols., Results: Using the developed method, somatic mutations and CNAs were detected in plasma DNA of HNSCC patients in 67% and 52%, respectively. HPV-DNA in plasma was detected in 100% of patients with HPV-positive tumors, and not in plasma of patients with HPV-negative tumors or non-cancer controls. Combined analysis increased the detection rate of tumor DNA in plasma to 78%. The detection rate was significantly associated with the stage of disease of the tumor. Neither HPV status nor location of the primary tumor influenced detection of CNAs or somatic mutations in plasma., Conclusions: This study demonstrates that the combined analysis of CNAs, HPV and somatic mutations in plasma of HNSCC patients is feasible and contributes to a higher sensitivity of the assay compared to single modality analyses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: The majority of patients with locally advanced larynx or hypopharynx squamous cell carcinoma are treated with organ-preserving chemoradiotherapy (CRT). Clinical outcome following CRT varies greatly. We hypothesized that tumor microRNA (miRNA) expression is predictive for outcome following CRT., Methods: Next-generation sequencing (NGS) miRNA profiling was performed on 37 formalin-fixed paraffin-embedded (FFPE) tumor samples. Patients with a recurrence-free survival (RFS) of less than 2 years and patients with late/no recurrence within 2 years were compared by differential expression analysis. Tumor-specific miRNAs were selected based on normal mucosa miRNA expression data from The Cancer Genome Atlas database. A model was constructed to predict outcome using group-regularized penalized logistic ridge regression. Candidate miRNAs were validated by RT-qPCR in the initial sample set as well as in 46 additional samples., Results: Thirteen miRNAs were differentially expressed (p < 0.05, FDR < 0.1) according to outcome group. Initial class prediction in the NGS cohort (n = 37) resulted in a model combining five miRNAs and disease stage, able to predict CRT outcome with an area under the curve (AUC) of 0.82. In the RT-qPCR cohort (n = 83), 25 patients (30%) experienced early recurrence (median RFS 8 months; median follow-up 42 months). Class prediction resulted in a model combining let-7i-5p, miR-192-5p and disease stage, able to discriminate patients with good versus poor clinical outcome (AUC:0.80)., Conclusion: The combined miRNA expression and disease stage prediction model for CRT outcome is superior to using either factor alone. This study indicates NGS miRNA profiling using FFPE specimens is feasible, resulting in clinically relevant biomarkers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The most widely applied algorithm for human papillomavirus (HPV) detection in formalin-fixed, paraffin-embedded (FFPE) specimens of oropharyngeal head and neck squamous cell carcinoma (HNSCC) consists of p16INK4A immunostaining followed by PCR-based detection of high-risk HPV DNA on p16INK4A-immunopositive samples. However, in nonoropharyngeal HNSCC this algorithm fails, hampering correct interpretation of the prevalence and prognosis of HPV in these cases. In this study, we developed and validated a molecular HPV detection method for FFPE specimens of oropharyngeal and nonoropharyngeal HNSCC. Sectioning of FFPE blocks was circumvented by using punch biopsies from tumor-enriched regions of FFPE tissue blocks, and combined extraction was applied to obtain high-quality DNA and RNA from the punch biopsy. Next, PCR-based detection of HPV DNA was performed for 15 high-risk HPV types with subsequent detection of E6 mRNA for validation. The combined DNA/RNA FFPE test of tissue cores was assessed in well-characterized cohorts with known HPV status based on earlier work, that is, a cohort of oropharyngeal HNSCC (n = 80) and oral cavity HNSCC (n = 25), and reached an accuracy of 97% and 100%, respectively. In conclusion, our method is rapid, simple, and shows an excellent diagnostic performance for detection of HPV type 16. Ultimately, it can be applied for large cohort studies to determine the etiologic fraction and prognostic implication of HPV in nonoropharyngeal HNSCC., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: Head and neck squamous cell carcinomas (HNSCC) arise in the mucosal lining of the upper aerodigestive tract. Risk factors are exogenous carcinogen exposure, human papillomavirus (HPV) infection, and genetic predisposition such as Fanconi anemia (FA). Clinically, tumors are stratified based on stage, site and HPV-status. The majority of HPV-positive and -negative HNSCC is characterized by frequent copy number (CN) changes and an abrogated p53-pathway. A third genetically-defined HPV-negative subclass of HNSCC is emerging: tumors that lack gross chromosomal changes (CN-silent), are mostly TP53-proficient, and have a relatively favorable prognosis., Methods: A representative panel of HPV-positive, HPV-negative and FA-HNSCC-derived cell lines was genetically characterized., Results: Despite apparent differences in etiology, FA-HNSCC cell lines show comparable genetic alterations as sporadic non-FA-HNSCC-derived cell lines. Furthermore, we identified a near diploid CN-silent HPV-negative HNSCC line: VU-SCC-040. Molecular characterization uncovers the absence of TP53 mutations, a functional p53-pathway and a CASP8 mutation. TP53 gene knockout using CRISPR-Cas9 resulted in resistance to MDM2 inhibition. Whereas p53-status is often proposed as a predictive biomarker for treatment response, TP53-knockout did not change sensitivity to cisplatin, Chk1 and Wee1 inhibition. Additionally, 84 CN-silent tumors were identified in the HNSCC PanCancer cohort and shown to be enriched for female gender, HRAS and CASP8 mutations., Conclusion: FA-derived HNSCC cell lines share comparable CN-profiles and mutation patterns as sporadic HPV-negative HNSCC. In contrast, a subclass of CN-silent, HPV-negative and TP53 wild-type HNSCC separates from the majority of HNSCC tumors. We show that VU-SCC-040 is a HNSCC cell model representative of this subclass., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Molecular markers are urgently needed to select non-small cell lung cancer (NSCLC) patients most likely to benefit from platinum-based chemotherapies. Of particular interest are proteins that can be found in biofluids like sputum for non-invasive detection. Therefore, we profiled the secretomes of 6 NSCLC cell lines with varying IC50-values for cisplatin, using label-free GeLC-MS/MS-based proteomics. Out of a total dataset of 2610 proteins, 304 proteins showed significant differences in expression levels between cisplatin sensitive and insensitive cell lines. Functional data mining revealed that the secretion of typically extracellular factors was associated with a higher sensitivity towards cisplatin, while cisplatin insensitivity correlated with increased secretion of theoretically intra-cellular proteins. Stringent statistical analysis and quantitative filtering yielded 58 biomarker candidates, 34 of which could be detected in clinical biofluids of lung cancer patients such as sputum using label-free LC-MS/MS-based proteomics. To assess performance of these biofluid biomarker candidates, we correlated protein expression with patient survival using a publically available clinical gene expression data set (GSE14814). We thus identified 3 top candidates with potential predictive value in determining cisplatin response (UGGT1, COL6A1 and MAP4) for future development as non-invasive biomarkers to guide treatment decisions. SIGNIFICANCE: Platinum-based chemotherapies are still the standard of care for NSCLC and other lung cancer types in the clinic today. However, due to chemoresistance, many patients suffer from the toxic side effects of these treatments without gaining any benefit in terms of survival. To date, no molecular biomarkers are available to predict clinical outcome of platinum-based chemotherapy. Because proteins present the functional read-out of genetic, epigenetic and translational events in the cell, a protein test is likely to be particularly suitable for response prediction. Of high relevance are proteins that are shed or secreted from cells, for example at primary tumor sites, and can be found in easily accessible biofluids like sputum for non-invasive detection. Here, we report the proteome profiling of the conditioned media (secretomes) of a panel of NSCLC cell lines in relation to cisplatin IC50 values, as a pre-clinical model, and of patient sputum as a clinical, lung cancer relevant biofluid. Using this approach in conjunction with exploration of the predictive potential in a transcriptome lung cancer patient dataset, we reveal biofluid biomarker candidates that, with further validation, may be used for non-invasive cisplatin response prediction in the future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV) infections are well known causes of anogenital cancers. Recent studies show that HPV also plays a role in oropharyngeal cancer (OPC). A review on the role of HPV vaccination in the prevention of head and neck squamous cell carcinoma (HNSCC) with special emphasis on OPC was conducted and available vaccines and vaccination strategies in HNSCC and OPC are discussed. Prophylactic vaccination is known to be effective for prevention of anogenital HPV infection and precursor lesions in the cervix and anus. While the value of vaccination for prevention of OPC and possibly as an adjuvant treatment is still an open question, evidence to date supports the possibility that HPV vaccination may prove to be effective in reducing the incidence of this malignancy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Relapses have a great impact on both the morbidity and mortality rates of oral squamous cell carcinoma (OSCC) patients. Current classification criteria are imprecise and need improvements. Recent advances in understanding of OSCC relapses on a molecular level provide new possibilities to better classify true recurrences and second primary tumors. This review discusses the limitations of the current OSCC relapse classification method and presents possible alternatives to improve this classification based on molecular techniques. Moreover, these molecular techniques add to the further understanding of these lesions and may provide tools for clinical management., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Recent studies support a role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (SCCs); however, the significance of HPV in non-oropharyngeal head and neck cancers is uncertain. The aim of this study was to determine the prevalence of HPV in a large cohort of laryngeal and hypopharyngeal SCCs in northern Spain., Materials and Methods: Clinical records and paraffin-embedded tumor specimens of 124 consecutive patients surgically treated for laryngeal (62 cases) and hypopharyngeal (62 cases) SCCs between 2002 and 2007 were retrieved. All cases were histologically evaluated, and presence of HPV was assessed by p16-immunohistochemistry followed by GP5+/6+-PCR-based DNA detection. Samples positive in both assays were subjected to HPV genotyping and HPV E6 transcript analysis., Results: Seventeen cases (14%) were positive for p16 immunostaining, of which 2 (1 larynx, 1 hypopharynx, 1.6% of total series) were found positive for HPV DNA by subsequent GP5+6+-PCR. Both SCCs contained HPV type 16 and showed HPV16 E6 mRNA expression., Conclusions: HPV is only occasionally involved in laryngeal and hypopharyngeal SCC patients in northern Spain., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objectives: Early detection and treatment of high risk premalignant mucosal changes of the oral cavity, will expectedly improve survival and reduce treatment-related morbidity. Aims of this study were to evaluate a non-invasive screening approach and to assess the value of molecular markers to identify patients at risk for oral cancer., Materials and Methods: Exfoliated cells and biopsies were obtained from oral leukoplakia lesions of 43 patients, of whom six developed oral cancer. All samples were investigated for loss of heterozygosity (LOH) at chromosomes 3p, 9p, 11q and 17p using microsatellite markers. On the biopsy specimen additional immunohistochemical staining for p53, TP53 mutation analysis and histopathological grading were performed., Results: The analytical sensitivity of the non-invasive assay using exfoliated cells to detect genetic changes present in the lesions was 45% (9 of 20), the specificity was 100% (19 of 19), and the positive predictive value was also 100% (9 of 9). LOH was present in 20 of 39 (51%) of the biopsies with uniformly LOH at 9p. Mutated TP53 and LOH at 9p in the biopsy, as single markers and in combination, were significant risk factors for malignant progression of leukoplakia to oral cancer (Kaplan-Meier analysis, p<0.05)., Conclusion: A non-invasive genetic screening approach using LOH in exfoliated cells has limited value for monitoring patients with leukoplakia. However, LOH at 9p, but also mutated TP53 in biopsies of oral leukoplakia have a significant association with malignant transformation and are promising candidate biomarkers to predict the risk for malignant progression., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Infection with high-risk types of human papillomavirus (hrHPV) is associated with cervical, anogenital, and oropharyngeal cancers. Since a causal contribution of hrHPV infection to lung cancer (LC) is still a matter of debate, a comprehensive study was performed to delineate hrHPV involvement in LC, using a Dutch study population., Methods: Archival tissue specimens from 223 patients (145 men, 78 women, median age 65 years, range 27-87 years), who presented with cancer in the lungs, were subjected to GP5+/6+ polymerase chain reaction and p16 immunohistochemistry. The series included primary lung carcinomas of patients without a history of cancer (n = 175), primary lung carcinomas of patients with an unrelated cancer in the past (n = 36), and carcinomas with primary presentation in the lungs of which the origin (i.e., primary or metastasis) was equivocal at the time of diagnosis (n = 12). GP5+/6+ polymerase chain reaction/p16 double-positive carcinomas were subjected to HPV genotyping, HPVE7 transcript analysis, loss of heterozygosity analysis, and array-comparative genomic hybridization., Results: Whereas all primary lung carcinomas were hrHPV-negative (211 of 211, 100%), three hrHPV-positive equivocal carcinomas (3 of 12, 25%) were identified. These patients (1 male, 2 females) had a history of hrHPV-associated disease; one tonsillar and two cervical carcinomas. A clonal relationship between individual tumor pairs was supported by identical hrHPV genotype, pattern of p16 expression, HPVE7 mRNA expression, and genomic aberrations., Conclusions: hrHPV presence in a tumor with primary presentation in the lungs signifies pulmonary metastasis from a primary hrHPV-positive cancer elsewhere in the body. No support was found for an attribution of hrHPV infection to the development of primary LC.

Objectives: Infection with the human papillomavirus (HPV) is an important risk factor for development of head and neck squamous cell carcinoma (HNSCC). Strikingly, HPV-positive HNSCCs have a more favorable prognosis than their HPV-negative counterparts. The current study was designed to explain this favorable prognosis of HPV-positive HNSCC., Materials and Methods: This was performed by investigating the response of four HPV-positive and fourteen HPV-negative HNSCC cell lines to cisplatin, cetuximab and radiation., Results: Analysis of the responses of this cell line panel indicated that HPV-positive cells are more resistant to cisplatin treatment than the HPV-negative HNSCCs, whereas the response to radiation and cetuximab did not differ., Conclusions: The current study suggests that the favorable prognosis for patients with HPV-positive HNSCC does not seem to be related to an intrinsic sensitivity of these tumor cells to chemotherapy or radiation in vitro., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: Management of the clinically N0 neck in oral cancer patients remains controversial. We describe the outcome of patients with T1-T2 oral cancer and N0 neck based on ultrasound guided fine needle aspiration cytology (USgFNAC) who were treated by transoral excision and followed by a 'wait and scan' policy (W&S)., Patients and Methods: This retrospective analysis included 285 consecutive patients of whom 234 were followed by W&S and 51 underwent elective neck dissection (END). Survival rates were compared between groups and correction for confounding factors was performed., Results: Of W&S patients, the 5-year disease-specific (DSS) and overall survival (OS) were 94.2% and 81.6% respectively. During follow-up 72.2% remained free of lymph node metastases and 27.8% developed delayed metastases. W&S patients with delayed metastases had a 5-year DSS and OS of 80.0% and 62.8%, respectively. In patients with positive END these rates were 81.3% and 64.2%, respectively. Between the groups, survival rates were not significantly different. Of the W&S patients with delayed metastases, 90.6% needed adjuvant radiotherapy versus 55.0% of patients with positive END., Conclusion: With regard to survival, in patients with early stage oral cancer and cN0 neck a 'wait and scan' policy using strict USgFNAC surveillance is justified as survival is not negatively influenced. Using a 'wait and scan' follow-up strategy instead of elective neck treatment, unnecessary neck dissection and its accompanying morbidity can be avoided in 72.2% of patients. However, for the small proportion of patients with delayed metastases, more extensive treatment with adjuvant radiotherapy is needed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Oral leukoplakia is a potentially malignant disorder that will develop into oral cancer at an estimated rate of 1-2% per year. Aim of the present study is to assess the possible predictive value of DNA ploidy for malignant progression of oral leukoplakia. A cohort of 62 leukoplakia patients was studied and their biopsy was examined with standard histopathology and DNA image cytometry. Cox regression analysis was performed to establish the relationship between progression-free survival and the DNA ploidy status. During the follow-up time (median of 69 months) 13 patients developed an oral squamous cell carcinoma (OSCC). DNA aneuploidy was observed in 27 (44%) patients and was significantly associated with a shorter progression-free survival [Hazard ratio of 3.7, 95% confidence intervals (CI) of 1.1 and 13.0 and a p-value of 0.04]. Sensitivity and specificity scores were 54% and 60%, respectively. Aneuploidy was not correlated with dysplasia grading (chi-square analysis). DNA aneuploidy in oral leukoplakia is associated with an increased risk of progression to OSCC. However, for the individual leukoplakia patient, DNA ploidy status as single biomarker has limited value to predict progression to cancer., (Copyright © 2011. Published by Elsevier Ltd.)

Cancer of the oral cavity accounts for almost 3% of cancer cases in the world. The incidence varies widely reflecting geographic differences in exposure to risk factors. The recent rise in younger age groups and females seen in many countries is of particular concern. Treatment and management of complications, locoregional recurrence and further primary tumors result in high morbidity and mortality especially when the disease is advanced stage at initial diagnosis. Progress in cancer research has provided abundant new knowledge about cellular processes and molecular biology underlying oral carcinogenesis and tumor progression. The present review attempts to summarize the current most widely-used research approaches and their application in the prevention, diagnosis, effective treatment, and improved outcome of oral cancer., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. Strong prognostic indicators that predict development of distant metastases are the presence and number of lymph node metastases in the neck, and extranodal spread. Recently, it was shown in several studies that also the presence of disseminated tumor cells (DTC) in the bone marrow predicts development of distant metastases. We have investigated whether protein signatures could be detected in primary HNSCC that distinguish tumors that disseminate into the bone marrow from those that do not. Therefore, DTC-positive and -negative primary HNSCC tumors were analyzed by 2D-DIGE. A signature consisting of 51 differential protein spots was identified upon stratification for bone marrow status, which allowed a correct classification of DTC-positive and DTC-negative HNSCC tumors in 95% of cases, using hierarchical clustering. The most prominent feature within this signature was the down-regulation of CK19 in DTC-positive tumors. Our data show that tumor cell dissemination to the bone marrow, the onset of hematogenic metastasis, can be deduced from the protein profile in the primary tumor. The highly significant down-regulation of CK19 supports a model of epithelial-mesenchymal transition for tumors that show a high proclivity for hematogenic dissemination., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common cancer worldwide. Despite improvements in diagnosis and treatment, the five-year-survival rate of advanced HNSCC has only moderately increased, which is largely due to the high proportion of patients that present with advanced disease stage and the frequent development of relapse and second primary tumors. Protein biomarkers allowing early detection of primary HNSCC or relapse may aid to improve clinical outcome. Screening for precursor changes in the mucosal linings preceding the development of invasive tumors and for accurate prediction of risk of malignant transformation, may be propitious opportunities, which are as yet difficult. This review summarizes recent results in HNSCC proteomics for biomarker research. Despite the wide diversity of experimental designs, a few common markers have been detected. Although some of these potential biomarkers are very promising, they still have to be further clinically validated. Finally, treatment of advanced cancers of several sites within the head and neck has shifted significantly during the last decade, and also, targeted drugs have entered the clinic. This has major consequences for the research questions in HNSCC research and accordingly for the future direction of proteome research in HNSCC biomarker discovery., (Copyright © 2010. Published by Elsevier B.V.)

The relatively modest survival of patients surgically treated for advanced HNSCC can partly be explained by the development of local relapse. It is important that surgeons are able to predict which patients are at high risk to develop local relapse, since clinical management can be tailored. Local relapse after resection of a primary HNSCC is easily explained, when tumour is detected in the surgical margins and thus residual tumour is likely to remain in the patient, but the pathobiology is more complex in cases where the margins are histologically tumour-free. Molecular studies indicate that there are two different mechanisms responsible in these cases. First, small clusters of residual tumour cells that are undetectable on routine histopathological examination (known as minimal residual cancer: MRC) proliferate and this forms the basis of recurring cancer. A second cause of relapse is a remaining field of preneoplastic cells that is struck by additional genetic hits leading to invasive cancer. It is likely that within this field, that can be over 7cm in diameter, the primary carcinoma has also emerged. Despite careful histopathological examination of the surgical margins of the primary carcinoma, it is at present not reliably possible to predict which patient will develop local relapse. Herein we focus on new developments regarding the analysis of margins, causes of local relapse, and how novel molecular techniques can be of help in a more accurate risk assessment. Critical analysis of the studies that have been published thus far shows that there is a list of promising markers, based on protein expression (immuno-histochemistry) and nucleic acid analysis. Further studies should be focused on validation and assessment of the clinical utility of these markers. Margin analysis should reveal whether one is dealing with residual cancer cells that might be treated by post-operative radiotherapy or with preneoplastic fields that remained behind. For this latter entity, there is no intervention available at present, except for a more intensive surveillance.

Oral leukoplakia is defined as a white patch in the oral cavity that cannot be diagnosed as any other known disorder. These lesions carry an increased risk of malignant progression, and approximately 2-3% per year do progress to cancer. At present biopsies are histopathologically graded for dysplasia to assess the risk of progression, but this grading is somewhat subjective and of limited use for the individual patient. In a previous study we discovered by a comprehensive proteomics approach that compared to normal mucosa, protein expression of cornulin, keratin 4 and keratin 13 is decreased in tumors and severe dysplasia, preneoplastic tissue with a high risk of malignant progression. Here, we studied whether loss of expression of these proteins can predict malignant transformation of oral leukoplakia. Biopsies of 12 progressing and 36 non-progressing leukoplakia lesions were analyzed for cornulin, keratin 4 and keratin 13 expression by immunohistochemistry, and graded for dysplasia. Kaplan-Meier analysis showed that loss of expression of neither cornulin (p=0.075), keratin 4 (p=0.789) nor keratin 13 (p=0.732) was significantly associated with malignant transformation of leukoplakia lesions. However, decreased expression of these proteins was significantly associated with the presence of hyperkeratosis. Only dysplasia grading correlated significantly with malignant progression of leukoplakia (p=0.024). Despite the promising outlook that decreased cornulin, keratin 4 and keratin 13 expression in the oral mucosa is associated with a premalignant state, these markers do not predict malignant transformation of leukoplakia lesions. The most likely explanation is that the aberrant differentiation state of hyperkeratotic leukoplakia lesions already causes a decreased expression, obscuring the putative association with malignant transformation. Our results support the significance of dysplasia grading for the prediction of malignant transformation.

The 5-year-survival rate of head and neck squamous cell carcinoma (HNSCC) has been only moderately improved over the last few decades. HNSCC develops in precursor fields of genetically altered mucosal cells, typically characterized by p53 pathway disruption, that mostly do not give any clinical symptoms. Patients present therefore often with invasive carcinomas in an advanced stage. After tumor resection, part of these fields frequently stays behind unnoticed, causing secondary tumors. Identification of these precursor fields would allow screening and early detection of both primary and secondary tumors. Our aim was to identify differential proteins related to p53 dysfunction. These proteins may serve as valuable biomarkers that can predict the presence of a precursor field. We used a squamous cell model for p53 inactivation, which was analyzed by 2D-DIGE and LC-MS/MS. This approach enabled us to identify a set of 74 proteins that were differentially expressed in cells with normal versus disrupted p53 function. For six proteins the major changes in expression were verified with immunohistochemical staining. The most promising result was the identification of peroxiredoxin-1 which allowed immunohistochemical discrimination between normal epithelium and precursor field tissue with a TP53 mutation.

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.