Serum samples of patients with head and neck squamous cell carcinoma (HNSCC) contained lower levels of IFN-γ (mean level, 6.08 pg/mL) compared with those of controls (mean level, 26.20 pg/mL) (P = .004). Error bars indicate standard error.
Serum samples of patients with head and neck squamous cell carcinoma (HNSCC) contained lower levels of IL-13 (mean level, 2.85 pg/mL) compared with those of controls (mean level, 7.23 pg/mL) (P = .02). Error bars indicate standard error.
Serum samples of patients with head and neck squamous cell carcinoma (HNSCC) contained lower levels of MIP-1β (mean level, 14.91 pg/mL) compared with those of controls (mean level, 28.98 pg/mL) (P = .004). Error bars indicate standard error.
Serum samples of patients with head and neck squamous cell carcinoma (HNSCC) contained elevated levels of IP-10 (mean level, 359.24 pg/mL) compared with those of controls (mean level, 216.40 pg/mL) (P = .04). Error bars indicate standard error.
Kaskas NM, Moore-Medlin T, McClure GB, Ekshyyan O, Vanchiere JA, Nathan CO. Serum Biomarkers in Head and Neck Squamous Cell Cancer. JAMA Otolaryngol Head Neck Surg. 2014;140(1):5-11. doi:10.1001/jamaoto.2013.5688
Serum biomarkers may be useful in the evaluation of suspected head and neck squamous cell cancer (HNSCC) and as indicators of treatment success or failure in adjuvant and chemopreventive clinical trials.
To determine serum cytokine and chemokine concentrations altered in patients with HNSCC compared with healthy volunteers to identify potential biomarkers.
Design, Setting, and Participants
A retrospective experimental laboratory study at Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport. Serum samples from 50 patients with stages II, III, and IV HNSCC and 20 healthy volunteers were available for study. Primary tumor sites represented in the patient group included the nasal cavity, oral cavity, oropharynx, hypopharynx, and larynx.
Following institutional review approval and written informed consent, blood samples were drawn from patients. No intervention, to include any kind of diagnostic workup or treatment, was provided to patients during the course of this study.
Main Outcomes and Measures
The main outcome measures were the quantification of cytokine and chemokine concentrations in serum samples. Luminex multiplex panel technology was used for simultaneous measurement of 18 analytes, including fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, growth-related oncogene, interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, inducible protein (IP)-10, soluble CD40 ligand, tumor necrosis factor, and vascular endothelial growth factor.
The serum samples of patients with HNSCC contained lower levels of IFN-γ (mean patient serum level, 6.08 pg/mL, compared with the mean control level, 26.20 pg/mL; P = .004), IL-13 (mean patient serum level, 2.85 pg/mL, compared with the control mean level, 7.23 pg/mL; P = .02), and macrophage inflammatory protein-1β (MIP-1β) (mean patient serum level, 14.91 pg/mL, compared with the mean control level, 28.98 pg/mL; P = .004), and elevated levels of IP-10 (mean patient serum level, 359.24 pg/mL, compared with mean control level, 216.40 pg/mL; P = .04). All other markers tested were not significantly different between patients with cancer and controls.
Conclusions and Relevance
This pilot study demonstrated a significant decrease in serum IFN-γ, IL-13, and MIP-1β levels and a significant elevation of serum IP-10 concentration in patients with HNSCC, irrespective of primary tumor site. If validated in larger, independent studies, these serum biomarkers may be useful in the diagnosis and treatment of HNSCC. In the future, a defined, multianalyte screening panel could facilitate early diagnosis of HNSCC, allowing for earlier treatment and thereby reducing patient mortality.
Head and neck cancer encompasses a broad range of epithelial malignant neoplasms, which include neoplasms of the oral cavity, nasal cavity, paranasal sinuses, pharynx, larynx, thyroid, and parathyroid. Over 90% of head and neck cancers arise from squamous epithelium.1 Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, affecting 600 000 new patients and causing over 300 000 deaths annually.2,3 HNSCC is a considerable public health problem in the United States, with over 50 000 cases and over 11 000 deaths per year.1 Furthermore, investigators have noted that there has been a notable increase in the incidence of squamous cell carcinoma (SCC) of the oral tongue, base of tongue, and tonsil in the past decade, particularly in young adults (<45 years).4 While the increase has been associated with rising human papillomavirus (HPV) infection rates, the dramatic rise is a cause for concern because oral tongue SCC has increased 5- and 6-fold among young men and women, respectively.4
Most patients with HNSCC have metastatic disease at the time of diagnosis: 43% present with regional lymph node metastasis, and 10% present with distant metastasis.1 This is a compelling concern because early diagnosis is imperative for a reduction in mortality. While early disease may be treated with single modality therapy, such as surgery or radiation, patients with advanced disease have a worse prognosis despite extensive cytotoxic treatment regimens involving surgery, radiation, chemotherapy, or a combination of all 3.3 For patients with locally advanced disease at the time of diagnosis, the 5-year survival rate is less than 40%.1 Advances in surgery, chemotherapy, and radiotherapy have not altered the survival rates of patients with HNSCC over the past few decades.5 HNSCC is associated with exposure to several known mutagens, including alcohol, smoking, high-risk HPV subtypes, and betel quid (particularly in Southeast Asia).3,6,7 Research aimed at developing targeted translational therapeutics has renewed an interest in further elucidating the multifactorial etiology of HNSCC.2,3
Cytokines, chemokines, and growth factors are cell-signaling proteins, which have been implicated in the physiologic and pathophysiologic mechanisms of immunity, inflammation, and hematopoiesis of tumorigenesis. Previous studies have examined plasma, serum, and tissue samples of patients with head and neck cancer to attempt to detect a biomarker of clinical significance.8,9 Identifying an aberrant cytokine profile that is consistent among patients with HNSCC may have utility in diagnosis through inclusion in a cancer screening panel. However, identifying informative tissue biomarkers has been challenging owing to the heterogeneous nature of HNSCC, with tumor sites arising from a variety of different locations in the upper aerodigestive tract, each with their own distinct histologic characteristics and lymphatic drainage.2 Identification of a serum biomarker may therefore have greater diagnostic value than a tissue marker. We used the multiplex magnetic bead assays to compare a large number of analytes in serum samples of a heterogeneous group of patients with HNSCC with those of healthy volunteers. We hypothesized that irrespective of tumor site there would be specific analytes that were elevated among patients with HNSCC compared with the healthy volunteers. Such biomarkers may provide information about the pathophysiologic mechanisms of HNSCC, facilitate early diagnosis, and serve as a marker of response for novel therapeutic strategies.
The study was approved by the institutional review board, and written informed consent was obtained in all cases.
Serum samples were obtained retrospectively from 50 patients diagnosed as having stage II, III, or IV HNSCC (Table 1). Staging was assigned based on the American Joint Committee on Cancer protocol for head and neck carcinoma, which categorizes stage II as T2/N0/M0, stage III as T3N0M0/T1N1M0/T2N1M0/T3N1M0, and stage IV (IVA-IVC) as up to any T/any N/M13 (Table 2). Most serum samples (45) were obtained from patients at initial presentation, with a minority (5) obtained following treatment (Table 3). Serum samples were obtained from 20 healthy volunteers with no known malignant neoplasms.
Luminex cytokine bead array technology was utilized for this study using a EMD Millipore HCTYOMAG-60K kit on a BioPlex analyzer (BioRad) according to the protocol established by Milliplex.10 Standards and serum samples were tested in duplicate. Premixed fluorescent beads specific for 18 analytes were added, including fibroblast growth factor 2 (FGF-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-related oncogene (GRO), interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1β, soluble CD40 ligand (sCD40L), tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF). Data were acquired using the Bioplex analyzer, and the contents of each well were examined using Luminex xPonent software. The data were delivered in a Microsoft Excel format.
Analyte concentrations were compared between patients with HNSCC and healthy volunteers using the InStat program for descriptive and analytic statistics. A standard unpaired 2-tailed t test was used to determine the significance of the differences between mean values for patients with HNSCC vs controls. Analysis of variance (ANOVA) tests were performed to determine if the analytes differed significantly based on the tumor stage. P < .05 was considered significant.
Sample demographics are summarized in Table 1. Serum samples from 36 male patients, 14 female patients, and 20 healthy volunteers were analyzed. Patient age ranged from 41 to 98 years with most in the 50- to 59-year-old category. Most patients (70%) were white, and 30% were African American.
The HNSCC tumor characteristics are summarized in Table 2. Primary tumor sites included nasal cavity (1 [2%]), oral cavity (7 [14%]), oropharynx (21 [42%]), hypopharynx (4 [8%]), and larynx (18 [36%]). Patients with multiple primary tumor sites had each site noted in the appropriate category. Patients who presented with base of tongue or tonsil cancer (n = 10) were tested for HPV; 8 were HPV positive. Sixty-four percent of patients (32) were classified as having stage T4 HNSCC, 76% (38) presented with lymph node involvement, and 98% (49) did not exhibit distant metastasis. Two percent and 12% of patients (1 and 6, respectively) presented with stage II and stage III HNSCC, respectively, while most patients were classified as having stage IV HNSCC: 76% (38) had stage IVA HNSCC; 8% (4), stage IVB HNSCC; and 2% (1), stage IVC HNSCC.
Detailed information regarding the 5 previously treated patients is summarized in Table 3. Previous treatment was defined to include any amount of prior radiation and/or chemotherapy. To determine if the patients receiving previous treatment presented confounding data, a standard, unpaired 2-tailed t test was analyzed for each of the significant analytes between the mean of the patients with previously treated HNSCC and those with HNSCC whose serum samples was obtained on initial presentation. The t tests confirmed that there was no significant difference between sample means of patients who received prior radiation and/or chemotherapy and patients who did not (IFN-γ level, P = .26; IL-13 level, P = .39; MIP-1β level, P = .61; IP-10 level, P = .63).
Cytokine and chemokine concentrations in serum samples of patients with HNSCC and in those of healthy volunteers were compared to determine if there was significant variation in the levels of FGF-2, GM-CSF, GRO, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IP-10, MCP-1, MIP-1β, sCD40L, TNF, and VEGF. The serum samples of patients with HNSCC contained lower levels of IFN-γ (patient serum mean level, 6.08 pg/mL, compared with control mean level, 26.20 pg/mL; P = .004) (Figure 1), IL-13 (patient serum mean level, 2.85 pg/mL, compared with the control mean level, 7.23 pg/mL; P = .02) (Figure 2), and MIP-1β (patient serum mean level, 14.91 pg/mL, compared with the control mean level, 28.98 pg/mL; P = .004) (Figure 3), and elevated levels of IP-10 (patient serum mean level, 359.24 pg/mL, compared with the control mean level, 216.40 pg/mL; P = .04) (Figure 4). All other markers tested were not significantly different between patients with cancer and control patients (data not shown). The ANOVA test demonstrated that cytokine levels did not differ significantly among patients with stage 2 HNSCC (n = 1), those with stage 3 HNSCC (n = 6), and those with stage 4 HNSCC (n = 43) (IFN-γ, P = .89; IL-13, P = .08; MIP-1β, P = .79; IP-10, P = .16) in our sample population. In our limited sample, the means of controls without a history of tobacco use (n = 13) did not differ significantly from the means of controls with a history of tobacco use (n = 7) based on standard unpaired 2-tailed t tests (MIP-1β, P = .27; IP-10, P = .25; IFN-γ, P = .16; IL-13, P = .83). In addition, the means of patients with HNSCC without a history of tobacco use (n = 5) did not differ significantly from the means of controls with a history of tobacco use (n = 45) based on standard unpaired 2-tailed t tests (MIP-1β, P = .41; IP-10, P = .51; IFN-γ, P = .30; IL-13, P = .48).
In terms of our patient demographics, χ2 test results show no statistically significant difference in terms of sex between patients and controls (Table 1). While age differed between patient and control groups, Kim et al11 have recently demonstrated that serum levels of IFN-γ, MIP-1β, and IP-10 do not differ significantly between healthy young adults and elderly individuals. Other studies have shown that serum levels of IL-13 do not vary significantly with increasing age.12 While our groups differed racially because we included 3 Asian patients in our control group, studies have demonstrated no significant difference in serum levels of IFN-γ or IP-10 between Asian and North American individuals.13,14 To our knowledge, no study has demonstrated significant differences in serum IL-13 or MIP-1β level among different racial groups. In regard to the 5 patients who received prior radiation and/or chemotherapy, analysis determined that the means of biomarkers in the serum samples of previously treated patients with HNSCC did not differ significantly from the means of biomarkers in serum samples obtained from patients at initial presentation in our sample population. We speculate that this could possibly be due to failure of combination chemotherapy and radiation to treat the tumor (in 4 of 5 of previously treated patients) or occurrence of a new primary tumor (in 1 of 5 of previously treated patients).
This pilot study demonstrated that patients with HNSCC exhibited significantly lower levels of IFN-γ (P = .004) than the controls. Lathers and Young8 correlated this finding, noting marked depressed levels of the TH1 cytokine IFN-γ in the plasma of 101 patients with HNSCC (P < .001) using enzyme-linked immunosorbent assay (ELISA). In addition, Malaspina et al9 determined decreased levels of IFN-γ in the gingival tissue of 11 patients with oral SCC using ELISA on supernatant of lesion samples.
Interferon-γ is a proinflammatory cytokine involved in both innate and adaptive immunity that plays an integral role in tumor surveillance. The mechanism of the downregulation of IFN-γ in HNSCC may be linked to the increased expression of Programmed Death-1 (PD-1) and Programmed Death-1 Ligand 1 (PD-L1).15 The interaction of PD-1 with PD-L1 leads to the inhibition of T-cell proliferation and downregulation of IFN-γ.9,15 PD-1:PD-L1 interaction may be a mechanism of tumor escape because initiation of antitumor response is observed on PD-1 blockade in animal studies.15 Malaspina et al9 recently demonstrated a significant increase in the PD-1 expression of oral SCC lesions, which may explain the decreased levels of IFN-γ in patients with HNSCC. While IFN-γ treatment has previously been found to inhibit the proliferation of murine renal carcinoma and human prostate carcinoma, a new study published by Liu et al16 demonstrated IFN-γ inhibited the growth of nasopharyngeal carcinoma.17,18 Therefore, IFN-γ may prove to be a promising therapeutic agent in the treatment of SCC in other sites of the upper aerodigestive tract.
Our data demonstrated a significantly decreased level of IL-13 (P = .02) among patients with HNSCC compared with healthy controls. The literature has indicated a pleiotropic role of IL-13 in disease and tumor evasion. While IL-13 has been implicated as the key effector in ulcerative colitis and asthma,19,20 it has also been previously shown to be a potent inhibitor of renal cell carcinoma and breast carcinoma.21,22 Interleukin-13 is produced primarily by activated TH2 cells and is involved in B-cell maturation and differentiation.
Interleukin-13 is known as one of the primary inducers of alternatively activated macrophages, which have been demonstrated to have substantial anti-inflammatory and tissue repair capacities23- 25 In contrast to classically activated macrophages, which are considered the M1 phenotype, alternatively activated macrophages are referred to as the M2 phenotype.24,25 Recent studies in breast and colon cancer have observed that the origin of the carcinoma dictates differential skewing to the M1 or M2 phenotype.26 Pettersen et al27 recently demonstrated through gene set enrichment analysis of cutaneous SCC that the M1 macrophage gene sets were significantly upregulated in the SCC genomic phenotype, whereas the M2 macrophage gene set was not. Furthermore, the study revealed that normal skin at steady state exhibited predominant alternatively activated macrophage expression.27 Therefore, IL-13 may be downregulated by a diversion of tumor microenvironment resources toward the M1 phenotype. Tumor microenvironment suppression of IL-13 may be a key mechanism of immune evasion: a new study found that IL-13 favorably modified atherosclerotic lesions by increasing collagen content and decreasing vascular cell adhesion molecule-1 (VCAM-1)-dependent monocyte recruitment through the induction of alternately activated macrophages.23 Downregulation of IL-13 may allow the carcinogenesis process to evade the protective effects of alternately activated macrophages.
We observed significantly decreased levels of MIP-1β, also known as CCL4 (P = .004). MIP-1β is a chemokine produced by macrophages, dendritic cells, and lymphocytes. Intratumoral expression of MIP-1β has been shown to induce antitumor responses through the chemoattraction of T and NK cells in colorectal adenocarcinoma cells.28 Furthermore, injection of MIP-1β into these tumors significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice.28 A more recent study found that mice transfected with human MIP-1β gene exhibited decreased tumorigenicity, indicating a preemptive protective effect of the chemokine.29 However, our data contradict the findings of Trellakis et al,30 who demonstrated higher concentrations of MIP-1β in the peripheral blood of patients with HNSCC. The study by Trellakis et al30 included stage I and stage II HNSCC samples, whereas we primarily included serum samples from patients with stage III and IV HNSCC.30 It may be that as carcinogenesis progresses, the antitumor activity of MIP-1β becomes overwhelmed or perhaps progressively suppressed through cross-talk with malignant cells. The downregulation of MIP-1β in advanced malignant disease contributes to the overall profound immunosuppressive state of HNSCC.
We found that HNSCC serum samples exhibited significantly elevated levels of IP-10/CXCL10 compared with those of healthy volunteers (P = .04). Inducible protein-10 is a proinflammatory pleiotropic chemokine that mediates biological activity through binding to G protein coupled receptors and glycosaminoglycans.31 Inducible protein-10 has been shown to be induced by both IFN-γ–dependent and IFN-γ–independent mechanisms.32 Previous studies have found elevated IP-10 levels in the tissue and peripheral blood of patients with nasopharyngeal carcinoma.33,34 Other studies in the literature have demonstrated increased levels of IP-10 in the oral fluid of patients with orolaryngeal cancer through ELISA.31 Furthermore, the concentration of IP-10 in the oral fluid of patients with orolaryngeal cancer returned to normal levels after therapy, suggesting a promising marker for following recurrence potential and response to therapy.31 Chemotaxis of cytotoxic cells at the tumor site in HNSCC is altered by dysfunctional interaction between IP-10 and its receptor, CXCR3.32 In addition, IP-10 has been shown to be a major mediator of mast cell proliferation of thyroid carcinoma in vitro, promoting survival and invasive ability of cancer cells.35 Chakraborty et al32 demonstrated that administering neem leaf glycoprotein downregulates CXCR3 in HNSCC, which returns IP-10 function to physiological homeostasis.
A potential limitation of this study is that we did not assess if there was an impact of medications on cytokine levels. While none of the serum samples were drawn from patients receiving chemotherapeutic or biologic agents, the population of patients with HNSCC generally could be taking medications for other conditions; a much larger sample size would have to be analyzed to determine if medications have an impact on the cytokine profile. Although this study includes a relatively small study size, to our knowledge this pilot study is the first to observe a significant decrease in serum IFN-γ, IL-13, and MIP-1β and a significant elevation of serum IP-10 concentration in patients with HNSCC, irrespective of primary tumor site. Future studies exploring the immune pathophysiologic characteristics of HNSCC carcinogenesis will be essential in understanding how these aberrant cytokines contribute to tumorigenesis.
In conclusion, our findings implicate aberrant inflammatory factor expression and suppressed immunosurveillance in the microenvironment of head and neck cancer. A future multianalyte screening panel including these cytokines may facilitate early diagnosis of HNSCC. In addition to promoting more favorable prognoses through early diagnosis, a screening panel may also be used in detecting early recurrence of initial disease or development of further primaries. Earlier treatment would be invaluable in reducing patient mortality. Assessment of the prognostic utility of serum biomarkers will require longitudinal studies of HNSCC in larger cohorts. Screening serum for biomarkers of oncologic disease is less invasive than tissue biopsy and may be more cost-effective and practical for widespread use. In addition, cytokine concentrations may parallel cancer progression and/or response to therapy and could serve as measures to assess the impact of treatment with novel therapeutics.
Submitted for Publication: May 3, 2013; final revision received July 29, 2013; accepted September 4, 2013.
Corresponding Author: Cherie-Ann O. Nathan, MD, Department of Otolaryngology–Head and Neck Surgery, Louisiana State University Health, 1501 Kings Hwy, Shreveport, LA 71130-3932 (firstname.lastname@example.org).
Published Online: November 14, 2013. doi:10.1001/jamaoto.2013.5688.
Author Contributions: Mss Kaskas and Moore-Medlin and Dr Nathan had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Kaskas, Moore-Medlin, Nathan.
Acquisition of data: Kaskas, McClure, Vanchiere.
Analysis and interpretation of data: Kaskas, Moore-Medlin, Ekshyyan, Vanchiere, Nathan.
Drafting of the manuscript: Kaskas, McClure.
Critical revision of the manuscript for important intellectual content: Kaskas, Moore-Medlin, Ekshyyan, Vanchiere, Nathan.
Statistical analysis: Kaskas, Ekshyyan.
Obtained funding: Nathan.
Administrative, technical, or material support: Moore-Medlin, McClure, Ekshyyan, Vanchiere, Nathan.
Study supervision: Vanchiere, Nathan.
Conflict of Interest Disclosures: None reported.
Funding/Support: Funding for this project was provided by the Department of Otolaryngology, Louisiana State University (LSU) Health, Shreveport, Feist-Weiller Cancer Center, and LSU Shreveport School of Medicine.
Role of the Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Previous Presentation: This study was a poster presented at the American Academy of Otolaryngology–Head and Neck Surgery Annual Spring Meeting; April 10-11, 2013; Orlando, Florida.