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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Cancer. 2013 Dec 30;120(7):992–1001. doi: 10.1002/cncr.28538

Elevated intrinsic cancer stem cell population in human papillomavirus-associated head and neck squamous cell carcinoma

Manchao Zhang 1,2, Bhavna Kumar 1,2, Longzhu Piao 1,2, Xiujie Xie 1,2, Alessandra Schmitt 3, Nicole Arradaza 4, Michael Cippola 1,2, Matthew Old 1,2, Amit Agrawal 1,2, Enver Ozer 1,2, David E Schuller 1,2, Theodoros N Teknos 1,2, Quintin Pan 1,2,*
PMCID: PMC3961512  NIHMSID: NIHMS549319  PMID: 24382806

Abstract

Background

Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), in particular, oropharyngeal SCC (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies and postulated to be responsible for disease recurrence and/or progression. Since the prognosis of HPV16-positive and HPV-negative HNSCC are distinct, we determine if differences in the CSC number may account for this clinical observation.

Methods

CSC population in HPV16-positive and HPV-negative HNSCC was assessed using the ALDEFLUOR assay, in vitro tumorsphere formation assay, and in vivo limiting cell dilution in NOD/SCID mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC.

Results

HPV16-positive HNSCC had a higher intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 is a major mechanism for elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using HPV16-positive and HPV-negative OPSCC patient tumors indicated that the CSC frequency is 62.5-fold higher in the HPV16-positive OPSCC tumor than in the HPV-negative OPSCC tumor. Primary tumors from HPV16-positive OPSCC patients were associated with elevated tumor ALDH1 staining further extending the association between HPV16 and CSC.

Conclusions

Our data and the clinical observation that HPV16-positive HNSCC patients respond more favorably than HPV-negative HNSCC patients to current treatment paradigms support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression.

Keywords: cancer initiating cells, cancer stem cells, head and neck cancer, human papillomavirus, ALDH1, prognostic biomarker

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with an annual incidence of approximately 600,000 cases worldwide 1. Alcohol and tobacco use are the hallmark etiological factors for HNSCC. However, the pathogenesis of HNSCC is changing due to the recognition of human papillomavirus (HPV) as a major risk factor for the development of HNSCC, in particular oropharyngeal squamous cell carcinoma (OPSCC). High-risk HPV16 is by far the most frequent HPV type detected in HNSCC 2. Epidemiological data indicate that the prevalence of HPV-associated HNSCC has rapidly increased by about 3-fold in the past three decades in the United States and Europe 3–5. In light of these observations, it has been suggested that an epidemic of HPV-positive HNSCC will emerge in the near future 5, 6.

Cancer stem cells (CSCs) are a small sub-set of cancer cells within the tumor with the exclusive capacity to divide and expand the CSC pool or to differentiate into heterogeneous non-tumorigenic cells that constitute the bulk of the tumor. There is emerging evidence that CSCs are refractory to chemotherapy and radiation suggesting that CSCs may be responsible for disease relapse and progression 7–11. To date, the effect of HPV16 infection on the CSC population is unknown. Our results demonstrate that HPV16-positive OPSCC have a higher intrinsic CSC population than HPV-negative HNSCC. Elevated CSC number in HPV16-positive OPSCC is partly due to E6-mediated p53 inactivation. In a large cohort of OPSCC patients, HPV16-positive OPSCC is associated with increased ALDH1 staining in the tumor and stromal cells. The clinical observation that HPV-positive OPSCC patients have better prognosis than HPV-negative OPSCC provides initial evidence that CSC phenotype may be more critical than CSC number as a determinant of patient outcome. Our work suggests that CSC phenotype is not static and challenges the current dogma of homogeneity in the CSC population.

Materials and Methods

Cell lines

SCC15 and CAL27 were purchased from ATCC (Manasses, VA). UMSCC-47 and UMSCC-74A were obtained from Dr. Thomas Carey at the University of Michigan. UD-SCC2 was provided by Dr. Henning Bier at Heinrich-Heine University, Dusseldorf, Germany. UPCI:SCC090 was provided by Dr. Susanne Gollin at the University of Pittsburgh. SCC15 cells were grown in a 1:1 mixture of Ham’s F-12 and DMEM supplemented with 0.4 mg/mL hydrocortisone, 10% FBS, 2 mM L-glutamine, 100 mg/mL streptomycin and 100 U/mL penicillin. UD-SCC2, CAL27, UMSCC-47, UMSCC-74A, and UPCI:SCC090 cells were grown in DMEM containing 10% FBS, 2 mM glutamine, 100 mg/mL streptomycin and 100 U/mL penicillin. Cell lines were authenticated using STR profile analysis.

HPV-negative and HPV16-positive HNSCC patient tumors

Fresh previously untreated HPV-negative (n=3) and HPV16-positive (n=3) OPSCC tumors were collected with a protocol approved from the Institutional Review Board at The Ohio State University. Informed consent was obtained from all patients included in the study. Tumors were digested in collagenase/hyaluronidase (Stem Cell Technologies) for 6–8 hours with gentle agitation at 30°C, followed by brief trypsinization, and filtered through a 40 μM cell strainer. Single cell suspensions were depleted for fibroblasts (Anti-Fibroblast microbeads, Miltenyi Biotec Inc., Auburn, CA), CD45 cells (Anti-CD45 microbeads, Miltenyi Biotec Inc.), and Lineage cells (Lineage cell depletion kit, Miltenyi Biotec Inc.) to enrich for tumor cells. Enriched HNSCC patient tumor cells were stained with propidium iodide and sorted by fluorescence activated cell sorting to eliminate dead cells. Subsequently, purified HNSCC patient tumor cells were assessed for CSCs using the ALDEFLUOR and tumorsphere formation efficiency assays.

ALDEFLUOR assay

Enriched HNSCC patient tumor cells and HNSCC cell lines were incubated in buffer containing ALDH substrate (BAAA) in the presence or absence of DEAB, a specific ALDH inhibitor, to serve as the negative control. The sorting gate for ALDHhigh CSCs was established using the negative control as the baseline. Fluorescence activated cell sorting (FACS) analyses were performed using BD FACS Calibur (BD Biosciences Corporation, Franklin Lakes, NJ) at The Ohio State University Comprehensive Cancer Center Analytical Cytometry Core.

Tumorsphere formation assay

Enriched HNSCC patient tumor cells and HNSCC cell lines were seeded on low-attachment plate in a defined, serum-free culture medium 12 at a density of 1000 cells/well. Tumorspheres were allowed to grow for 7 days. Tumorsphere formation efficiency was calculated as the number of tumorspheres formed divided by the original number of cells seeded.

Hetero-transplantation OPSCC NOD/SCID lines

A fresh previously untreated HPV-negative (n=1) and HPV16-positive (n=1) OPSCC tumor were collected with a protocol approved from the Institutional Review Board at The Ohio State University. Informed consent was obtained from all patients included in the study. All mouse procedures were approved by the Animal Care and Use Committee at The Ohio State University. The HPV-negative and HPV16-positive HNSCC hetero-transplantation lines were established by subcutaneous implantation of solid tissue fragments in 6- to 8-week old NOD/SCID mice (National Cancer Institute, Bethesda, MD). HNSCC tumors were minced into small fragments and implanted subcutaneously on the dorsal flank of NOD/SCID mice. Recipient NOD/SCID mice were anesthetized with ketamine-xylazine mixture prior to tumor implantation. Once tumors reached a volume of around 500 mm3, tumors were resected and serially passaged in naïve NOD/SCID mice using the same protocol as described above.

In vivo limiting cell dilution assay

HPV-negative and HPV16-positive tumors were resected from NOD/SCID mice and digested in collagenase/hyaluronidase (Stem Cell Technologies) for 6–8 hours with gentle agitation at 30°C, followed by brief trypsinization, and filtered through a 40 μM cell strainer. Single cell suspensions were depleted for mouse cells (Anti-H2Kd, BD Pharmingen, San Diego, CA), fibroblasts (Anti-Fibroblast microbeads, Miltenyi Biotec Inc., Auburn, CA), CD45 cells (Anti-CD45 microbeads, Miltenyi Biotec Inc.), and Lineage cells (Lineage cell depletion kit, Miltenyi Biotec Inc.) to enrich for human tumor cells. Purified HNSCC patient tumor cells were stained with propidium iodide and sorted by fluorescence activated cell sorting to eliminate dead cells. The indicated number of tumor cells was suspended in DMEM (50:50 Matrigel) and implanted subcutaneously in the flanks of 6-week old NOD/SCID. Tumor incidence was observed over a 60 day period. CSC frequency was calculated using the L-Calc program (Stem Cell Technologies).

Immunohistochemistry

A tissue microarray consisting of 236 OPSCC tumors from patients seen at The Ohio State University Medical Center was used for this study. All patients were treated surgically (primary tumor resection and neck dissection) followed by adjuvant radiation and/or chemotherapy. The Ohio State University Institutional Review Board approved this retrospective study. Tissue microarrays slides were deparaffinized and hydrated. Antigen retrieval was done using citrate buffer for 20 min in a decloacking chamber. Endogenous peroxidase and alkaline phosphatase were blocked by incubation with Dual Endogenous Enzyme Block (Dako) for 10 min. Vetastain Elite ABC Kit (Vector Laboratories) was used to perform ALDH1 immunostaining (BD Biosciences; 1:100). ALDH1 staining intensity was visualized using AEC. HPV16 status was determined using in situ hybridization (GenPoint HPV DNA Probe; Dako). ALDH1 levels in the tumor and stroma were scored by a board-certified pathologist. Each core was assigned a quick score by multiplying the stain proportion and intensity. Due to multiple sample cores per patient, quick scores were averaged across all the cores for each patient to create one overall measurement. To define negative or positive expression of ALDH1, the mean quick scores were divided into quartiles. Mean quick scores at or below the second quartile were classified as negative. Mean quick scores greater than the second quartile were classified as positive. Relationships between ALDH1 and HPV16 or p16 status for the OPSCC patients were assessed using χ2 tests. P<0.05 were considered statistically significant. Statistical analyses were performed using SAS version 9.2 (SAS Institute). Overall survival curves were calculated using the product-limit estimate (Kaplan-Meier method).

Results

CSC population is elevated in HPV16-associated HNSCC

Numerous studies have validated the use of the ALDEFLUOR assay as a marker to identify the CSCs from primary tumors or established cancer cell lines in various solid tumors, including HNSCC 13–17. As shown in Figure 1a, HPV16-positive OPSCC patients showed a 2.4-fold (P=0.04) increase in ALDHhigh cells compared to HPV-negative OPSCC patients. The mean ALDHhigh cell population was 1.2% for three HPV-negative OPSCC patients and 2.8% for three HPV16-positive OPSCC patients. Tumorsphere forming ability under non-adherent culture conditions has been widely used as a functional in vitro assay to assess normal stem cells and CSCs 18–20. Recent work from our group demonstrated that a single tumorsphere derived from a HNSCC cell line is sufficient to generate a bulky tumor in a NOD/SCID mouse 12. This observation shows that tumorspheres are indeed enriched for functional CSCs with in vivo cancer initiating potential. Similar to the ALDEFLUOR data, tumorsphere formation efficiency was 2-fold (P=0.04) higher for tumor cells enriched from HPV16-positive OPSCC tumors than from HPV-negative OPSCC tumors (Figure 1b). Next, we determined the CSC population in three HPV-negative and three HPV16-positive HNSCC cell lines. HPV16-positive HNSCC cell lines had a 2.9-fold (P=0.001) increase in ALDHhigh cells than HPV-negative HNSCC cell lines (Figure 1c). The percentage of ALDHhigh CSCs in HPV-negative HNSCC cell lines was 6.2% for SCC15, 8.1% for CAL27, and 3.2% for UMSCC-74A. In contrast, the percentage of ALDHhigh CSCs in HPV16-positive HNSCC cell lines was 19.1% for UD-SCC2, 18.3% for UMSCC-47, and 17.2% for UPCI:SCC090. SCC15 and UMSCC-74A had tumorsphere formation efficiencies of 2.3% and 1.0% whereas UMSCC-47 and UPCI:SCC090 had tumorsphere formation efficiencies of 4.7% and 3.9% (Figure 1d).

Figure 1. CSC population is elevated in HPV16-associated HNSCC.

Figure 1

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(a and b) CSCs in HPV-negative and HPV16-positive OPSCC patient tumors. ALDHhigh cells were assessed using the ALDEFLUOR assay. Tumorsphere formation efficiency (TFE) was calculated as the number of tumorspheres formed divided by the initial number of cells seeded. Data is presented as mean ± SEM. *P<0.05, HPV-negative vs. HPV16-positive OPSCC patients. (c and d) CSCs in HPV-negative and HPV-positive HNSCC cell lines. CSCs were quantitated using the ALDEFLUOR and tumorsphere formation efficiency assay as described above. *P<0.01 HPV-negative vs. HPV16-positive HNSCC cell lines. (e) Establishment of hetero-transplantation HPV-negative and HPV-positive OPSCC NOD/SCID lines. Standard immunohistochemistry was used to detect EGFR and p16 in the hetero-transplanted HPV-negative and HPV16-positive OPSCC tumors. (f) in vivo CSC frequency in HPV-negative and HPV16-positive HNSCC patient tumor. The indicated number of tumor cells was implanted subcutaneously in the flanks of 6-week old NOD/SCID. Tumor incidence (palpable tumor of any size) was observed over a 60 day period. CSC frequency was calculated using the L-Calc program (Stem Cell Technologies).

To further explore the association between HPV16 and CSC, we developed a HPV-negative and a HPV16-positive OPSCC hetero-transplantation NOD/SCID mouse line. Primary tumor fragments from a newly diagnosed, previously untreated HPV-negative and HPV16-positive OPSCC patient were cultivated and serially transplanted in NOD/SCID mice. Two independent groups reported a high degree of similarity in gene expression profile between the original patient tumors and their corresponding hetero-transplantation tumors 21, 22. These two studies demonstrate that the genetic composition is maintained and thus, tumors serially grown in mice recapitulate the phenotype of the original patient tumors. Immunohistochemical analyses showed that HPV-negative/Patient #1 NOD/SCID line is EGFR-positive and p16-negative while the HPV16-positive/Patient #1 NOD/SCID line is EGFR-negative and p16-positive (Figure 1e). HPV16 E6 and E7 oncogenes were detected by qPCR in the HPV16-positive/Patient #1 tumor but not in the HPV-negative/Patient #1 tumor. Both HPV-negative/Patient #1 and HPV16-positive/Patient #1 tumors were serially transplanted for 1 generation before experimental use. In vivo limiting-dilution analyses showed that the CSC frequency is 62.6-fold higher in the HPV16-positive/Patient #1 tumor (1/438) than the HPV-negative/Patient #1 tumor (1/27,403) (Figure 1f). Our in vitro and in vivo results indicate that HPV16-positive OPSCC have a higher intrinsic CSC pool than HPV-negative OPSCC.

p53 regulates the CSC pool in HPV16-positive HNSCC

HPV16 E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p53 acetylation and transcriptional activity. Previous work from our group showed that over-expression of the CH1 domain of p300 inhibits E6-p300 interaction, reactivates p53, and reduces the CSC population in HPV16-positive HNSCC cells 12. To confirm that p53 plays an essential role in regulating the CSC pool, p53 was ablated using a shRNA approach in CH1-overexpressing HPV16-positive UMSCC-47 and UPCI:SCC090 HNSCC cells (Figure 2a). Knockdown of p53, as expected, decreased the expression of p53-regulated genes, miR-34c and miR-200a (P<0.01, n=3). Using the tumorsphere formation and ALDEFLUOR assays, genetic ablation of p53 completely rescued the CSC population-defect in p53-reactivated UMSCC-47/CH1 and UPCI:SCC090/CH1 cells (Figures 2c and 2d).

Figure 2. p53 regulates the CSC pool in HPV16-positive HNSCC.

Figure 2

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CH1-overexpressing UMSCC-47 and UPCI:SCC090 HNSCC cells were transduced with shRNA-control or shRNA-p53 lentiviral particles and selected in antibiotics to generate polyclonal populations. (a) p53 levels. Cell lysates were immunoblotted with anti-p53 or anti-GAPDH antibodies. (b) p53-regulated miRs. miR-34a and miR-200c expression was determined using qPCR with validated TaqMan assays. Data was normalized to empty cells and presented as mean ± SEM. *P<0.01, n=3. (c) Tumorsphere formation efficiency. (d) ALDEFLUOR assay. Tumorsphere formation efficiency and ALDEFLUOR assay were quantitated as described in Figure 1. *P<0.01, n=3.

ALDH1 staining is associated with HPV16-positive OPSCC

To further extend the association between HPV16 and CSCs, primary tumors from surgically-treated HPV16-positive (n=124) and HPV-negative (n=97) OPSCC patients were subjected to immunohistochemical (IHC) analysis for aldehyde dehydrogenase 1 (ALDH1). ALDH1 has been used as a surrogate marker for the identification of CSC in tumor sections and extensively studied as a prognostic biomarker in solid malignancies, such as breast and lung carcinomas 14, 16, 23, 24. In Figure 3a, ALDH1 staining is shown for representative HPV-negative and HPV16-positive OPSCC tumor cores spotted on a high-density tissue microarray. HPV16-positive tumors were associated with positive ALDH1 staining in the tumor (P=0.0108) (Figure 3b). This finding validates our in vitro and in vivo results to demonstrate that HPV16-positive OPSCC has elevated intrinsic CSC population compared to HPV-negative OPSCC. p16 is a biomarker often used as a surrogate for HPV-associated HNSCC. In our OPSCC cohort, p16 is highly associated with HPV16 status (P<0.0001, χ2 test). Consistent with our in situ HPV16 dataset, p16-positive OPSCC tumors tend to have positive ALDH1 staining in the tumor cells (P=0.0033). As shown in Table 1, tumor ALDH1 staining correlated with age but was not associated with other clinicopathologic parameters in OPSCC patients. Interestingly, ALDH1 was detected in the stroma of HPV-negative and HPV16-positive OPSCC tumors. HPV16-positive or p16-positive OPSCC tumors are associated with positive ALDH1 staining in the stromal cells (P=0.0003 for HPV16 and P<0.0001 for p16). In our highly uniform OPSCC study cohort, tumor ALDH1 was not a prognostic biomarker for overall survival in HPV-negative or HPV16-positive OPSCC (Figure 4a). Moreover, tumor ALDH1 was not associated with patient outcome in p16-negative or p16-positive OPSCC (Figure 4b). We also noted that stromal ALDH1 was not associated with survival in OPSCC, regardless of HPV16 (P=0.5458) or p16 status (P=0.9251).

Figure 3. ALDH1 staining is associated with HPV16-positive OPSCC.

Figure 3

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(a) Representative HPV-negative and HPV16-positive OPSCC tumor cores. Higher magnification images of ALDH-positive cases are shown on the bottom panel. (b) ALDH1 staining is associated with HPV16-positive or p16-positive HNSCC.

Table 1.

Association of ALDH1 with clinicopathological parameters in OPSCC.

Clinicopathologic Parameters Tumor ALDH1− (n=113) Tumor ALDH1+ (n=109) P-value
Average age (SD), years 59.3 (8.9) 56.1 (9.9} 0.0118
Sex 0.8313
 Male 91 89
 Female 22 20
Lymph node status 0.9900*
 N0 19 19
 N1 20 21
 N2 69 65
 N3 5 4
T stage 0.0997
 T1 16 28
 T2 47 47
 T3 26 18
 T4 24 16
TNM stage 0.6978
 I 5 6
 II 10 7
 III 20 25
 IV 78 71
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*

Fisher’s Exact Test

Figure 4. ALDH1 is not prognostic biomarker in a surgical OPSCC cohort.

Figure 4

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(a) HPV-negative and HPV16-positive OPSCC. (b) p16-negative and p16-positive OPSCC. Overall survival curves were calculated using the product-limit estimate (Kaplan-Meier method).

Discussion

The current dogma in the CSC field is that CSCs are homogeneous and resistant to conventional therapy. Furthermore, there is emerging evidence to indicate that CSCs are responsible for disease relapse and progression 10. Based on these observations, we hypothesized that HPV16-positive OPSCC has better clinical outcome possibly due to the fact that HPV16-positive OPSCC has a lower number of CSCs than HPV-negative OPSCC. Contradictory to our initial hypothesis, our results showed that HPV16-positive OPSCC has elevated intrinsic CSC pool compared to HPV-negative OPSCC. These results are surprising and counterintuitive suggesting that CSC phenotype may be more important than absolute CSC number as a marker of aggressive disease. Inactivation of p53 was shown to be an essential mechanism to enhance the CSC population in HPV16-positive OPSCC. This finding is consistent with published reports linking p53 to stem cell expansion and/or self-renewal. Blocking p53 dramatically enhances the efficiency of transforming differentiated cells into induced pluripotent stem cells 25–27. In addition, loss of p53 expands the stem cell population by favoring symmetric cell division of mammary stem cells 28.

There is scant literature on the clinical utility of ALDH1 as a prognostic biomarker in HNSCC. A recent study compared the use of standard IHC to quantum dot-based immunohistofluorescence (QD-IHF) to assess ALDH1 levels in surgically-treated HNSCC patients (n=96) 29. A significant correlation between IHC and QD-IHF for ALDH1 staining intensity was demonstrated. HNSCC patients with high ALDH1 as determined by QD-IHF had inferior 5-year overall survival compared to HNSCC patients with low ALDH1. However, this survival association was not observed using IHC to quantify ALDH1 intensity. It was suggested that QD-IHF is a more sensitive and less subjective method than IHC to quantitate ALDH1 levels 29. Another group reported that high ALDH1 staining using IHC was associated with superior local progression-free survival in recurrent/inoperable HNSCC patients (n=68) treated with platinum-based chemoradiation (CRT) 30. Several key experimental differences may explain the discrepant results between these two studies; use of different ALDH1 antibodies and different HNSCC patient populations (surgical resection in previously untreated patients vs. CRT in recurrent/inoperable patients). A limitation of the recurrent/inoperable study is that the patients were not treated uniformly since a subset (n=15) received cetuximab and CRT. A caveat in both studies is the inclusion of HNSCC patients from various sub-sites, including OPSCC, and thus, disease sub-site and HPV status were not controlled.

Our study cohort was homogenous and consists of newly diagnosed OPSCC patients treated uniformly with surgical intervention (primary tumor resection and neck dissection) and adjuvant radiation and/or chemotherapy. We used a different ALDH1 antibody than the other two HNSCC studies. The ALDH1 antibody utilized in our study was validated for specificity by immunoblot analysis and widely used by other research groups to assess ALDH1 as a prognostic biomarker 14, 24, 31–33. Also, HPV16 and p16 status were assessed in our entire study population using in situ hydridization and IHC, respectively. In our uniform OPSCC cohort, tumor ALDH1 was not a prognostic biomarker for overall survival in HPV-negative or HPV16-positive OPSCC. It should be noted that ALDH1 was detected in the stroma of HPV-negative and HPV16-positive OPSCC tumors. Other studies have reported ALDH1 staining in the stromal cells of breast, lung, and salivary carcinomas 33–35. It has been suggested that the ALDH1-positive stromal cells may be the tumor cells that have undergone epithelial-to-mesenchymal transition to possess CSC characteristics 35. Interestingly, tumor ALDH1 staining was associated with stromal ALDH1 staining (P=0.0225) in our study suggesting that the interaction between the ALDH1-positive stromal and epithelial cells may provide an optimal microenvironment to favor the accumulation and/or expansion of the CSC pool. This notion is supported by a report demonstrating that ALDHhigh, but not ALDHlow, mesenchymal stem cells modulated CSC self-renewal resulting in an expansion of the CSC pool in breast carcinoma 36.

Several possibilities can be put forth to explain our ALDH1 outcomes dataset. ALDH1 IHC may not fully discriminate a CSC from a non-CSC in formalin-fixed paraffin-embedded (FFPE) tumor cores. CSCs are posited to be rare and constitute only a minor population in the bulk tumor. Surprisingly, we observed that select HPV-negative and HPV16-positive OPSCC tumor cores have ALDH1 staining in >50% of the tumor cells. This finding is in line with other reports showing that ALDH1 staining is detected in a majority of tumor cells in select primary tumor cores from lung and head and neck carcinoma patients 30, 33. In direct support of this notion, ALDH1 as a single biomarker was not associated with clinical outcome whereas a multi-marker co-expression combination of ALDH1, CD44, and cytokeratin was shown to be an independent poor prognostic biomarker in a breast carcinoma cohort 31. To our knowledge, the use of multi-markers to selectively identify CSCs in FFPE HNSCC, including OPSCC, tumors has not been reported. An alternate explanation for the high percentage of ALDH1-positive tumor cells is that each tumor core covers a limited area of a primary tumor specimen and therefore, is it possible that the select cores with >50% ALDH1-positive tumor cells contain a discrete CSC niche with a high proportion of CSCs by happenstance.

Similar to the seminal work in breast carcinoma, several studies reported that HNSCC cells with CSC-like properties are resistant to conventional chemotherapy in vitro 17, 37–39. However, there is no direct clinical evidence demonstrating that HNSCC, including OPSCC, CSCs are refractory to chemotherapeutics. Therefore, it is of high importance to determine if the proportion of CSCs is increased between pre-treatment and post-treatment biopsies of CRT-treated HPV-negative and HPV16-positive OPSCC patients. This information will provide the key evidence to ascertain whether OPSCC CSCs are intrinsically resistant to conventional modalities. Another intriguing question is whether ALDH1 is a prognostic biomarker in first-line CRT-treated OPSCC patients. If CSCs are universally resistant to chemotherapy and/or radiation as believed, then in a CRT-treated OPSCC cohort, ALDH1-positive patients would be expected to have poorer outcome than ALDH1-negative patients. In a retrospective study from a randomized clinical trial, HPV16-positive HNSCC patients were reported to have better progression-free and overall survival than HPV-negative HNSCC patients in response to CRT 40. This clinical observation and our data showing that HPV16-positive OPSCC has higher CSC frequency than HPV-negative OPSCC seemingly contradict the dogma that CSCs are universally refractory to chemotherapy and radiation. A provocative hypothesis is that HPV16-positive CSCs may be phenotypically distinct from HPV-negative CSCs and perhaps more responsive to CRT. An extension to our corollary is that CSC phenotype may be more critical than absolute CSC number as a predictive biomarker for disease recurrence and/or progression. Further work to understand the phenotypic differences between HPV16-positive and HPV-negative CSCs in OPSCC should be prioritized.

In conclusion, HPV16-positive OPSCC was shown to have a higher intrinsic CSC pool than HPV-negative OPSCC. This intriguing finding and the fact that HPV16-positive OPSCC patients respond more favorably than HPV-negative OPSCC patients to current treatment paradigms support the suggestion that CSC phenotype is not homogeneous.

Acknowledgments

Work was supported in part by National Institutes of Health grant R01CA135096; Mary E. and John W. Alford Cancer Research Endowment Fund; The Michelle Theado Memorial Grant from the Joan Bisesi Fund for Head and Neck Oncology Research; and Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center.

Footnotes

Conflict of Interest: The authors declare no conflict of interest.

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