Clin Oral Invest DOI 10.1007/s00784-015-1518-0
ORIGINAL ARTICLE
Evaluation of a biomarker based blood test for monitoring surgical resection of oral squamous cell carcinomas Martin Grimm 1 & Wiebke Kraut 1 & Sebastian Hoefert 1 & Michael Krimmel 1 & Thorsten Biegner 2 & Peter Teriete 3 & Marcel Cetindis 1 & Joachim Polligkeit 1 & Susanne Kluba 1 & Adelheid Munz 1 & Siegmar Reinert 1
Received: 12 January 2015 / Accepted: 24 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Introduction The potential use of determination of biomarkers in blood for the monitoring of surgical removal of oral squamous cell carcinomas (OSCC) was evaluated using the epitope detection in monocytes (EDIM) technology. Materials and methods In tumor specimen, elevated Apo10 and transketolase-like 1 (TKTL1) expression was analyzed by immunohistochemistry. Apo10 and TKTL1 biomarkers have been used prospectively for EDIM blood test in patients with primary and/or recurrent OSCC (n = 92) before surgery and after curative tumor resection (n = 45). Results There were highly significant (p < 0.0001) correlations found between EDIM blood scores and the tissue expression of both biomarkers measured by immunohistochemistry (Apo10: n = 89/92, 97 %; TKTL1: n = 90/92, 98 %). EDIM Apo10 and EDIM-TKTL1 scores were positive in 92 % (EDIM-Apo10: n = 85/92) and 93 % (EDIM-TKTL1: n = 86/92), respectively, in patients with OSCC before surgery. The combined score EDIM-Apo10/EDIM-TKTL1 increased significantly the detection rate of tumors to 97 % (n = 89/92). After surgery, the EDIM-TKTL1 and EDIM Electronic supplementary material The online version of this article (doi:10.1007/s00784-015-1518-0) contains supplementary material, which is available to authorized users. * Martin Grimm [email protected] 1
Department of Oral and Maxillofacial Surgery, University Hospital Tuebingen, Osianderstrasse 2-8, 72076 Tuebingen, Germany
2
Department of Pathology, University Hospital Tuebingen, Liebermeisterstrasse 8, 72076 Tuebingen, Germany
3
Cancer Research Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
Apo10 scores significantly decreased in 75.6 and 86.7 % of the patients (p < 0.0001), respectively, in the aftercare. Conclusions The correlation of TKTL1 and Apo10 immunohistochemistry with the blood test results indicates that the EDIM blood test could serve as a non-invasive diagnostic tool (liquid biopsy) to assess surgical removal of OSCC by determination of two biomarkers. Clinical relevance This is the first study that has been demonstrated a reliable and successful monitoring of OSCC cancer patients by a blood test. The specific and significant decrease of EDIM-TKTL1 and EDIM-Apo10 scores after surgery could serve as a new tool for monitoring surgical removal of OSCC. Keywords Oral squamous cell carcinoma . Tumor metabolism . Biomarker . DNaseX . Apo10 . TKTL1 . EDIM blood test . Early detection and diagnosis
Introduction Although some progress in the early detection and treatment of oral squamous cell carcinoma (OSCC) has been achieved [1], a high proportion (~50 % [2]) of patients with OSCC are subjected to metastatic disease at the time of initial diagnosis or in the aftercare, leading to a generally unfavorable prognosis. In recurrent disease, OSCC may originate from residual microscopic malignancy, not evident at the time of surgery. Therefore, early detection of primary and/or recurrent OSCC by routine laboratory tools is required [3]. As of yet, most serum or blood biomarkers lack a high degree of specificity and sensitivity to detect OSCC [4]. The ability to monitor the response to successful surgical therapy by serial measurement of biomarkers showing increased pretreatment levels and decreased posttreatment levels is much needed [3]. Moreover,
Clin Oral Invest
the usage of new tumor blood biomarkers may allow an earlier detection of the primary tumor, and/or tumor recurrence, and/ or metastasis, and may serve as a possible predictor of prognosis. In general, the diagnostic accuracy of a test is enhanced by the measurement of more than one biomarker [5], and more importantly, markers that show significant correlations with various clinicopathological factors in OSCC cancer patients [5, 6]. For detecting biomarkers in OSCC, several markers and methods have been described, e.g., the measurement of serum placenta growth factor [7], spin electron paramagnetic resonance of albumin [8], free DNA [9], zinc finger protein [10], or cytokines like interleukins (IL)-6, IL-8 [11]. Recently, we identified two independent fundamental biophysical processes by the two biomarkers Apo10 and transketolase-like 1 (TKTL1), which allow a sensitive and specific detection of OSCC [5]. Endonuclease DNaseX (DNaseI-like 1) is a biochemical suicide molecule that has been used to identify the Apo10 protein epitope and marks tumor cells in many solid tumors [5] with abnormal apoptosis and proliferation activity. Apo10 overexpression was associated with metastasis of the colon [12] and OSCC [5]. The TKTL1 enzyme is centrally involved in the sugar metabolism linking the activated pentose phosphate pathway (PPP) with altered tumor metabolism, malignant transformation, and tumor progression [5]. Overexpression of TKTL1 in cancer cells has been correlated with a more malignant cancer phenotype [13], increased rate of metastasis [5, 14–17], and poor prognosis in patients with OSCC [5], esophageal [18], lung [19], urothelium (bladder/renal) [17, 20], ovarian [15], colorectal [17, 21] tumors, and resistance to chemo- and radiation therapy [21]. In addition to the detection in tissue sections, both biomarkers have been detected intracellularly in monocytes of blood samples using the epitope detection in monocytes (EDIM) technique [5, 12, 22–25]. Even in the presence of low tumor mass, the EDIM technique allows sensitive and specific as well as non-invasive detection of OSCC by blood samples [5] utilizing the fact that activated monocytes phagocytize tumor-related tissue [26]. Those activated monocytes contain intracellular Apo10 and TKTL1 tumor epitopes and can be detected by CD14 and CD16 specific antibodies using flow cytometry [5, 12, 22, 23, 25]. A previous study by Feyen et al. demonstrated a high correlation of EDIM-TKTL1 blood test with F-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) results in patients with malignancies [22]. Clinically, non-invasive molecular imaging, especially PET, may be used to evaluate tumor hypoxia, increased glucose uptake, and upregulation of proteins and enzymes for glucose metabolism (e.g. TKTL1) [22, 27]. In addition, our previous study demonstrated Apo10 and TKTL1 expression levels, measured by immunohistochemistry, as independent prognostic factors for reduced tumor-specific survival in OSCC [5]. In general, using both
biomarkers in routine immunohistochemical analysis of tumor samples may additionally allow to distinguish between benign and malign tumors and enables the identification of tumors eligible for targeted therapies addressing TKTL1 glucose metabolism by small compounds or metabolic treatments. Therefore, in the present study, we analyzed prospectively the potential of Apo10 and TKTL1 expression as pretreatment and posttreatment monitoring parameters by using EDIMApo10 and EDIM-TKTL1 blood test in patients with primary and/or recurrent OSCC. Moreover, we introduce and describe a clinical course in the cases of elevated EDIM-Apo10 and EDIM-TKTL1 values in the aftercare of OSCC.
Materials and methods Patients, blood samples, and EDIM blood tests Written informed consent to participate was obtained prospectively from all patients (Ethics Committee Tuebingen, Germany, approval number: 562-2013BO2). Overall, n = 92 patients with histopathologically confirmed primary and/or recurrent OSCC were prospectively enrolled in this study (Fig. 1). The diagnosis of squamous cell carcinoma was confirmed by the Department of Pathology, University Hospital Tübingen. Clinical staging of OSCC patients involved of clinical examination, ultrasonography of the head and neck, whole body computed tomography scans, and diagnostic Bpanendoscopy^ (laryngoscopy, bronchoscopy, esophagogastro-duodenoscopy). Moreover, 3-ml blood samples were collected in EDTA tubes prior to surgery and from the sixth postoperative week after curative R0 tumor resection, anonymized and then processed within 24 h, blinded to the clinical data. The monthly follow-up of OSCC treatment consisted of clinical examination and ultrasonography of the head and neck. Computed tomography (CT) and/or magnetic resonance imaging (MRI) scans were used for routine followup after 6 months postoperative or when necessary to clarify tumor recurrence. Blood was taken when patients presented tumor free either clinically or by imaging techniques (CT/ MRI) and showed no indications for malignancy (e.g., suspect precancerous lesions, tumor recurrence, second primary tumor, or metastasis) or an acute inflammatory reaction/disease (e.g., wound healing disorder). In 47 out of 92 cases (n = 47/92, 51 %), postoperative blood was not available as patients did not undergo regular follow-up because of several reasons (e.g., palliative therapy, aftercare elsewhere, non-compliance, tumor conditional death, personal reasons of the patient, etc.). Immunocytochemical staining and flow cytometric analysis of blood samples were performed as described previously [5, 12, 22, 24]. Fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated Apo10/TKTL1 antibodies were provided by TAVARTIS GmbH (Hainburg, Germany).
Clin Oral Invest
Fig. 1 Flow chart of OSCC patients’ enrolment. The flow chart demonstrates the enrolment of patients with oral cavity cancer, including the assessment for eligibility, the allocation, and the analysis of groups based on the posttreatment EDIM blood test results in the follow-up time period. After the mean follow-up time of 22.2 months,
the result of each group is given in the boxes at the bottom. OSCC, oral squamous cell carcinoma; EDIM, epitope detection in monocytes; FDGPET/CT: F-18 fluorodeoxyglucose positron emission tomography/ computed tomography
FITC-conjugated CD14 and allophycocyanin (APC)-conjugated CD16 antibodies were purchased from BD Biosciences (Heidelberg, Germany). An aliquot of the cell suspension was incubated with fluorophore-conjugated rat or mouse immunoglobulins (BD Biosciences) as a negative control to reveal background staining by the conjugated primary antibody. Samples were analyzed by fluorescence-activated cell sorting using three fluorescence-activated cell sorting devices (CantoII, BD Biosciences; Calibur, BD Biosciences; FC500, Beckman Coulter) [22]. EDIM scores were determined as described previously [5]. The result of the EDIM test is given as a relative score indicating the relative amount of CD14/CD16 positive monocytes harboring Apo10 or TKTL1 compared to the total amount of CD14/CD16 positive monocytes multiplied by 10 [5].
immunoperoxidase method was used for immunostaining of Apo10 and TKTL1 in FFPE tumor tissue sections, which were evaluated as described previously [5]. For immunohistochemical analysis, monoclonal antibodies against Apo10 (TAVARTIS GmbH, Hainburg, Germany, rat anti-human mAb, 5 μg/ml [5]), TKTL1 antibody (TAVARTIS GmbH, Hainburg, Germany, mouse anti-human mAb, 5 μg/ml clone JFC12T10 [28]), and isotype control antibodies (BD Pharmingen, Heidelberg, Germany) were used. The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tissue area, was semi-quantitatively scored on a scale of 0 to 3 as the following: 0, 60 %. The intensities of the signals were scored as 1+, 2+, and 3+. Then, a combined score (0–9) for each specimen was calculated by multiplying the values of these two categories [29]. Cases were classified as negative, 0 points, positive, 1–9 points.
Immunohistochemistry (IHC) Archival formalin-fixed, paraffin-embedded (FFPE) tissue from routine histopathological work-up was obtained from the Department of Pathology, University Hospital Tuebingen from 2012 to 2014. The standard streptavidin biotin
Statistical analysis Statistical analysis was performed with MedCalc Software, Version 14.10.2 (Mariakerke, Belgium). McNemar’s chi-
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Area under the curve (AUC) analysis was determined for quality measurement of EDIM-Apo10 and EDIM-TKTL1 expression.
square (binomial test) test for paired categorical data test was used to analyze the difference of pre- and posttreatment EDIM values. Non-parametric Kendall’s tau (т) correlation coefficient was measured to assess the correlation between EDIM blood test and tissue expression of both biomarkers. All pvalues presented were two-sided and p < 0.05 was considered statistically significant. In our previous work, when screening for EDIM-Apo10 and EDIM-TKTL1 expression [5], the cut-off point for healthy individuals (blood donors) compared with a cancer group was determined by receiver operating characteristics (ROC) analysis, as the value corresponding with the highest diagnostic average of sensitivity and specificity (highest diagnostic accuracy). Cut-off points [5] showing high sensitivity (Apo10: 90 %; TKTL1: 92 %; combined EDIM-Apo10 and EDIM-TKTL1 expression: 94 %) and high specificity (Apo10: 94.6 %; TKTL1: 95.9 %; combined EDIM-Apo10 and EDIM-TKTL1 expression: 97.3 %) for detection of malignancy compared with healthy blood donors were determined at the score of >109 for EDIM-Apo10, at the score of >117 for EDIM-TKTL1, and at the score of >227 for the combined EDIM-Apo10 and EDIMTKTL1 expression (summation of both scores) corresponding with highest Youden index. ROC analysis was additionally performed to analyze differences in the EDIM-Apo10 and EDIM-TKTL1 scores of different clinicopathological characteristics in cancer patients.
Prospectively, EDIM blood tests (Figs. 1 and 2) were assessed in OSCC patients with primary and/or recurrent OSCC. Preoperative 85 out of 92 patients (n = 85/92, 92 %) with OSCC (Supplementary Table 1) showed positive EDIMApo10 scores, and 86 out of 92 patients showed positive EDIM-TKTL1 scores (n = 86/92, 93 %). Only two patients (n = 2/92, 2 %) were negative for both values. Using the combined score (EDIM-Apo10/EDIM-TKTL1 > 227), 89 OSCC out of 92 patients (n = 89/92, 97 %) were positively detected (Supplementary Table 1). Measured in 92 OSCC patients, the preoperative mean value of EDIM-Apo10 score was 143 (95 % CI, confidence interval 138 to 148), the EDIM-TKTL1 score was 142 (95 % CI: 138 to 146), and the combined score EDIM-Apo10/EDIMTKTL1 was 285 (95 % CI: 278 to 292).
Fig. 2 Pre- and posttreatment EDIM dotplots of Apo10 and TKTL1 staining. Dotplots show pretreatment/preoperative values (OSCC, a Apo10; b TKTL1) and posttreatment/postoperative values (after curative tumor resection, c Apo10; d TKTL1) and (representative)
isotype controls (background staining, e Apo10; f TKTL1). Score values indicate the relative amount of positive macrophages. FITC-A (fluorescein isothiocyanate area) and PE-A (phycoerythrin area), red population; APC-A (allophycocyanin area) blue population
Results EDIM-Apo10 and EDIM-TKTL1 blood tests are diagnostic tools to identify and to monitor OSCC patients
15%b 5/45 (11 %)
In 45 out of 92 cases (n = 45/92, 49 %), postoperative blood was available. Mean follow-up time to the first posttreatment evaluation of EDIM scores in 45 patients was 9.8 months (95 % CI: 8.7 to 10.8 months). Preoperative 41 out of these 45 patients with OSCC (n = 41/45, 91 %) showed positive EDIM-Apo10 scores and 44 out of these 45 patients showed positive EDIM-TKTL1 scores (n = 44/45, 98 %). Using the combined score, 44 OSCC patients (n = 44/45, 98 %) were positively graded (Table 1). To discriminate OSCC patients between BEDIM negative^ (Group 1) and BEDIM positive^ (Group 2 and 3) values in the aftercare (Fig. 1), preoperative mean values of EDIM-Apo10 score, EDIM-TKTL1 score, and the combined score EDIMApo10/EDIM-TKTL1 were used (above). Authors determined the mean EDIM scores as indicative for tumor recurrence (Group 3). EDIM scores below the mean EDIM scores (Group 2) were also regarded as EDIM positive if not dedicated to EDIM negative (Group 1) (Fig. 1). Compared with pretreatment EDIM values, posttreatment EDIM values demonstrated a significant (p < 0.0001, McNemar’s chi-square; Table 1, Fig. 3) decrease after curative tumor resection. EDIM-Apo10 score decreased significantly in 82.2 % (95 % CI: 66.6 to 82.2 %), EDIM-TKTL1 in 75.6 % (95 % CI: 60.0 to 75.6 %), and combined EDIM-Apo10/ EDIM-TKTL1 score in 86.7 % (95 % CI: 71.0 to 86.7 %) of the patients (Table 1, Figs. 2 and 3). Based on the EDIM data, we then describe a clinical course (detailed shown in Fig. 1) and recommended either regular clinical visits (n = 39/45, 87 % of the cases), re-evaluation of EDIM blood test in 3 months (n = 1/45, 2 % of the cases), or FDG-PET/CT (n = 5/45, 11 % of the cases) to analyze (to confirm or to exclude) locoregional tumor recurrence or second primary malignancy. In cases of strongly elevated EDIM values (Group 3, Fig. 1), FDG-PET/CT was not specifically indicative for locoregional tumor recurrence but showed inflammatory reactions of complications after surgery (n = 2/5) or tooth extraction (n = 1/5) or gave no indication for tumor recurrence (n = 2/5) at the time of investigation. In all OSCC patients (n = 92), Apo10 and TKTL1 expression had been analyzed by immunohistochemistry (Fig. 4). Eighty-nine out of 92 OSCCs were positively stained for Apo10 (n = 89/92, 97 %), and 90 out of 92 individuals were positive for TKTL1 (n = 90/92, 98 %).
Compared with healthy individuals (blood donors, [5])
Definition is given in Fig. 1 b
OSCC oral squamous cell carcinoma
a
20%b 4/45 (9 %) Preoperative negativea 4/45 (9 %) Postoperative negative or ≤20%b 41/45 (91 %)
Preoperative negativea 1/45 (2 %) Postoperative negative or ≤10%b 35/45 (78 %)
Preoperative positivea 44/45 (98 %) Postoperative positive >10%b 10/45 (22 %)
133 ≤149 >131 ≤139 ≤123 >131 ≤139 ≤318 EDIM value Sensitivity 72.97 84.62 77.78 80.0 62.16 2.56 94.44 54.55 81.08 Specificity 38.18 43.40 44.59 45.95 60.0 83.02 35.14 59.46 9.09
pNb
Gc
UICCd
0.56 0.32 >274
0.58 0.27 >268
0.56 0.39 >267
82.05 47.17
88.89 33.78
80.0 40.54
Abbrevations: AUC area under the ROC curve, T tumor size, N cervical lymph node metastasis, G grading, UICC International Union Against Cancer a
pT1/2 vs. pT3/4
b
pN0 vs. pN+
c
G1/2 vs. G3/4
d
UICC stage I/II vs. UICC III/IV
analyzed oral precursor lesions [30, 31], we first introduced and described a clinical course in the cases of persistently elevated/increasing EDIM-Apo10 and EDIM-TKTL1 values in the aftercare of patients with OSCC. Nevertheless, two of the three criterions (elevated EDIM-Apo10, EDIM-TKTL1, or combined EDIM-Apo10 and EDIM-TKTL1 score) in each group (mild, moderate, strong, Table 1) must be met to enable the following suggested clinical course as two independent fundamental biophysical processes are measured. The authors suggest that patients with postoperative persistently but mildly elevated levels of either EDIM-Apo10 score (about ≤130, ≤ 20 %) or EDIM-TKTL1 score (about ≤130, ≤ 10 %) or combined EDIM-Apo10 and EDIM-TKTL1 score (about ≤260, ≤ 15 %) patients may follow regular clinical visits according to clinical guidelines [32]. Although curative tumor resection has been performed, the physical condition of OSCC cancer patients is not comparable with Bhealthy^ blood donors as a control group due to several reasons. Therefore, authors decided to adapt the cut-off value in the aftercare for the decision of EDIM negative or EDIM positive adapted to the specific clinical situation of OSCC cancer patients. In this context, it seems unreasonable to associate posttreatment/postoperative decreased EDIM values, which have not fully reached Bnormal EDIM levels^ of healthy blood donors [5] with tumor recurrence although persistent elevated markers could be indicative for early tumor recurrence. Only a long-rise analysis may clarify this question as our mean follow-up time in this pilot study was just 22.2 months. Concerning OSCC, statistically, ~25 % of the patients will suffer from tumor recurrence (including metastasis) within the first 5 years [33]. However, in our study, we do not assume that 32 out of 45 patients (n = 32/45, 71 %, Supplementary Table 1) will suffer from tumor recurrence since they exhibit elevated EDIM levels compared to the normal EDIM levels of healthy blood donors [5]. The majority of the patients’ EDIM values have been clearly decreased after treatment, indicating that relative levels
bear prognostic importance. Moreover, in our current studies [30, 31], we found immunohistochemically a constant increase of Apo10/TKTL1 expression during multistep carcinogenesis (from simple hyperplasia to invasive OSCC). Therefore, mild (persistent) elevated levels of both EDIM scores may also be attributed to oral (or topographically other) precursor lesions (e.g., in the upper aerodigestive tract), which are primarily not associated with disease recurrence. In addition, from our current evidence, little is known about other predisposal factors marginal influencing Apo10/TKTL1 expression in blood samples, like general or focal inflammatory disorders (e.g., oral lichen planus), or coincidental benign tumors of the oral cavity (e.g., fibroma), or topographically other benign lesions with few or without potential of malignant transformation. As an example, single cases of so far unpublished data of patients with acute inflammatory diseases (e.g., abscess of the jaw) or large benign tumors (e.g., epulis fibromatosa) showed either a normal or mild elevated EDIM values. Moreover, remodeling processes after the operation (tumor resection, neck dissection, considerably reconstruction of hard and soft tissue of maxillofacial defects with microvascular transplants) may take several months. There can be an overlap between mildly elevated EDIM values and natural occurring microscopic wound healing processes. Remodeling processes with involvement of matrix degeneration in wound healing processes may lead to (mild) persistent elevated TKTL1 positivity. Extensive surgical procedures as indicated above for OSCC are therefore not comparable with surgical procedures, e.g., in prostate and breast cancer, which is accompanied by a faster wound healing as only soft tissue is treated. Therefore, authors recommend postoperative blood withdrawal from the twelfth postoperative week or longer adapted to the clinical situation of patients with OSCC. In the case of postoperative persistently but moderately elevated levels of either EDIM-Apo10 score (about >130 to 20 % but 130
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to 10 % but 260 to 280, >15 % but
ORIGINAL ARTICLE
Evaluation of a biomarker based blood test for monitoring surgical resection of oral squamous cell carcinomas Martin Grimm 1 & Wiebke Kraut 1 & Sebastian Hoefert 1 & Michael Krimmel 1 & Thorsten Biegner 2 & Peter Teriete 3 & Marcel Cetindis 1 & Joachim Polligkeit 1 & Susanne Kluba 1 & Adelheid Munz 1 & Siegmar Reinert 1
Received: 12 January 2015 / Accepted: 24 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Introduction The potential use of determination of biomarkers in blood for the monitoring of surgical removal of oral squamous cell carcinomas (OSCC) was evaluated using the epitope detection in monocytes (EDIM) technology. Materials and methods In tumor specimen, elevated Apo10 and transketolase-like 1 (TKTL1) expression was analyzed by immunohistochemistry. Apo10 and TKTL1 biomarkers have been used prospectively for EDIM blood test in patients with primary and/or recurrent OSCC (n = 92) before surgery and after curative tumor resection (n = 45). Results There were highly significant (p < 0.0001) correlations found between EDIM blood scores and the tissue expression of both biomarkers measured by immunohistochemistry (Apo10: n = 89/92, 97 %; TKTL1: n = 90/92, 98 %). EDIM Apo10 and EDIM-TKTL1 scores were positive in 92 % (EDIM-Apo10: n = 85/92) and 93 % (EDIM-TKTL1: n = 86/92), respectively, in patients with OSCC before surgery. The combined score EDIM-Apo10/EDIM-TKTL1 increased significantly the detection rate of tumors to 97 % (n = 89/92). After surgery, the EDIM-TKTL1 and EDIM Electronic supplementary material The online version of this article (doi:10.1007/s00784-015-1518-0) contains supplementary material, which is available to authorized users. * Martin Grimm [email protected] 1
Department of Oral and Maxillofacial Surgery, University Hospital Tuebingen, Osianderstrasse 2-8, 72076 Tuebingen, Germany
2
Department of Pathology, University Hospital Tuebingen, Liebermeisterstrasse 8, 72076 Tuebingen, Germany
3
Cancer Research Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
Apo10 scores significantly decreased in 75.6 and 86.7 % of the patients (p < 0.0001), respectively, in the aftercare. Conclusions The correlation of TKTL1 and Apo10 immunohistochemistry with the blood test results indicates that the EDIM blood test could serve as a non-invasive diagnostic tool (liquid biopsy) to assess surgical removal of OSCC by determination of two biomarkers. Clinical relevance This is the first study that has been demonstrated a reliable and successful monitoring of OSCC cancer patients by a blood test. The specific and significant decrease of EDIM-TKTL1 and EDIM-Apo10 scores after surgery could serve as a new tool for monitoring surgical removal of OSCC. Keywords Oral squamous cell carcinoma . Tumor metabolism . Biomarker . DNaseX . Apo10 . TKTL1 . EDIM blood test . Early detection and diagnosis
Introduction Although some progress in the early detection and treatment of oral squamous cell carcinoma (OSCC) has been achieved [1], a high proportion (~50 % [2]) of patients with OSCC are subjected to metastatic disease at the time of initial diagnosis or in the aftercare, leading to a generally unfavorable prognosis. In recurrent disease, OSCC may originate from residual microscopic malignancy, not evident at the time of surgery. Therefore, early detection of primary and/or recurrent OSCC by routine laboratory tools is required [3]. As of yet, most serum or blood biomarkers lack a high degree of specificity and sensitivity to detect OSCC [4]. The ability to monitor the response to successful surgical therapy by serial measurement of biomarkers showing increased pretreatment levels and decreased posttreatment levels is much needed [3]. Moreover,
Clin Oral Invest
the usage of new tumor blood biomarkers may allow an earlier detection of the primary tumor, and/or tumor recurrence, and/ or metastasis, and may serve as a possible predictor of prognosis. In general, the diagnostic accuracy of a test is enhanced by the measurement of more than one biomarker [5], and more importantly, markers that show significant correlations with various clinicopathological factors in OSCC cancer patients [5, 6]. For detecting biomarkers in OSCC, several markers and methods have been described, e.g., the measurement of serum placenta growth factor [7], spin electron paramagnetic resonance of albumin [8], free DNA [9], zinc finger protein [10], or cytokines like interleukins (IL)-6, IL-8 [11]. Recently, we identified two independent fundamental biophysical processes by the two biomarkers Apo10 and transketolase-like 1 (TKTL1), which allow a sensitive and specific detection of OSCC [5]. Endonuclease DNaseX (DNaseI-like 1) is a biochemical suicide molecule that has been used to identify the Apo10 protein epitope and marks tumor cells in many solid tumors [5] with abnormal apoptosis and proliferation activity. Apo10 overexpression was associated with metastasis of the colon [12] and OSCC [5]. The TKTL1 enzyme is centrally involved in the sugar metabolism linking the activated pentose phosphate pathway (PPP) with altered tumor metabolism, malignant transformation, and tumor progression [5]. Overexpression of TKTL1 in cancer cells has been correlated with a more malignant cancer phenotype [13], increased rate of metastasis [5, 14–17], and poor prognosis in patients with OSCC [5], esophageal [18], lung [19], urothelium (bladder/renal) [17, 20], ovarian [15], colorectal [17, 21] tumors, and resistance to chemo- and radiation therapy [21]. In addition to the detection in tissue sections, both biomarkers have been detected intracellularly in monocytes of blood samples using the epitope detection in monocytes (EDIM) technique [5, 12, 22–25]. Even in the presence of low tumor mass, the EDIM technique allows sensitive and specific as well as non-invasive detection of OSCC by blood samples [5] utilizing the fact that activated monocytes phagocytize tumor-related tissue [26]. Those activated monocytes contain intracellular Apo10 and TKTL1 tumor epitopes and can be detected by CD14 and CD16 specific antibodies using flow cytometry [5, 12, 22, 23, 25]. A previous study by Feyen et al. demonstrated a high correlation of EDIM-TKTL1 blood test with F-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) results in patients with malignancies [22]. Clinically, non-invasive molecular imaging, especially PET, may be used to evaluate tumor hypoxia, increased glucose uptake, and upregulation of proteins and enzymes for glucose metabolism (e.g. TKTL1) [22, 27]. In addition, our previous study demonstrated Apo10 and TKTL1 expression levels, measured by immunohistochemistry, as independent prognostic factors for reduced tumor-specific survival in OSCC [5]. In general, using both
biomarkers in routine immunohistochemical analysis of tumor samples may additionally allow to distinguish between benign and malign tumors and enables the identification of tumors eligible for targeted therapies addressing TKTL1 glucose metabolism by small compounds or metabolic treatments. Therefore, in the present study, we analyzed prospectively the potential of Apo10 and TKTL1 expression as pretreatment and posttreatment monitoring parameters by using EDIMApo10 and EDIM-TKTL1 blood test in patients with primary and/or recurrent OSCC. Moreover, we introduce and describe a clinical course in the cases of elevated EDIM-Apo10 and EDIM-TKTL1 values in the aftercare of OSCC.
Materials and methods Patients, blood samples, and EDIM blood tests Written informed consent to participate was obtained prospectively from all patients (Ethics Committee Tuebingen, Germany, approval number: 562-2013BO2). Overall, n = 92 patients with histopathologically confirmed primary and/or recurrent OSCC were prospectively enrolled in this study (Fig. 1). The diagnosis of squamous cell carcinoma was confirmed by the Department of Pathology, University Hospital Tübingen. Clinical staging of OSCC patients involved of clinical examination, ultrasonography of the head and neck, whole body computed tomography scans, and diagnostic Bpanendoscopy^ (laryngoscopy, bronchoscopy, esophagogastro-duodenoscopy). Moreover, 3-ml blood samples were collected in EDTA tubes prior to surgery and from the sixth postoperative week after curative R0 tumor resection, anonymized and then processed within 24 h, blinded to the clinical data. The monthly follow-up of OSCC treatment consisted of clinical examination and ultrasonography of the head and neck. Computed tomography (CT) and/or magnetic resonance imaging (MRI) scans were used for routine followup after 6 months postoperative or when necessary to clarify tumor recurrence. Blood was taken when patients presented tumor free either clinically or by imaging techniques (CT/ MRI) and showed no indications for malignancy (e.g., suspect precancerous lesions, tumor recurrence, second primary tumor, or metastasis) or an acute inflammatory reaction/disease (e.g., wound healing disorder). In 47 out of 92 cases (n = 47/92, 51 %), postoperative blood was not available as patients did not undergo regular follow-up because of several reasons (e.g., palliative therapy, aftercare elsewhere, non-compliance, tumor conditional death, personal reasons of the patient, etc.). Immunocytochemical staining and flow cytometric analysis of blood samples were performed as described previously [5, 12, 22, 24]. Fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated Apo10/TKTL1 antibodies were provided by TAVARTIS GmbH (Hainburg, Germany).
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Fig. 1 Flow chart of OSCC patients’ enrolment. The flow chart demonstrates the enrolment of patients with oral cavity cancer, including the assessment for eligibility, the allocation, and the analysis of groups based on the posttreatment EDIM blood test results in the follow-up time period. After the mean follow-up time of 22.2 months,
the result of each group is given in the boxes at the bottom. OSCC, oral squamous cell carcinoma; EDIM, epitope detection in monocytes; FDGPET/CT: F-18 fluorodeoxyglucose positron emission tomography/ computed tomography
FITC-conjugated CD14 and allophycocyanin (APC)-conjugated CD16 antibodies were purchased from BD Biosciences (Heidelberg, Germany). An aliquot of the cell suspension was incubated with fluorophore-conjugated rat or mouse immunoglobulins (BD Biosciences) as a negative control to reveal background staining by the conjugated primary antibody. Samples were analyzed by fluorescence-activated cell sorting using three fluorescence-activated cell sorting devices (CantoII, BD Biosciences; Calibur, BD Biosciences; FC500, Beckman Coulter) [22]. EDIM scores were determined as described previously [5]. The result of the EDIM test is given as a relative score indicating the relative amount of CD14/CD16 positive monocytes harboring Apo10 or TKTL1 compared to the total amount of CD14/CD16 positive monocytes multiplied by 10 [5].
immunoperoxidase method was used for immunostaining of Apo10 and TKTL1 in FFPE tumor tissue sections, which were evaluated as described previously [5]. For immunohistochemical analysis, monoclonal antibodies against Apo10 (TAVARTIS GmbH, Hainburg, Germany, rat anti-human mAb, 5 μg/ml [5]), TKTL1 antibody (TAVARTIS GmbH, Hainburg, Germany, mouse anti-human mAb, 5 μg/ml clone JFC12T10 [28]), and isotype control antibodies (BD Pharmingen, Heidelberg, Germany) were used. The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tissue area, was semi-quantitatively scored on a scale of 0 to 3 as the following: 0, 60 %. The intensities of the signals were scored as 1+, 2+, and 3+. Then, a combined score (0–9) for each specimen was calculated by multiplying the values of these two categories [29]. Cases were classified as negative, 0 points, positive, 1–9 points.
Immunohistochemistry (IHC) Archival formalin-fixed, paraffin-embedded (FFPE) tissue from routine histopathological work-up was obtained from the Department of Pathology, University Hospital Tuebingen from 2012 to 2014. The standard streptavidin biotin
Statistical analysis Statistical analysis was performed with MedCalc Software, Version 14.10.2 (Mariakerke, Belgium). McNemar’s chi-
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Area under the curve (AUC) analysis was determined for quality measurement of EDIM-Apo10 and EDIM-TKTL1 expression.
square (binomial test) test for paired categorical data test was used to analyze the difference of pre- and posttreatment EDIM values. Non-parametric Kendall’s tau (т) correlation coefficient was measured to assess the correlation between EDIM blood test and tissue expression of both biomarkers. All pvalues presented were two-sided and p < 0.05 was considered statistically significant. In our previous work, when screening for EDIM-Apo10 and EDIM-TKTL1 expression [5], the cut-off point for healthy individuals (blood donors) compared with a cancer group was determined by receiver operating characteristics (ROC) analysis, as the value corresponding with the highest diagnostic average of sensitivity and specificity (highest diagnostic accuracy). Cut-off points [5] showing high sensitivity (Apo10: 90 %; TKTL1: 92 %; combined EDIM-Apo10 and EDIM-TKTL1 expression: 94 %) and high specificity (Apo10: 94.6 %; TKTL1: 95.9 %; combined EDIM-Apo10 and EDIM-TKTL1 expression: 97.3 %) for detection of malignancy compared with healthy blood donors were determined at the score of >109 for EDIM-Apo10, at the score of >117 for EDIM-TKTL1, and at the score of >227 for the combined EDIM-Apo10 and EDIMTKTL1 expression (summation of both scores) corresponding with highest Youden index. ROC analysis was additionally performed to analyze differences in the EDIM-Apo10 and EDIM-TKTL1 scores of different clinicopathological characteristics in cancer patients.
Prospectively, EDIM blood tests (Figs. 1 and 2) were assessed in OSCC patients with primary and/or recurrent OSCC. Preoperative 85 out of 92 patients (n = 85/92, 92 %) with OSCC (Supplementary Table 1) showed positive EDIMApo10 scores, and 86 out of 92 patients showed positive EDIM-TKTL1 scores (n = 86/92, 93 %). Only two patients (n = 2/92, 2 %) were negative for both values. Using the combined score (EDIM-Apo10/EDIM-TKTL1 > 227), 89 OSCC out of 92 patients (n = 89/92, 97 %) were positively detected (Supplementary Table 1). Measured in 92 OSCC patients, the preoperative mean value of EDIM-Apo10 score was 143 (95 % CI, confidence interval 138 to 148), the EDIM-TKTL1 score was 142 (95 % CI: 138 to 146), and the combined score EDIM-Apo10/EDIMTKTL1 was 285 (95 % CI: 278 to 292).
Fig. 2 Pre- and posttreatment EDIM dotplots of Apo10 and TKTL1 staining. Dotplots show pretreatment/preoperative values (OSCC, a Apo10; b TKTL1) and posttreatment/postoperative values (after curative tumor resection, c Apo10; d TKTL1) and (representative)
isotype controls (background staining, e Apo10; f TKTL1). Score values indicate the relative amount of positive macrophages. FITC-A (fluorescein isothiocyanate area) and PE-A (phycoerythrin area), red population; APC-A (allophycocyanin area) blue population
Results EDIM-Apo10 and EDIM-TKTL1 blood tests are diagnostic tools to identify and to monitor OSCC patients
15%b 5/45 (11 %)
In 45 out of 92 cases (n = 45/92, 49 %), postoperative blood was available. Mean follow-up time to the first posttreatment evaluation of EDIM scores in 45 patients was 9.8 months (95 % CI: 8.7 to 10.8 months). Preoperative 41 out of these 45 patients with OSCC (n = 41/45, 91 %) showed positive EDIM-Apo10 scores and 44 out of these 45 patients showed positive EDIM-TKTL1 scores (n = 44/45, 98 %). Using the combined score, 44 OSCC patients (n = 44/45, 98 %) were positively graded (Table 1). To discriminate OSCC patients between BEDIM negative^ (Group 1) and BEDIM positive^ (Group 2 and 3) values in the aftercare (Fig. 1), preoperative mean values of EDIM-Apo10 score, EDIM-TKTL1 score, and the combined score EDIMApo10/EDIM-TKTL1 were used (above). Authors determined the mean EDIM scores as indicative for tumor recurrence (Group 3). EDIM scores below the mean EDIM scores (Group 2) were also regarded as EDIM positive if not dedicated to EDIM negative (Group 1) (Fig. 1). Compared with pretreatment EDIM values, posttreatment EDIM values demonstrated a significant (p < 0.0001, McNemar’s chi-square; Table 1, Fig. 3) decrease after curative tumor resection. EDIM-Apo10 score decreased significantly in 82.2 % (95 % CI: 66.6 to 82.2 %), EDIM-TKTL1 in 75.6 % (95 % CI: 60.0 to 75.6 %), and combined EDIM-Apo10/ EDIM-TKTL1 score in 86.7 % (95 % CI: 71.0 to 86.7 %) of the patients (Table 1, Figs. 2 and 3). Based on the EDIM data, we then describe a clinical course (detailed shown in Fig. 1) and recommended either regular clinical visits (n = 39/45, 87 % of the cases), re-evaluation of EDIM blood test in 3 months (n = 1/45, 2 % of the cases), or FDG-PET/CT (n = 5/45, 11 % of the cases) to analyze (to confirm or to exclude) locoregional tumor recurrence or second primary malignancy. In cases of strongly elevated EDIM values (Group 3, Fig. 1), FDG-PET/CT was not specifically indicative for locoregional tumor recurrence but showed inflammatory reactions of complications after surgery (n = 2/5) or tooth extraction (n = 1/5) or gave no indication for tumor recurrence (n = 2/5) at the time of investigation. In all OSCC patients (n = 92), Apo10 and TKTL1 expression had been analyzed by immunohistochemistry (Fig. 4). Eighty-nine out of 92 OSCCs were positively stained for Apo10 (n = 89/92, 97 %), and 90 out of 92 individuals were positive for TKTL1 (n = 90/92, 98 %).
Compared with healthy individuals (blood donors, [5])
Definition is given in Fig. 1 b
OSCC oral squamous cell carcinoma
a
20%b 4/45 (9 %) Preoperative negativea 4/45 (9 %) Postoperative negative or ≤20%b 41/45 (91 %)
Preoperative negativea 1/45 (2 %) Postoperative negative or ≤10%b 35/45 (78 %)
Preoperative positivea 44/45 (98 %) Postoperative positive >10%b 10/45 (22 %)
133 ≤149 >131 ≤139 ≤123 >131 ≤139 ≤318 EDIM value Sensitivity 72.97 84.62 77.78 80.0 62.16 2.56 94.44 54.55 81.08 Specificity 38.18 43.40 44.59 45.95 60.0 83.02 35.14 59.46 9.09
pNb
Gc
UICCd
0.56 0.32 >274
0.58 0.27 >268
0.56 0.39 >267
82.05 47.17
88.89 33.78
80.0 40.54
Abbrevations: AUC area under the ROC curve, T tumor size, N cervical lymph node metastasis, G grading, UICC International Union Against Cancer a
pT1/2 vs. pT3/4
b
pN0 vs. pN+
c
G1/2 vs. G3/4
d
UICC stage I/II vs. UICC III/IV
analyzed oral precursor lesions [30, 31], we first introduced and described a clinical course in the cases of persistently elevated/increasing EDIM-Apo10 and EDIM-TKTL1 values in the aftercare of patients with OSCC. Nevertheless, two of the three criterions (elevated EDIM-Apo10, EDIM-TKTL1, or combined EDIM-Apo10 and EDIM-TKTL1 score) in each group (mild, moderate, strong, Table 1) must be met to enable the following suggested clinical course as two independent fundamental biophysical processes are measured. The authors suggest that patients with postoperative persistently but mildly elevated levels of either EDIM-Apo10 score (about ≤130, ≤ 20 %) or EDIM-TKTL1 score (about ≤130, ≤ 10 %) or combined EDIM-Apo10 and EDIM-TKTL1 score (about ≤260, ≤ 15 %) patients may follow regular clinical visits according to clinical guidelines [32]. Although curative tumor resection has been performed, the physical condition of OSCC cancer patients is not comparable with Bhealthy^ blood donors as a control group due to several reasons. Therefore, authors decided to adapt the cut-off value in the aftercare for the decision of EDIM negative or EDIM positive adapted to the specific clinical situation of OSCC cancer patients. In this context, it seems unreasonable to associate posttreatment/postoperative decreased EDIM values, which have not fully reached Bnormal EDIM levels^ of healthy blood donors [5] with tumor recurrence although persistent elevated markers could be indicative for early tumor recurrence. Only a long-rise analysis may clarify this question as our mean follow-up time in this pilot study was just 22.2 months. Concerning OSCC, statistically, ~25 % of the patients will suffer from tumor recurrence (including metastasis) within the first 5 years [33]. However, in our study, we do not assume that 32 out of 45 patients (n = 32/45, 71 %, Supplementary Table 1) will suffer from tumor recurrence since they exhibit elevated EDIM levels compared to the normal EDIM levels of healthy blood donors [5]. The majority of the patients’ EDIM values have been clearly decreased after treatment, indicating that relative levels
bear prognostic importance. Moreover, in our current studies [30, 31], we found immunohistochemically a constant increase of Apo10/TKTL1 expression during multistep carcinogenesis (from simple hyperplasia to invasive OSCC). Therefore, mild (persistent) elevated levels of both EDIM scores may also be attributed to oral (or topographically other) precursor lesions (e.g., in the upper aerodigestive tract), which are primarily not associated with disease recurrence. In addition, from our current evidence, little is known about other predisposal factors marginal influencing Apo10/TKTL1 expression in blood samples, like general or focal inflammatory disorders (e.g., oral lichen planus), or coincidental benign tumors of the oral cavity (e.g., fibroma), or topographically other benign lesions with few or without potential of malignant transformation. As an example, single cases of so far unpublished data of patients with acute inflammatory diseases (e.g., abscess of the jaw) or large benign tumors (e.g., epulis fibromatosa) showed either a normal or mild elevated EDIM values. Moreover, remodeling processes after the operation (tumor resection, neck dissection, considerably reconstruction of hard and soft tissue of maxillofacial defects with microvascular transplants) may take several months. There can be an overlap between mildly elevated EDIM values and natural occurring microscopic wound healing processes. Remodeling processes with involvement of matrix degeneration in wound healing processes may lead to (mild) persistent elevated TKTL1 positivity. Extensive surgical procedures as indicated above for OSCC are therefore not comparable with surgical procedures, e.g., in prostate and breast cancer, which is accompanied by a faster wound healing as only soft tissue is treated. Therefore, authors recommend postoperative blood withdrawal from the twelfth postoperative week or longer adapted to the clinical situation of patients with OSCC. In the case of postoperative persistently but moderately elevated levels of either EDIM-Apo10 score (about >130 to 20 % but 130
Clin Oral Invest
to 10 % but 260 to 280, >15 % but
