Prognostic Value of Circulating Tumor Cells with Podoplanin Expression in Patients with Locally Advanced or Metastatic Head and Neck Squamous Cell Carcinoma Jason Chia-Hsun Hsieh, M.D.1, 2*, Hung-Chih Lin, M.S.3*, Chi-Ya Huang, M.S.4, Hsung-Ling Hsu, B.S.4, Tyler Min-Hsien Wu, Ph.D.5, Chia-Lin, Lee, M.D.6,7, Min-Chi Chen, Ph.D.8, Hung-Ming Wang, M.D.1,9#, Ching-Ping Tseng, Ph.D.3,4,10# 1
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Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan 333, Taiwan, ROC 2 Department of Chemical and Materials Engineering, Chang Gung University, Taoyuan 333, Taiwan, ROC 3 Graduate Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC 4 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC 5 Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 333, Taiwan, ROC 6 Division of Endocrinology and Metabolism, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung 407, Taiwan, ROC 7 Department of Public Health, College of Public Health, China Medical University, Taichung 404, Taiwan, ROC 8 Department of Public Health and Biostatistics Consulting Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 9 Department of Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC 10 Molecular Medicine Research Center, Chang Gung University, Taoyuan 333, Taiwan, ROC
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*
Hsieh CH and Lin HC contribute equally to this study and are considered as co-first authors.
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#Correspondence to: Professor Ching-Ping Tseng, Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan 333, Republic of China; Tel: +886-3-2118800 ext. 5202; Fax: +886-3-2118355; E-mail: [email protected]; Professor Hung-Ming Wang, Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan 333, Taiwan, Republic of China; Tel: +886-3-2118800 ext 3599; Fax: +886-3-2118668; E-mail: [email protected]. Key words: Circulating tumor cells, podoplanin, head and neck squamous cell carcinoma, prognosis, and biomarker. Running title: Podoplanin-CTCs and HNSCC ACKNOWLEDGMENT This work was supported by the National Science Council Grants NSC 99-2632-B-182-001-MY3, 99-2628-B-182-001-MY3, 102-2628-B-182-009-MY3 and 102-2628-B-182-010-MY3, Chang Gung Memorial Hospital Grants CMRPD180423, CMRPD1B0392, CMRPD1C0551, and CMRPG3B0971~3, and Chang Gung Molecular Medicine Research Center Grants EMRPD1C0121 to C.P.T. This trial (CTCHNSCC01 trial) was also registered on www.clinicaltrials.gov (NCT01884129).
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/hed.23779
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ABSTRACT Background Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear. Methods PowerMag system was used to enumerate CTCs from 53 pre-chemotherapy HNSCC patients and 61 healthy donors. PDPN expression was determined by immunofluorescence staining. Results were correlated with clinicopathological parameters and clinical outcome such as patient survival by ROC, univariate and multivariate analyses.
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Results PDPN was expressed in a subset of CTCs. Both EpCAM+-CTCs and PDPN+-CTCs counts were statistically different between disease and non-disease groups (P20% was a significant prognostic factor for 6-months death
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(P=0.011) and was correlated with poor PFS (P=0.016) and OS (P=0.015). Conclusions PDPN+/EpCAM+-CTCs ratio is a prognostic factor and defining the ratio in
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HNSCC patients might be valuable to clinical management.
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INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is a common malignant disease worldwide. The 5-year survival rate for all stages of HNSCC is approximately 40-50% (1). In locally advanced diseases, which account for about 60% of all HNSCC settings, 20-30% of the patients eventually relapse despite aggressive protocols in the standard first-line treatment such as the combinations of curative surgery, adjuvant radiotherapy or chemoradiotherapy (CRT) and definitive concurrent chemoradiotherapy (CCRT). Even in survival after treatment, the patients are usually impaired in social, speaking and eating abilities. Early detection of
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recurrence by any examination is thereby crucial for HNSCC patient care. A number of clinical features of HNSCC patients such as TNM staging, comorbidity, sites of origin,
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treatment modality (2, 3), and immunohistochemistry staining markers (4-6) have been proposed for prediction of survivals. New imaging techniques (7) and specialized tests for immunologic responses-associated markers
(8)
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and genetic/epigenetic alteration
developed.
(9)
have also been
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Circulating tumor cells (CTCs) are an emerging “liquid biopsy” that provide prognostic value for various types of solid cancer
(10, 11)
. However, the clinical significance of CTCs in
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HNSCC is still under debate. Only few studies have been reported and limited conclusions for the prognostic value of CTCs in HNSCC are reached
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(1, 12-16)
. Moreover, it is clear from
several studies that the number of CTCs alone does not provide sufficient information for assessing
the
status
of
cancer
patients
(15)
.
Protein
expression
related
to
epithelial-mesenchymal transition (EMT) in CTCs has been shown to provide add-on values when the clinical status of cancer patients was assessed (17, 18). Podoplanin (PDPN) is a transmembrane protein that has multiple functions in lymphatic vessel formation and cellular cytoskeleton remodeling. PDPN is involved in disease progression of various cancer types
(19)
by mediating tumor cell-platelet interactions and
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promoting tumor cell survival in bloodstream. In HNSCC, PDPN has been demonstrated as a novel biomarker for cancer migration/invasion
(20)
, early lymphatic dissemination, poor
prognosis and low response to neoadjuvant chemoradiotherapy
(21, 22)
. However, whether
PDPN is expressed in CTCs from the HNSCC patients and whether PDPN+-CTCs have prognostic value have not yet been elucidated. In this study, we address these issues by analyzing the baseline CTCs from 53 locally advanced and metastatic HNSCC patients prior to chemotherapy. We report for the first time that PDPN is expressed in the CTCs from HNSCC patients. In addition, the ratio of CTCs with PDPN expression but not the absolute
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CTCs count is a prognostic factor of survival in HNSCC. The significance of these findings is discussed.
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METHODS Study Design This was a prospective single-center study approved by the Institute Review Board of Chang Gung Memorial Hospital with the approval ID of 100-4644A3, 100-4623C and 101-2161C. Locally advanced, recurrent or initially metastatic HNSCC patients that confirmed histologically or cytopathologically were enrolled in this study. Other criteria of enrollment included (1) age ≥ 20 years old; (2) patients who could understand and signed the informed consents by their own will; and (3) adequate liver and renal function and white
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blood cell counts before anti-cancer therapy. Patients with synchronous cancer or prior cancers within 5 years were excluded with exception of curative treated non-melanoma skin
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cancer and cervical carcinoma in situ which are barely recurrent within 5 years after treatment. Disease stage and management of the patients followed the institutional guidelines and the
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standard treatment protocols. Enrolled patients were classified into three distinct treatment subgroups: (1) group A: definitive CCRT for the patients with advanced disease; (2) group B:
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curative surgery followed by adjuvant CRT for the patients with pathologic features such as positive margin (none in this study), pN2, and extracapsular spread (ECS) of involved lymph
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nodes indicating early relapse according to recommendation from NCCN guidelines; and (3)
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group C: palliative chemotherapy for the patients with distant metastasis or with poor general condition for definitive CCRT. Blood samples were collected within 7 days prior to the first dose of chemotherapy. The 6-months survival after chemotherapy, progression-free survival (PFS) and overall survival (OS) were used as the endpoints for this study. Study results were reported following REMARK recommendations (23). Enrichment and isolation of CTCs Enrichment and isolation of CTCs were performed using the PowerMag system which has been published and validated as described previously
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(24)
. Briefly, PowerMag is a
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negative selection method for effective leukocyte depletion and enhanced detection of viable EpCAM positive and negative CTCs from patients with various cancer types including HNSCC and colorectal cancer (24). Immunofluorescence staining and CTCs enumeration For immunofluorescence staining, the cell filtrate after depletion of CD45+ cells was incubated with the anti-EpCAM antibody (Abcam Inc, Cambridge, England), anti-PDPN antibody and Hoechst 33342 DNA staining dye (Invitrogen Inc, Carsbad, CA) at room temperature for 1 h followed by several washes and centrifugation to remove the supernatant,.
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The use of second epithelial cell marker such as cytokeratin (CK) did not provide add-on (24)
value for CTCs identification and enumeration
. Hence, immunofluorescence staining
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using anti-pan-CK antibody was not performed in this study. The Alexa Fluor 488-conjugated donkey anti-mouse antibody and Alexa Fluor 555-conjugated goat anti-rat
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antibody (Invitrogen Inc, Carsbad, CA) were then added to the cell suspension and kept in the dark for 30 min. After removing the unbound antibody, immunofluorescent images were
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captured by fluorescent microscopy (Zeiss Axiovert 200M). CTCs were defined as the cells that were positive for Hochest 33342 and EpCAM. Repeated depletion of CD45+ cells by
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PowerMag removed almost all CD45+ cells with only few (< 5 cells/ml) remained in the cell
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filtrate. None of these cells was positive for both CD45 and EpCAM (24). Single cell real-time RT-PCR analysis
The EpCAM+ cells were picked individually under fluorescent microscopy. Single cell real-time RT-PCR was performed according to the protocol provided by Fluidigm Corporation (South San Francisco, CA, USA)
(25, 26)
. The expression of PDPN and the
internal control genes β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined using the BioMark HDTM System. Statistical analysis
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The CTCs count in healthy donors and HNSCC patients was compared using boxplot and Kruskal-Wallis test for all groups and Mann-Whitney U test between any two groups with 2-sided significance. The impacts for the baseline CTCs and PDPN+-CTCs count, the ratio for the PDPN+-CTCs count over total CTCs count (PDPN+/EpCAM+-CTCs ratio) and the factors reported to shorten or prolong survival such as age, body mass index (BMI), performance status (PS) and distant metastasis at initial presentation on PFS and OS were assessed by univariate Cox proportional hazards regression analysis. Parameters with significance in univariate analysis were subject to multivariate Cox regression analysis. PFS
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and OS were measured from the date of baseline CTC to the date of confirmed clinical progression, death, or censoring at the last follow-up. Statistical analysis was performed
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using SPSS for Windows (version 18, SPSS, Chicago, IL). A P value of 0.05 was considered statistically significant.
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RESULTS Basic characteristics of enrolled patients A total of 53 patients with locally advanced or metastatic HNSCC and 61 healthy donors were enrolled in the Division of Medical Oncology, Chang Gung Memorial Hospital at Linkou and Chang Gung University between October 2011 and August 2012. Clinical information and survival data were updated until February 2013. Basic characteristics of the enrolled patients are shown in Table 1 with further details in Supplementary Table 1. The median age was 52 with a range from 35 to 80. Primary tumor sites were mainly oral cavity
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(56.6%) followed by oropharynx (17%) and hypopharynx (17%). Forty-two (79.2%) patients had locally advanced stage IV status (including 12 patients with distant metastasis at initial
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presentation, stage IVc) according to the TNM staging system of American Joint Committee on Cancer (AJCC) 7th edition. Forty-two patients (79.2%) had performance status 0-1.
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Thirty-seven patients (69.8%) had a BMI less than 25. The number of CTCs and PDPN+-CTCs differentiates patient groups from healthy donors
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The CTCs (EpCAM+-CTCs) counts between different treatment groups (A, B, and C as
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shown in Table 1) and healthy donors were statistically different even for those who
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underwent curative surgery but had microscopic risk of relapse (Figure 1A and Supplementary Table 2). As revealed by ROC analysis, CTCs enumeration elicited a sensitivity of 96.2% and a specificity of 86.9% with the area under curve (AUC) equivalent to 0.983 when 5 CTCs/ml was used as the cutoff (Figure 1B). Positive PDPN staining was only observed in EpCAM+ cells and was barely found in EpCAM- cells. Hence, CTCs can be either PDPN positive (referred to as PDPN+-CTCs) or negative (Supplementary Figure 1). Consistent with these findings, real-time RT-PCR analysis of 87 EpCAM+ single cells obtained from 10 HNSCC patients revealed that PDPN
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was differentially expressed in each single cell with no PDPN expression in some of the cells (Supplementary Table 3). According to immunofluorescence staining, PDPN+-CTCs counts between different treatment groups (Groups A, B and C) and healthy donors were statistically different and were able to differentiate HNSCC patients from healthy individuals (Figure 1C). ROC analysis revealed that the sensitivity of the assay was 83.3% and the specificity was 81.5% with the AUC equivalent to 0.916 when 3 PDPN+-CTCs/ml was used as the cutoff (Figure 1D). The PDPN+/EpCAM+-CTCs ratio is a prognostic factor for patient survival of HNSCC
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For the whole cohort, the median PFS was 8.6 (95% CI: 4.781-12.419) months and the median OS has not reached 50% after the follow-up duration of 10.5 (6.6-18.5) months. At
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the time of analysis, 23 patients died of cancer and 26 patients experienced disease progression. Because the patients in this trial all have 6-months confirmed dead or alive
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status, we first assessed whether the CTCs and PDPN+-CTCs count, or the PDPN+/EpCAM+-CTCs ratio is able to predict patients with poor clinical outcome and
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therefore disease progression or death at 6 months after anticancer therapy. ROC analysis revealed that the AUC was 0.489, 0.596 and 0.718 for CTCs count, PDPN+-CTCs count and
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PDPN+/EpCAM+-CTCs ratio, respectively (Figure 2A-C). The PDPN+/EpCAM+-CTCs ratio
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but not the CTCs or PDPN+-CTCs counts had the merit for prediction of 6-month survival in HNSCC patients (p = 0.011). With the cutoff at 20%, the predictive sensitive and specificity was 94.7% and 51.7%, respectively (Figure 2D). Univariate Cox proportional hazards regression analysis was performed to identify factors with prognostic value in HNSCC patients. Among the conventional factors that have been reported to shorten or prolong survival, ECOG-PS, distant metastasis at initial presentation, AJCC stage at enrollment and treatment groups all had clinical impacts on PFS. The factors that had statistical impact on OS included patients’ age at enrollment, ECOG-PS,
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distant metastasis at initial presentation and treatment groups. Regarding the CTCs-related factors, neither CTCs count nor PDPN+-CTCs count had statistical impact on PFS and OS (Table 2). Notably, the survival status was highly correlated with the PDPN+/EpCAM+-CTCs ratio in HNSCC patients. The hazard ratio (HR) for OS was 6.186 (95% CI: 1.206 - 31.733, P = 0.029) and a similar trend for PFS was observed (P = 0.113). Moreover, with the cutoff at 20% for PDPN+/EpCAM+-CTCs ratio, the HR for PFS was 4.666 with a 95% CI of 1.373 to 15.856 (P = 0.016) and the HR for OS was 12.400 with a 95% CI of 1.643 to 93.570 (P = 0.015).
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Multivariate Cox regression analysis further revealed that ECOG-PS, treatment group and PDPN+/EpCAM+-CTCs ratio were independent prognostic factors to OS as well as
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ECOG-PS and the treatment group to PFS (Table 2). For the PDPN+/EpCAM+-CTCs ratio, the HR for OS was 10.969 (95% CI: 1.448 - 83.068, P = 0.018) while a similar trend for PFS
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was observed (P = 0.081). Kaplan-Meier survival curve further confirmed that CTCs and PDPN+-CTCs counts did not show any association with PFS and OS (Figure 3A-D). Notably,
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the PDPN+/EpCAM+-CTCs ratio with the cutoff at 20% clearly differentiates the patients with good vs. poor PFS (P = 0.006) and (P = 0.008) (Figure 3E- F). The conclusion is also
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valid even when the patient group with palliative treatment was excluded from the analysis (P = 0.026 for PFS and P = 0.043 for OS; data not shown)
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DISCUSSION Several important features of CTCs in locally advance or metastatic HNSCC are unveiled in the present study. PDPN, a previously defined prognostic factor for HNSCC (20-22, 27, 28)
, is confirmed to express in a subset of CTCs. The number of CTCs and PDPN+-CTCs
clearly differentiates HNSCC patients from healthy control but fails to show prognostic value. Notably, the PDPN+/EpCAM+-CTCs ratio is an independent prognostic factor regardless of treatment modality and tumor staging. With the cutoff at 20%, the PDPN+/EpCAM+-CTCs ratio clearly differentiates HNSCC patients with good prognosis (≤ 20%) from those with
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poor prognosis (> 20%). This study represents the first report to define the subset of CTCs with PDPN expression contributes mainly to poor clinical outcome in HNSCC patients.
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The clinical significance of CTCs in HNSCC has been addressed in a limited number of studies. Buglione et al. demonstrated that there is no correlation between CTCs positivity at diagnosis and survival or relapse
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(15)
. Similarly, Nichols et al. reported 4 out of 5 patients
(80%) with detectable CTCs developed recurrent and progressive diseases but PFS is not related to the number of CTCs
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(13)
. In contrary to these studies, Jatana et al. reported in a
prospective study of 48 post-operation HNSCC patients that the higher the CTCs counts, the worst the disease-free survival
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(29)
. Due to the diverse study design and relatively small
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sample size for these aforementioned studies, it remains controversial whether CTCs count is a confounding factor for poor prognosis in HNSCC patients. In the present study, we reveal that CTCs count with the cutoff at 5 cells/ml clearly differentiates HNSCC patients from healthy individuals but has no prognostic value in the prediction of clinical outcome. While CTCs are highly heterogeneous (30), it is likely that not every CTC has major roles in disease progression. Unveiling the subset(s) of CTCs with aggressive features is both clinical and translational importance. In human squamous cell carcinoma, PDPN expression is generally heterogeneous and is
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often detectable in the one-cell layer at the invasive edge of the tumors
(31)
. This type of
setting facilitates tumor cell invasion in a collective cell migration and invasion independent of EMT (31). PDPN is also involved in single cell invasion contributing to EMT-mediated cell motility (19, 20). By analysis of HNSCC cancer tissues, Kreppel et al. reported that PDPN is a prognostic factor for regional lymph node metastases
(21)
and neoadjuvant platinum-based
CRT (27), and is a prognostic factor for OS and loco-regional control (27). In the present study, we revealed by univariate and multivariate analyses that the PDPN+/EpCAM+-CTCs ratio is an independent prognostic factor for HNSCC which clearly differentiates HNSCC patients
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with good prognosis (ratio ≤ 20%) from those with poor prognosis (ratio > 20%). Kaplan-Meier survival curve further confirms the patients with PDPN+/EpCAM+-CTC ratio ≤
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20% have better survival which is still applicable in a recent update of our clinical data (data not shown). This study thereby provides conceptual advance that the ratio of a specific CTCs
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population (in this case the PDPN+-CTCs) but not the number of CTCs is important in determining the degree of malignancy. Similar concepts have been revealed in several recent
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work indicating that CTCs with stem cell marker CD133 and known EMT regulators play a role in chemo-resistance and in the blood-borne dissemination of human breast cancer,
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respectively (32).
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With the cutoff at 20%, ROC analysis reveals that the sensitivity and specificity of PDPN+/EpCAM+-CTC ratio in the prediction of patients with early disease progression or death at 6 months after chemotherapy was 94.7% and 51.7%, respectively. Despite a useful biomarker is usually expected to have high specificity and high sensitivity, tumor markers with lower specificity may still be useful in high-risk populations because the high prevalence in the population greatly increases the positive predictive value of the assay Because PDPN is known as a poor prognostic factor in HNSCC
(33)
.
(20, 21, 27, 34)
, the moderately
low specificity of PDPN+/EpCAM+-CTC probably has minimal impact on its usage as a
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prognostic marker. Alternatively, this factor could be a valuable supplement marker complementary with other clinical factors or markers. This notion is supported by a report demonstrating that the squamous cell carcinoma antigen which had sensitivity of 84% and specificity of 46% was complementary with α-fetoprotein in detecting hepatocellular carcinoma (HCC) (35) and was a valuable supplement marker for the diagnosis of HCC (36). To the best of our knowledge, this is the first tool to offer sufficient statistical power to predict early progression or death within 6-months of treatment for HNSCC patients. Despite the mean and median CTCs counts for the patients in adjuvant setting (group B)
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was in fact lower than the patients with metastatic lesions (groups C) (Supplementary Table 2), the CTC counts for the patients in groups B and C were not statistically different. It is
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likely that the patients in group B of this study mainly have microscopic risk for relapse such as pN2 or ECS. This high-risk group has been shown to have poor clinical outcome when
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compared to those not having pN2 or ECS (37). Hence, residual cancer cells could still present in the circulation even after surgical operation. It is also likely that the relatively small sample
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size (18 patients for group B vs. 20 patients for group C) could contribute to the no significant difference of CTCs counts between the two groups of patients. Increasing the case number in future trials should be able to clarify this issue.
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In conclusion, this study demonstrates that CTCs enumeration effectively differentiates HNSCC patients from healthy donors but fails to correlate with the prognosis of patients. Moreover, the PDPN+/EpCAM+-CTC ratio is a prognostic factor for 6-month survival after treatment, PFS and OS in HNSCC patients.
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carcinomas. Int J Cancer 2005;113:899-910. 20. Raica M, Cimpean AM, Ribatti D. The role of podoplanin in tumor progression and metastasis. Anticancer Res 2008;28:2997-3006. 21. Kreppel M, Krakowezki A, Kreppel B, et al. Podoplanin expression in cutaneous head and neck squamous cell carcinoma--prognostic value and clinicopathologic implications. J Surg Oncol 2013;107:376-383. 22. Vormittag L, Thurnher D, Geleff S, et al. Co-expression of Bmi-1 and podoplanin predicts overall survival in patients with squamous cell carcinoma of the head and neck
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treated with radio(chemo)therapy. Int J Radiat Oncol Biol Phys 2009;73:913-918. 23. McShane LM, Altman DG, Sauerbrei W, et al. REporting recommendations for tumour
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MARKer prognostic studies (REMARK). Br J Cancer 2005;93:387-391. 24. Lin HC, Hsu HC, Hsieh CH, et al. A negative selection system PowerMag for effective
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leukocyte depletion and enhanced detection of EpCAM positive and negative circulating tumor cells. Clin Chim Acta 2013;419:77-84.
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25. Citri A, Pang ZP, Sudhof TC, Wernig M, Malenka RC. Comprehensive qPCR profiling of gene expression in single neuronal cells. Nat Protoc 2012;7:118-127.
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26. Strati A, Markou A, Parisi C, et al. Gene expression profile of circulating tumor cells in
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breast cancer by RT-qPCR. BMC Cancer 2011;11:422.
27. Kreppel M, Drebber U, Wedemeyer I, et al. Podoplanin expression predicts prognosis in patients with oral squamous cell carcinoma treated with neoadjuvant radiochemotherapy. Oral Oncol 2011;47:873-878. 28. Yuan P, Temam S, El-Naggar A, et al. Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer 2006;107:563-569. 29. Jatana KR, Balasubramanian P, Lang JC, et al. Significance of circulating tumor cells in patients with squamous cell carcinoma of the head and neck: initial results. Arch
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Otolaryngol Head Neck Surg 2010;136:1274-1279. 30. van de Stolpe A, Pantel K, Sleijfer S, Terstappen LW, den Toonder JM. Circulating tumor cell isolation and diagnostics: toward routine clinical use. Cancer Res 2011;71:5955-5960. 31. Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell 2006;9:261-272. 32. Yu M, Bardia A, Wittner BS, et al. Circulating breast tumor cells exhibit dynamic
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changes in epithelial and mesenchymal composition. Science 2013;339:580-584. 33. King TC. Elsevier’s Integrated Pathology. Philadelphia: Mosby Inc.; 2007. p111-143.
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34. Foschini MP, Leonardi E, Eusebi LH, et al. Podoplanin and E-cadherin expression in preoperative incisional biopsies of oral squamous cell carcinoma is related to lymph
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node metastases. Int J Surg Pathol 2013;21:133-141. 35. Giannelli G, Marinosci F, Sgarra C, Lupo L, Dentico P, Antonaci S. Clinical role of
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tissue and serum levels of SCCA antigen in hepatocellular carcinoma. Int J Cancer 2005;116:579-583.
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36. Zhao YJ, Ju Q, Li GC. Tumor markers for hepatocellular carcinoma. Mol Clin Oncol
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2013;1:593-598.
37. Liao CT, Lee LY, Huang SF, et al. Outcome analysis of patients with oral cavity cancer and extracapsular spread in neck lymph nodes. Int J Radiat Oncol Biol Phys 2011;81:930-937.
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FIGURE LEGENDS Figure 1. Boxplots and ROC curves of the whole cohorts and healthy donors. Panels A and C show the boxplots for the number of CTCs and PDPN+-CTCs among groups. Panels B and D demonstrate the area under curve for CTCs and PDPN+-CTCs respectively. In Panels A and C, Kruskal-Wallis test and Mann-Whitney U test are used for statistical analyses. Groups A, B and C are defined in Table 1, whereas healthy donors are classified as Group 0.
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Figure 2. ROC curves to predict 6-month death. ROC curves with AUC values for the CTCs (panel A) and PDPN+-CTCs (panel B) count and its use in prediction of 6-month death
was
demonstrated
without
statistical
significance.
In
contrast,
the
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PDPN+/EpCAM+-CTC ratio has an acceptable AUC (0.718) with a P value of 0.011, indicating that the ratio could be successfully used to predict 6-month survival status (panel
er
C). Several cutoff values were listed in panel D of which the ratio at 20% has the sensitivity of 94.7% and specificity of 51.7% and is a better cutoff point.
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Figure 3. Kaplan-Meier survival curves reveal that the PDPN+/EpCAM+-CTCs ratio but not the CTCs or PDPN+-CTCs count has prognostic value for patient survival. The
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number of CTCs and PDPN+-CTCs failed to show statistical significance to patient survival,
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even though the Kaplan-Meier survival curves demonstrate a trend that the lower CTCs (panels A-B) and PDPN+-CTCs (panels C-D) count, the longer survival time for the patients. In contrast, the PDPN+/EpCAM+-CTCs ratio at 20% clearly differentiates the patients with good vs poor PFS (P = 0.006) and OS (P = 0.008) (panels E-F).
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Table 1. Basic Characteristics of Enrolled Patients
Age , Median (range), years
n
%
52
(35-80)
Gender, Male/Female
50/3
Follow-up duration, median (range), months
10.5 (6.6-18.5)
Tumor Site Oral Cavity
30
56.6%
Oropharynx
9
17.0%
Hypopharynx
9
17.0%
Larynx
4
7.5%
1
1.9%
T0-2
23
43.4%
T3-4
30
56.6%
N0-1
18
34.0%
35
66.0%
41
77.4%
12
22.6%
11
20.8%
42
79.2%
42
79.2%
11
20.8%
37
69.8%
13
24.5%
3
5.7%
A: Primary CCRT with/without induction chemotherapy
15
28.3%
B: Post-surgery status with high risk of recurrence, needing adjuvant CCRT
18
34.0%
C: Palliative chemotherapy only
20
37.7%
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Occult Primary Site T classification N classification
N2-3 M classification
M0
7 AJCC stage
II-III IV
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M1 th
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Eastern Cooperative Oncology Group Performance Status (ECOG-PS) 0-1 2-4
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Body mass index
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30 Treatment after Enrollment (Group A, B, C)
Abbreviations: AJCC = American Joint Committee on Cancer; CCRT = concurrent chemoradiotherapy
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Table 2. Univariate and Multivariate Analysis of Progression-free and Overall Survival Rates Progression-free survival Univariate
Multivariate
HR (95% CI)
P
Age
1.031(0.984-1.080)
0.203
ECOG PS
1.889(1.416-2.522)
0.000*
BMI category (>25)
0.405(0.154-1.063)
0.066
Oral cavity origin
1.386(0.616-3.115)
0.430
Initial M1 vs M0
3.252(1.314-8.046)
0.011*
Stage at enrollment
1.809(1.191-2.750)
0.005*
CTC (cells/ml)
0.999(0.998-1.001)
0.281
0.997(0.991-1.002)
0.251
3.599(0.739-17.532)
0.113
+
PDPN -CTC (cells/ml) +
+
+
+
PDPN /EpCAM ratio
Overall survival
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HR (95% CI)
P
rP
1.624(1.126-2.341)
ee
0.009*
rR
1.078(0.673-1.729)
7.192(0.782-66.105)
PDPN /EpCAM >20% 4.666(1.373-15.856) 0.016* Treatment Group
Univariate
0.754
0.035*
0.002*
HR (95% CI)
P
1.053(1.001-1.107)
0.047*
2.015(1.499-2.709)
0.000*
0.608(0.220-1.6836)
0.338
1.231(0.510-2.972)
0.643
4.490(1.875-10.753)
0.001*
2.155(1.341-3.463)
0.002*
0.999(0.998-1.001)
0.363
0.998(0.992-1.003)
0.413
6.186(1.206-31.733)
iew
0.029*
12.400(1.643-93.570)
0.015*
ev
0.081
Multivariate
HR (95% CI)
P
1.717(1.212-2.431)
0.002*
1.127(0.715-1.777)
0.608
10.969(1.448-83.068) 0.018*
0.001*
0.033*
A: Primary CCRT
0.088(0.020-0.391)
0.001*
0.113(0.021-0.613)
0.011*
0.054(0.007-0.410)
0.005*
0.076(0.009-0.661)
0.020*
B: Adjuvant CCRT
0.386(0.166-0.901)
0.028*
0.829(0.279-2.467)
0.736
0.217(0.078-0.605)
0.004*
0.350(0.106-1.157)
0.085
Reference
-
Reference
-
Reference
-
Reference
-
C: Palliative CT
Abbreviations: BMI = body mass index; CT = chemotherapy; ECOG = Eastern Cooperative Oncology Group; CCRT = concurrent chemoradiotherapy; HR = hazard ratio; OS = overall survival; PDPN = podoplanin; PFS = progression-free survival; PS = performance status; M = metastasis.
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Pe Figure 1 130x94mm (300 x 300 DPI)
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Figure 3 211x236mm (300 x 300 DPI)
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Supplementary Table and Figures Supplementary Figure 1.
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Supplementary Figure 1. Isolation and Characterization of CTCs from Patients with HNSCC. CTC was isolated by PowerMag system and analyzed by immunoflurescence staining using anti-EpCAM (green) and anti-PDPN (red) antibodies. CTCs were identified with two major populations, PDPN+-CTC and
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PDPN--CTC from HNSCC patients.
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Supplementary Table 1. Subgrouping by Status at Enrollment Treatment Arms
n
Group A (Primary CCRT ± induction chemotherapy for advanced status) Oral cavity cancer
15 3
Unresectable status, s/p primary CCRT ± induction chemotherapy Loco-regional recurrence after primary surgery Oropharyngeal cancer Hypopharyngeal cancer Laryngeal cancer Occult primary cancer, presenting as neck squamous cell carcinoma
2 1 4 6 1 1 18 17 1 20 10
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Group B (Post-surgery status with high risk of recurrence, needing adjuvant CCRT) Oral cavity cancer, post-surgery, with high risk for recurrence, such as ECS+ Laryngeal cancer with neck LAP, only local palliative surgery Group C (Palliative chemotherapy only) Oral cavity cancer, with distant recurrence, not suitable for another surgery Oropharyngeal cancer Recurrence with initial distant metastasis Hypopharyngeal cancer Recurrence with distant metastasis
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with initial distant metastasis Too weak to undergo standard CCRT Laryngeal cancer with recurrence, not suitable for another surgery
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Abbreviations: CCRT = concurrent chemoradiotherapy; ECS = extracapsular spread of involved lymph node; OC = oral cavity.
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Supplementary Table 2. CTC Numbers among Different Groups Treatment Arms
n
Mean ± SD Median, range 640.1 ± 1085.6
Group A (Primary CCRT ± induction 15 chemotherapy for advanced status)
P valuea
147.3 (5.2-3440.5)
Group B (Post-surgery with high risk of recurrence, needing adjuvant 18 59.9 ± 87.4 19.7 (4.0-315.3) CCRT) Group C (Palliative chemotherapy only)
20 108.0 ± 131.2 64.0 (5.0-460.8)
Group 0 (Healthy donors)
61
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Comparison between Groups Group 0 versus Group A Group 0 versus Group B Group 0 versus Group C Group A versus Group B Group A versus Group C Group B versus Group C
3.0 ± 2.8
2.5 (0.0-13.0)
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Treatment Arms by Surgery
35 336.0 ± 752.5
P value
75.0 (5.0-3440.5)
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needing primary CCRT ± induction chemotherapy or palliative chemotherapy alone)
Mean ± SD Median, range
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Group 1 (No surgery group; due to advanced disease or distant metastasis,
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Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan 333, Taiwan, ROC 2 Department of Chemical and Materials Engineering, Chang Gung University, Taoyuan 333, Taiwan, ROC 3 Graduate Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC 4 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC 5 Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 333, Taiwan, ROC 6 Division of Endocrinology and Metabolism, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung 407, Taiwan, ROC 7 Department of Public Health, College of Public Health, China Medical University, Taichung 404, Taiwan, ROC 8 Department of Public Health and Biostatistics Consulting Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 9 Department of Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC 10 Molecular Medicine Research Center, Chang Gung University, Taoyuan 333, Taiwan, ROC
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*
Hsieh CH and Lin HC contribute equally to this study and are considered as co-first authors.
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#Correspondence to: Professor Ching-Ping Tseng, Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan 333, Republic of China; Tel: +886-3-2118800 ext. 5202; Fax: +886-3-2118355; E-mail: [email protected]; Professor Hung-Ming Wang, Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan 333, Taiwan, Republic of China; Tel: +886-3-2118800 ext 3599; Fax: +886-3-2118668; E-mail: [email protected]. Key words: Circulating tumor cells, podoplanin, head and neck squamous cell carcinoma, prognosis, and biomarker. Running title: Podoplanin-CTCs and HNSCC ACKNOWLEDGMENT This work was supported by the National Science Council Grants NSC 99-2632-B-182-001-MY3, 99-2628-B-182-001-MY3, 102-2628-B-182-009-MY3 and 102-2628-B-182-010-MY3, Chang Gung Memorial Hospital Grants CMRPD180423, CMRPD1B0392, CMRPD1C0551, and CMRPG3B0971~3, and Chang Gung Molecular Medicine Research Center Grants EMRPD1C0121 to C.P.T. This trial (CTCHNSCC01 trial) was also registered on www.clinicaltrials.gov (NCT01884129).
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/hed.23779
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ABSTRACT Background Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear. Methods PowerMag system was used to enumerate CTCs from 53 pre-chemotherapy HNSCC patients and 61 healthy donors. PDPN expression was determined by immunofluorescence staining. Results were correlated with clinicopathological parameters and clinical outcome such as patient survival by ROC, univariate and multivariate analyses.
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Results PDPN was expressed in a subset of CTCs. Both EpCAM+-CTCs and PDPN+-CTCs counts were statistically different between disease and non-disease groups (P20% was a significant prognostic factor for 6-months death
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(P=0.011) and was correlated with poor PFS (P=0.016) and OS (P=0.015). Conclusions PDPN+/EpCAM+-CTCs ratio is a prognostic factor and defining the ratio in
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HNSCC patients might be valuable to clinical management.
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INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is a common malignant disease worldwide. The 5-year survival rate for all stages of HNSCC is approximately 40-50% (1). In locally advanced diseases, which account for about 60% of all HNSCC settings, 20-30% of the patients eventually relapse despite aggressive protocols in the standard first-line treatment such as the combinations of curative surgery, adjuvant radiotherapy or chemoradiotherapy (CRT) and definitive concurrent chemoradiotherapy (CCRT). Even in survival after treatment, the patients are usually impaired in social, speaking and eating abilities. Early detection of
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recurrence by any examination is thereby crucial for HNSCC patient care. A number of clinical features of HNSCC patients such as TNM staging, comorbidity, sites of origin,
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treatment modality (2, 3), and immunohistochemistry staining markers (4-6) have been proposed for prediction of survivals. New imaging techniques (7) and specialized tests for immunologic responses-associated markers
(8)
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and genetic/epigenetic alteration
developed.
(9)
have also been
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Circulating tumor cells (CTCs) are an emerging “liquid biopsy” that provide prognostic value for various types of solid cancer
(10, 11)
. However, the clinical significance of CTCs in
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HNSCC is still under debate. Only few studies have been reported and limited conclusions for the prognostic value of CTCs in HNSCC are reached
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(1, 12-16)
. Moreover, it is clear from
several studies that the number of CTCs alone does not provide sufficient information for assessing
the
status
of
cancer
patients
(15)
.
Protein
expression
related
to
epithelial-mesenchymal transition (EMT) in CTCs has been shown to provide add-on values when the clinical status of cancer patients was assessed (17, 18). Podoplanin (PDPN) is a transmembrane protein that has multiple functions in lymphatic vessel formation and cellular cytoskeleton remodeling. PDPN is involved in disease progression of various cancer types
(19)
by mediating tumor cell-platelet interactions and
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promoting tumor cell survival in bloodstream. In HNSCC, PDPN has been demonstrated as a novel biomarker for cancer migration/invasion
(20)
, early lymphatic dissemination, poor
prognosis and low response to neoadjuvant chemoradiotherapy
(21, 22)
. However, whether
PDPN is expressed in CTCs from the HNSCC patients and whether PDPN+-CTCs have prognostic value have not yet been elucidated. In this study, we address these issues by analyzing the baseline CTCs from 53 locally advanced and metastatic HNSCC patients prior to chemotherapy. We report for the first time that PDPN is expressed in the CTCs from HNSCC patients. In addition, the ratio of CTCs with PDPN expression but not the absolute
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CTCs count is a prognostic factor of survival in HNSCC. The significance of these findings is discussed.
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METHODS Study Design This was a prospective single-center study approved by the Institute Review Board of Chang Gung Memorial Hospital with the approval ID of 100-4644A3, 100-4623C and 101-2161C. Locally advanced, recurrent or initially metastatic HNSCC patients that confirmed histologically or cytopathologically were enrolled in this study. Other criteria of enrollment included (1) age ≥ 20 years old; (2) patients who could understand and signed the informed consents by their own will; and (3) adequate liver and renal function and white
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blood cell counts before anti-cancer therapy. Patients with synchronous cancer or prior cancers within 5 years were excluded with exception of curative treated non-melanoma skin
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cancer and cervical carcinoma in situ which are barely recurrent within 5 years after treatment. Disease stage and management of the patients followed the institutional guidelines and the
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standard treatment protocols. Enrolled patients were classified into three distinct treatment subgroups: (1) group A: definitive CCRT for the patients with advanced disease; (2) group B:
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curative surgery followed by adjuvant CRT for the patients with pathologic features such as positive margin (none in this study), pN2, and extracapsular spread (ECS) of involved lymph
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nodes indicating early relapse according to recommendation from NCCN guidelines; and (3)
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group C: palliative chemotherapy for the patients with distant metastasis or with poor general condition for definitive CCRT. Blood samples were collected within 7 days prior to the first dose of chemotherapy. The 6-months survival after chemotherapy, progression-free survival (PFS) and overall survival (OS) were used as the endpoints for this study. Study results were reported following REMARK recommendations (23). Enrichment and isolation of CTCs Enrichment and isolation of CTCs were performed using the PowerMag system which has been published and validated as described previously
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(24)
. Briefly, PowerMag is a
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negative selection method for effective leukocyte depletion and enhanced detection of viable EpCAM positive and negative CTCs from patients with various cancer types including HNSCC and colorectal cancer (24). Immunofluorescence staining and CTCs enumeration For immunofluorescence staining, the cell filtrate after depletion of CD45+ cells was incubated with the anti-EpCAM antibody (Abcam Inc, Cambridge, England), anti-PDPN antibody and Hoechst 33342 DNA staining dye (Invitrogen Inc, Carsbad, CA) at room temperature for 1 h followed by several washes and centrifugation to remove the supernatant,.
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The use of second epithelial cell marker such as cytokeratin (CK) did not provide add-on (24)
value for CTCs identification and enumeration
. Hence, immunofluorescence staining
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using anti-pan-CK antibody was not performed in this study. The Alexa Fluor 488-conjugated donkey anti-mouse antibody and Alexa Fluor 555-conjugated goat anti-rat
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antibody (Invitrogen Inc, Carsbad, CA) were then added to the cell suspension and kept in the dark for 30 min. After removing the unbound antibody, immunofluorescent images were
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captured by fluorescent microscopy (Zeiss Axiovert 200M). CTCs were defined as the cells that were positive for Hochest 33342 and EpCAM. Repeated depletion of CD45+ cells by
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PowerMag removed almost all CD45+ cells with only few (< 5 cells/ml) remained in the cell
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filtrate. None of these cells was positive for both CD45 and EpCAM (24). Single cell real-time RT-PCR analysis
The EpCAM+ cells were picked individually under fluorescent microscopy. Single cell real-time RT-PCR was performed according to the protocol provided by Fluidigm Corporation (South San Francisco, CA, USA)
(25, 26)
. The expression of PDPN and the
internal control genes β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined using the BioMark HDTM System. Statistical analysis
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The CTCs count in healthy donors and HNSCC patients was compared using boxplot and Kruskal-Wallis test for all groups and Mann-Whitney U test between any two groups with 2-sided significance. The impacts for the baseline CTCs and PDPN+-CTCs count, the ratio for the PDPN+-CTCs count over total CTCs count (PDPN+/EpCAM+-CTCs ratio) and the factors reported to shorten or prolong survival such as age, body mass index (BMI), performance status (PS) and distant metastasis at initial presentation on PFS and OS were assessed by univariate Cox proportional hazards regression analysis. Parameters with significance in univariate analysis were subject to multivariate Cox regression analysis. PFS
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and OS were measured from the date of baseline CTC to the date of confirmed clinical progression, death, or censoring at the last follow-up. Statistical analysis was performed
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using SPSS for Windows (version 18, SPSS, Chicago, IL). A P value of 0.05 was considered statistically significant.
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RESULTS Basic characteristics of enrolled patients A total of 53 patients with locally advanced or metastatic HNSCC and 61 healthy donors were enrolled in the Division of Medical Oncology, Chang Gung Memorial Hospital at Linkou and Chang Gung University between October 2011 and August 2012. Clinical information and survival data were updated until February 2013. Basic characteristics of the enrolled patients are shown in Table 1 with further details in Supplementary Table 1. The median age was 52 with a range from 35 to 80. Primary tumor sites were mainly oral cavity
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(56.6%) followed by oropharynx (17%) and hypopharynx (17%). Forty-two (79.2%) patients had locally advanced stage IV status (including 12 patients with distant metastasis at initial
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presentation, stage IVc) according to the TNM staging system of American Joint Committee on Cancer (AJCC) 7th edition. Forty-two patients (79.2%) had performance status 0-1.
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Thirty-seven patients (69.8%) had a BMI less than 25. The number of CTCs and PDPN+-CTCs differentiates patient groups from healthy donors
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The CTCs (EpCAM+-CTCs) counts between different treatment groups (A, B, and C as
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shown in Table 1) and healthy donors were statistically different even for those who
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underwent curative surgery but had microscopic risk of relapse (Figure 1A and Supplementary Table 2). As revealed by ROC analysis, CTCs enumeration elicited a sensitivity of 96.2% and a specificity of 86.9% with the area under curve (AUC) equivalent to 0.983 when 5 CTCs/ml was used as the cutoff (Figure 1B). Positive PDPN staining was only observed in EpCAM+ cells and was barely found in EpCAM- cells. Hence, CTCs can be either PDPN positive (referred to as PDPN+-CTCs) or negative (Supplementary Figure 1). Consistent with these findings, real-time RT-PCR analysis of 87 EpCAM+ single cells obtained from 10 HNSCC patients revealed that PDPN
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was differentially expressed in each single cell with no PDPN expression in some of the cells (Supplementary Table 3). According to immunofluorescence staining, PDPN+-CTCs counts between different treatment groups (Groups A, B and C) and healthy donors were statistically different and were able to differentiate HNSCC patients from healthy individuals (Figure 1C). ROC analysis revealed that the sensitivity of the assay was 83.3% and the specificity was 81.5% with the AUC equivalent to 0.916 when 3 PDPN+-CTCs/ml was used as the cutoff (Figure 1D). The PDPN+/EpCAM+-CTCs ratio is a prognostic factor for patient survival of HNSCC
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For the whole cohort, the median PFS was 8.6 (95% CI: 4.781-12.419) months and the median OS has not reached 50% after the follow-up duration of 10.5 (6.6-18.5) months. At
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the time of analysis, 23 patients died of cancer and 26 patients experienced disease progression. Because the patients in this trial all have 6-months confirmed dead or alive
er
status, we first assessed whether the CTCs and PDPN+-CTCs count, or the PDPN+/EpCAM+-CTCs ratio is able to predict patients with poor clinical outcome and
Re
therefore disease progression or death at 6 months after anticancer therapy. ROC analysis revealed that the AUC was 0.489, 0.596 and 0.718 for CTCs count, PDPN+-CTCs count and
vi
PDPN+/EpCAM+-CTCs ratio, respectively (Figure 2A-C). The PDPN+/EpCAM+-CTCs ratio
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but not the CTCs or PDPN+-CTCs counts had the merit for prediction of 6-month survival in HNSCC patients (p = 0.011). With the cutoff at 20%, the predictive sensitive and specificity was 94.7% and 51.7%, respectively (Figure 2D). Univariate Cox proportional hazards regression analysis was performed to identify factors with prognostic value in HNSCC patients. Among the conventional factors that have been reported to shorten or prolong survival, ECOG-PS, distant metastasis at initial presentation, AJCC stage at enrollment and treatment groups all had clinical impacts on PFS. The factors that had statistical impact on OS included patients’ age at enrollment, ECOG-PS,
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distant metastasis at initial presentation and treatment groups. Regarding the CTCs-related factors, neither CTCs count nor PDPN+-CTCs count had statistical impact on PFS and OS (Table 2). Notably, the survival status was highly correlated with the PDPN+/EpCAM+-CTCs ratio in HNSCC patients. The hazard ratio (HR) for OS was 6.186 (95% CI: 1.206 - 31.733, P = 0.029) and a similar trend for PFS was observed (P = 0.113). Moreover, with the cutoff at 20% for PDPN+/EpCAM+-CTCs ratio, the HR for PFS was 4.666 with a 95% CI of 1.373 to 15.856 (P = 0.016) and the HR for OS was 12.400 with a 95% CI of 1.643 to 93.570 (P = 0.015).
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Multivariate Cox regression analysis further revealed that ECOG-PS, treatment group and PDPN+/EpCAM+-CTCs ratio were independent prognostic factors to OS as well as
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ECOG-PS and the treatment group to PFS (Table 2). For the PDPN+/EpCAM+-CTCs ratio, the HR for OS was 10.969 (95% CI: 1.448 - 83.068, P = 0.018) while a similar trend for PFS
er
was observed (P = 0.081). Kaplan-Meier survival curve further confirmed that CTCs and PDPN+-CTCs counts did not show any association with PFS and OS (Figure 3A-D). Notably,
Re
the PDPN+/EpCAM+-CTCs ratio with the cutoff at 20% clearly differentiates the patients with good vs. poor PFS (P = 0.006) and (P = 0.008) (Figure 3E- F). The conclusion is also
vi
valid even when the patient group with palliative treatment was excluded from the analysis (P = 0.026 for PFS and P = 0.043 for OS; data not shown)
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DISCUSSION Several important features of CTCs in locally advance or metastatic HNSCC are unveiled in the present study. PDPN, a previously defined prognostic factor for HNSCC (20-22, 27, 28)
, is confirmed to express in a subset of CTCs. The number of CTCs and PDPN+-CTCs
clearly differentiates HNSCC patients from healthy control but fails to show prognostic value. Notably, the PDPN+/EpCAM+-CTCs ratio is an independent prognostic factor regardless of treatment modality and tumor staging. With the cutoff at 20%, the PDPN+/EpCAM+-CTCs ratio clearly differentiates HNSCC patients with good prognosis (≤ 20%) from those with
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poor prognosis (> 20%). This study represents the first report to define the subset of CTCs with PDPN expression contributes mainly to poor clinical outcome in HNSCC patients.
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The clinical significance of CTCs in HNSCC has been addressed in a limited number of studies. Buglione et al. demonstrated that there is no correlation between CTCs positivity at diagnosis and survival or relapse
er
(15)
. Similarly, Nichols et al. reported 4 out of 5 patients
(80%) with detectable CTCs developed recurrent and progressive diseases but PFS is not related to the number of CTCs
Re
(13)
. In contrary to these studies, Jatana et al. reported in a
prospective study of 48 post-operation HNSCC patients that the higher the CTCs counts, the worst the disease-free survival
vi
(29)
. Due to the diverse study design and relatively small
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sample size for these aforementioned studies, it remains controversial whether CTCs count is a confounding factor for poor prognosis in HNSCC patients. In the present study, we reveal that CTCs count with the cutoff at 5 cells/ml clearly differentiates HNSCC patients from healthy individuals but has no prognostic value in the prediction of clinical outcome. While CTCs are highly heterogeneous (30), it is likely that not every CTC has major roles in disease progression. Unveiling the subset(s) of CTCs with aggressive features is both clinical and translational importance. In human squamous cell carcinoma, PDPN expression is generally heterogeneous and is
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often detectable in the one-cell layer at the invasive edge of the tumors
(31)
. This type of
setting facilitates tumor cell invasion in a collective cell migration and invasion independent of EMT (31). PDPN is also involved in single cell invasion contributing to EMT-mediated cell motility (19, 20). By analysis of HNSCC cancer tissues, Kreppel et al. reported that PDPN is a prognostic factor for regional lymph node metastases
(21)
and neoadjuvant platinum-based
CRT (27), and is a prognostic factor for OS and loco-regional control (27). In the present study, we revealed by univariate and multivariate analyses that the PDPN+/EpCAM+-CTCs ratio is an independent prognostic factor for HNSCC which clearly differentiates HNSCC patients
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with good prognosis (ratio ≤ 20%) from those with poor prognosis (ratio > 20%). Kaplan-Meier survival curve further confirms the patients with PDPN+/EpCAM+-CTC ratio ≤
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20% have better survival which is still applicable in a recent update of our clinical data (data not shown). This study thereby provides conceptual advance that the ratio of a specific CTCs
er
population (in this case the PDPN+-CTCs) but not the number of CTCs is important in determining the degree of malignancy. Similar concepts have been revealed in several recent
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work indicating that CTCs with stem cell marker CD133 and known EMT regulators play a role in chemo-resistance and in the blood-borne dissemination of human breast cancer,
vi
respectively (32).
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Page 12 of 27
With the cutoff at 20%, ROC analysis reveals that the sensitivity and specificity of PDPN+/EpCAM+-CTC ratio in the prediction of patients with early disease progression or death at 6 months after chemotherapy was 94.7% and 51.7%, respectively. Despite a useful biomarker is usually expected to have high specificity and high sensitivity, tumor markers with lower specificity may still be useful in high-risk populations because the high prevalence in the population greatly increases the positive predictive value of the assay Because PDPN is known as a poor prognostic factor in HNSCC
(33)
.
(20, 21, 27, 34)
, the moderately
low specificity of PDPN+/EpCAM+-CTC probably has minimal impact on its usage as a
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prognostic marker. Alternatively, this factor could be a valuable supplement marker complementary with other clinical factors or markers. This notion is supported by a report demonstrating that the squamous cell carcinoma antigen which had sensitivity of 84% and specificity of 46% was complementary with α-fetoprotein in detecting hepatocellular carcinoma (HCC) (35) and was a valuable supplement marker for the diagnosis of HCC (36). To the best of our knowledge, this is the first tool to offer sufficient statistical power to predict early progression or death within 6-months of treatment for HNSCC patients. Despite the mean and median CTCs counts for the patients in adjuvant setting (group B)
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was in fact lower than the patients with metastatic lesions (groups C) (Supplementary Table 2), the CTC counts for the patients in groups B and C were not statistically different. It is
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likely that the patients in group B of this study mainly have microscopic risk for relapse such as pN2 or ECS. This high-risk group has been shown to have poor clinical outcome when
er
compared to those not having pN2 or ECS (37). Hence, residual cancer cells could still present in the circulation even after surgical operation. It is also likely that the relatively small sample
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size (18 patients for group B vs. 20 patients for group C) could contribute to the no significant difference of CTCs counts between the two groups of patients. Increasing the case number in future trials should be able to clarify this issue.
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In conclusion, this study demonstrates that CTCs enumeration effectively differentiates HNSCC patients from healthy donors but fails to correlate with the prognosis of patients. Moreover, the PDPN+/EpCAM+-CTC ratio is a prognostic factor for 6-month survival after treatment, PFS and OS in HNSCC patients.
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34. Foschini MP, Leonardi E, Eusebi LH, et al. Podoplanin and E-cadherin expression in preoperative incisional biopsies of oral squamous cell carcinoma is related to lymph
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37. Liao CT, Lee LY, Huang SF, et al. Outcome analysis of patients with oral cavity cancer and extracapsular spread in neck lymph nodes. Int J Radiat Oncol Biol Phys 2011;81:930-937.
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FIGURE LEGENDS Figure 1. Boxplots and ROC curves of the whole cohorts and healthy donors. Panels A and C show the boxplots for the number of CTCs and PDPN+-CTCs among groups. Panels B and D demonstrate the area under curve for CTCs and PDPN+-CTCs respectively. In Panels A and C, Kruskal-Wallis test and Mann-Whitney U test are used for statistical analyses. Groups A, B and C are defined in Table 1, whereas healthy donors are classified as Group 0.
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Figure 2. ROC curves to predict 6-month death. ROC curves with AUC values for the CTCs (panel A) and PDPN+-CTCs (panel B) count and its use in prediction of 6-month death
was
demonstrated
without
statistical
significance.
In
contrast,
the
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PDPN+/EpCAM+-CTC ratio has an acceptable AUC (0.718) with a P value of 0.011, indicating that the ratio could be successfully used to predict 6-month survival status (panel
er
C). Several cutoff values were listed in panel D of which the ratio at 20% has the sensitivity of 94.7% and specificity of 51.7% and is a better cutoff point.
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Figure 3. Kaplan-Meier survival curves reveal that the PDPN+/EpCAM+-CTCs ratio but not the CTCs or PDPN+-CTCs count has prognostic value for patient survival. The
vi
number of CTCs and PDPN+-CTCs failed to show statistical significance to patient survival,
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even though the Kaplan-Meier survival curves demonstrate a trend that the lower CTCs (panels A-B) and PDPN+-CTCs (panels C-D) count, the longer survival time for the patients. In contrast, the PDPN+/EpCAM+-CTCs ratio at 20% clearly differentiates the patients with good vs poor PFS (P = 0.006) and OS (P = 0.008) (panels E-F).
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Table 1. Basic Characteristics of Enrolled Patients
Age , Median (range), years
n
%
52
(35-80)
Gender, Male/Female
50/3
Follow-up duration, median (range), months
10.5 (6.6-18.5)
Tumor Site Oral Cavity
30
56.6%
Oropharynx
9
17.0%
Hypopharynx
9
17.0%
Larynx
4
7.5%
1
1.9%
T0-2
23
43.4%
T3-4
30
56.6%
N0-1
18
34.0%
35
66.0%
41
77.4%
12
22.6%
11
20.8%
42
79.2%
42
79.2%
11
20.8%
37
69.8%
13
24.5%
3
5.7%
A: Primary CCRT with/without induction chemotherapy
15
28.3%
B: Post-surgery status with high risk of recurrence, needing adjuvant CCRT
18
34.0%
C: Palliative chemotherapy only
20
37.7%
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Occult Primary Site T classification N classification
N2-3 M classification
M0
7 AJCC stage
II-III IV
er
M1 th
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Eastern Cooperative Oncology Group Performance Status (ECOG-PS) 0-1 2-4
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Body mass index
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30 Treatment after Enrollment (Group A, B, C)
Abbreviations: AJCC = American Joint Committee on Cancer; CCRT = concurrent chemoradiotherapy
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Table 2. Univariate and Multivariate Analysis of Progression-free and Overall Survival Rates Progression-free survival Univariate
Multivariate
HR (95% CI)
P
Age
1.031(0.984-1.080)
0.203
ECOG PS
1.889(1.416-2.522)
0.000*
BMI category (>25)
0.405(0.154-1.063)
0.066
Oral cavity origin
1.386(0.616-3.115)
0.430
Initial M1 vs M0
3.252(1.314-8.046)
0.011*
Stage at enrollment
1.809(1.191-2.750)
0.005*
CTC (cells/ml)
0.999(0.998-1.001)
0.281
0.997(0.991-1.002)
0.251
3.599(0.739-17.532)
0.113
+
PDPN -CTC (cells/ml) +
+
+
+
PDPN /EpCAM ratio
Overall survival
Fo
HR (95% CI)
P
rP
1.624(1.126-2.341)
ee
0.009*
rR
1.078(0.673-1.729)
7.192(0.782-66.105)
PDPN /EpCAM >20% 4.666(1.373-15.856) 0.016* Treatment Group
Univariate
0.754
0.035*
0.002*
HR (95% CI)
P
1.053(1.001-1.107)
0.047*
2.015(1.499-2.709)
0.000*
0.608(0.220-1.6836)
0.338
1.231(0.510-2.972)
0.643
4.490(1.875-10.753)
0.001*
2.155(1.341-3.463)
0.002*
0.999(0.998-1.001)
0.363
0.998(0.992-1.003)
0.413
6.186(1.206-31.733)
iew
0.029*
12.400(1.643-93.570)
0.015*
ev
0.081
Multivariate
HR (95% CI)
P
1.717(1.212-2.431)
0.002*
1.127(0.715-1.777)
0.608
10.969(1.448-83.068) 0.018*
0.001*
0.033*
A: Primary CCRT
0.088(0.020-0.391)
0.001*
0.113(0.021-0.613)
0.011*
0.054(0.007-0.410)
0.005*
0.076(0.009-0.661)
0.020*
B: Adjuvant CCRT
0.386(0.166-0.901)
0.028*
0.829(0.279-2.467)
0.736
0.217(0.078-0.605)
0.004*
0.350(0.106-1.157)
0.085
Reference
-
Reference
-
Reference
-
Reference
-
C: Palliative CT
Abbreviations: BMI = body mass index; CT = chemotherapy; ECOG = Eastern Cooperative Oncology Group; CCRT = concurrent chemoradiotherapy; HR = hazard ratio; OS = overall survival; PDPN = podoplanin; PFS = progression-free survival; PS = performance status; M = metastasis.
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Figure 3 211x236mm (300 x 300 DPI)
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Supplementary Table and Figures Supplementary Figure 1.
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Supplementary Figure 1. Isolation and Characterization of CTCs from Patients with HNSCC. CTC was isolated by PowerMag system and analyzed by immunoflurescence staining using anti-EpCAM (green) and anti-PDPN (red) antibodies. CTCs were identified with two major populations, PDPN+-CTC and
er
PDPN--CTC from HNSCC patients.
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Supplementary Table 1. Subgrouping by Status at Enrollment Treatment Arms
n
Group A (Primary CCRT ± induction chemotherapy for advanced status) Oral cavity cancer
15 3
Unresectable status, s/p primary CCRT ± induction chemotherapy Loco-regional recurrence after primary surgery Oropharyngeal cancer Hypopharyngeal cancer Laryngeal cancer Occult primary cancer, presenting as neck squamous cell carcinoma
2 1 4 6 1 1 18 17 1 20 10
r Fo
Group B (Post-surgery status with high risk of recurrence, needing adjuvant CCRT) Oral cavity cancer, post-surgery, with high risk for recurrence, such as ECS+ Laryngeal cancer with neck LAP, only local palliative surgery Group C (Palliative chemotherapy only) Oral cavity cancer, with distant recurrence, not suitable for another surgery Oropharyngeal cancer Recurrence with initial distant metastasis Hypopharyngeal cancer Recurrence with distant metastasis
er
Pe
Re
with initial distant metastasis Too weak to undergo standard CCRT Laryngeal cancer with recurrence, not suitable for another surgery
vi
Abbreviations: CCRT = concurrent chemoradiotherapy; ECS = extracapsular spread of involved lymph node; OC = oral cavity.
ew
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Head & Neck
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4 1 1 1 1 2
Head & Neck
Supplementary Table 2. CTC Numbers among Different Groups Treatment Arms
n
Mean ± SD Median, range 640.1 ± 1085.6
Group A (Primary CCRT ± induction 15 chemotherapy for advanced status)
P valuea
147.3 (5.2-3440.5)
Group B (Post-surgery with high risk of recurrence, needing adjuvant 18 59.9 ± 87.4 19.7 (4.0-315.3) CCRT) Group C (Palliative chemotherapy only)
20 108.0 ± 131.2 64.0 (5.0-460.8)
Group 0 (Healthy donors)
61
r Fo
Comparison between Groups Group 0 versus Group A Group 0 versus Group B Group 0 versus Group C Group A versus Group B Group A versus Group C Group B versus Group C
3.0 ± 2.8
2.5 (0.0-13.0)
er n
Treatment Arms by Surgery
35 336.0 ± 752.5
P value
75.0 (5.0-3440.5)
vi
needing primary CCRT ± induction chemotherapy or palliative chemotherapy alone)
Mean ± SD Median, range
Re
Group 1 (No surgery group; due to advanced disease or distant metastasis,
