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Fluorescence imaging of invasive head and neck carcinoma cells with integrin αvβ6-targeting RGD-peptides: an approach to a fluorescence-assisted intraoperative cytological assessment of bony resection margins
Available online at www.sciencedirect.com
ScienceDirect British Journal of Oral and Maxillofacial Surgery 56 (2018) 972–978
Fluorescence imaging of invasive head and neck carcinoma cells with integrin ␣v6-targeting RGD-peptides: an approach to a fluorescence-assisted intraoperative cytological assessment of bony resection margins M. Nieberler a,∗,1 , U. Reuning b , H. Kessler c , F. Reichart c , G. Weirich d,2 , K.-D. Wolff a,1 a
Department of Oral and Maxillofacial Surgery, University Hospital rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81679 Munich, Germany b Klinische Forschergruppe der Frauenklinik, University Hospital rechts der Isar, Technischen Universität München, Ismaninger Strasse 22, 81675 München, Germany c Institute for Advanced Study and Center for Integrated Protein Science (CIPSM), Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany d Institute of Pathology, Technische Universität München, Trogerstr. 18, 81675 Munich, Germany Accepted 5 November 2018 Available online 28 November 2018
Abstract We assessed the use of peptides containing arginylglycylaspartic acid (RGD) that target integrin ␣v6 as a potential approach for a fluorescenceassisted intraoperative cytological assessment of bony resection margins (F-AICAB) in patients who had bone-infiltrating squamous cell carcinoma (SCC) of the head and neck. This was assessed to demarcate invasive carcinoma cells that stained for ␣v6. Specimens from bony resection margins (n=362) were defined as either malignant or benign according to the results of cytological and histological examinations. Integrin ␣v6-targeting fluorescence-labelled RGD peptides were added to the cytological samples and the accuracy of the resulting signal assessed by comparing it with the cytological findings. The value of F-AICAB was evaluated to find out if it could help to improve future diagnoses, tests, and treatments. Integrin ␣v6 was strongly expressed in invasive SCC cells and qualified as a marker for bone-infiltrating carcinoma cells. It showed a high affinity to bind to invasive SCC cells and enabled swift and specific demarcation of ␣v6-stained carcinoma cells. It was also diagnostic, with a sensitivity of 100% (95% CI 81.3% to 99.3%), specificity of 98.3% (95% CI 94.4% to 99.0%), positive predictive value of 92% (95% CI 70.2% to 94.3%), and negative predictive value of 100% (95% CI 96.9% to 99.9%), compared with the cytological findings. The targeting of specific integrin subtypes with selective, synthetic ligands, adapted for multimodal imaging, is a promising new approach to diagnosis. Further studies are necessary to provide more evidence for successful clinical translation and to establish the impact on clinical procedures. ﾩ 2018 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Head and neck squamous cell carcinoma; Intraoperative margin control; Cytology; RGD peptide; Fluorescence imaging; Integrins; ␣v6 integrin
∗ Corresponding author. Tel.: +49 89 4140 5916 or +49 163 4714383, Fax: +49 89 4140 4993. E-mail address: [email protected] (M. Nieberler). 1 Tel.: +49 89 4140 2921; Fax: +49 89 4140 4993. 2 Tel.: +49 89 4140 4161; Fax. +49 89 4140 4865.
https://doi.org/10.1016/j.bjoms.2018.11.003 0266-4356/ﾩ 2018 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
M. Nieberler et al. / British Journal of Oral and Maxillofacial Surgery 56 (2018) 972–978
Introduction For patients with squamous cell carcinoma (SCC) of the head and neck, the life-limiting factors are recurrence and metastases. Prognosis correlates with incomplete resection (R1-status), undetected second primary carcinomas, and occult metastases. Despite elaborate guidelines, advances in preoperative and intraoperative diagnostics and curative therapeutic strategies, the five-year survival rate remains at about 50% .1 Consequently, additional approaches to diagnosis and treatment should be considered. The technique of the resection deserves special consideration because it is under the direct control of the surgeon. Adequacy of resection is judged from frozen sections, but because of the technical limitations of processing calcified tissue, the intraoperative histological assessment of the bony margins is not possible. To bridge this diagnostic gap, the intraoperative cytological assessment of bony resection margins (ICAB) was introduced.2–4 Although the assessment has diagnostic value, a targeted fluorescent imaging of invasive malignant cells may provide diagnostic information beyond cytomorphology. In the search for diagnostic markers, the cell adhesion and signalling receptors of the integrin superfamily are well recognised as biologically relevant targets for malignant cells.5 Integrins are heterodimeric transmembrane receptors for extracellular matrix ligands, consisting of non-covalent, associated subunits (␣ and ). The 18 ␣ subunits can interact with eight  subunits, resulting in 24 different integrin heterodimers. The combination of the subunits ␣ and  defines the integrin-binding specificity of the ligands and occurs within distinct pathophysiological units of cell-cell and cellextracellular matrix interactions that communicate essential oncological functions.6 As well as their adhesive functions when they bind to the ligands, integrins initiate bidirectional signalling across cell membranes (“outside-in” and “insideout”), and have an impact on adhesion, invasion, migration, proliferation, survival, and apoptosis of cells.7,8 As receptors within the cell membrane, integrins are easily accessible for diagnostic purposes, such as imaging with targeted fluorescence-labelled, integrin subtype-selective, synthetic ligands.6 Integrin ␣v6 is a member of the integrin subfamily, which specifically recognises the tripeptide arginylglycylaspartic acid (RGD) within extracellular matrix protein ligands, including fibronectin, osteopontin, vitronectin, tenascin-C, together with the latency-associated protein of transforming growth factor- (TGF-). This keeps TGF- in an inactive state.9–12 Within the integrin subfamily, ␣v6 exerts unique features. It is primarily expressed during the epithelialmesenchymal transition during embryonic development, but downregulated in healthy, differentiated tissues.6,13 However, it becomes upregulated again with transition-associated processes such as tissue remodelling, wound healing, and the growth of invasive tumours.14,15 Considering that ␣v6 is specifically expressed as a high-affinity receptor in malignant
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cells, it qualifies not only as a promising diagnostic, but also as a therapeutic target.16,17 For use in imaging we exploited its binding specificity to the RGD-motif of extracellular matrix proteins. A new stable and ␣v6-selective RGD-peptide with subnanomolar binding affinity IC50 (inhibitory concentration50 ) of 0.26 nmol towards ␣v6 has recently been designed and developed.18 In this study we evaluated the use of this peptide, functionalised with Cy5.5, for fluorescent cytological imaging of bone-infiltrating carcinoma cells, as an aid to the intraoperative cytological assessment of bone resection margins (ICAB).
Patients and methods Patients with infiltrating SCC of the head and neck (n=122; 39 women; 83 men; mean (range) age 62 (39-89) years) who presented between March 2009 and January 2015 at the department of oral and maxillofacial surgery, University Hospital rechts der Isar, Technische Universität, München, were recruited. The primary tumour, including suspected involved bone, was resected. The extent of resection was defined preoperatively by clinical and computed tomographic (CT)/magnetic resonance (MRI) morphological staging. We then made a cytological assessment of the margins of resected bone.2–4 All patients provided written informed consent. The study was approved by the ethics committee of the Medical Faculty of the Technische Universität München (Ethics approval number: 2435/09). Tissue samples Specimens were taken from the bony resection margins, and, for positive controls, from bone that had been histologically confirmed as infiltrated with carcinoma (n = 362). Specimens from healthy bone were used as negative controls. Cytological preparations were imaged by fluorescent cytology. In cases of complex tissues, samples were mechanically disintegrated, suspended, and filtered before assessment (Supplemental Fig. S1 in the online version at DOI: 10.1016/j.bjoms.2018.11.003). Corresponding histological slides were prepared from each site according to standard protocols. Cytological assessment Cytological preparations were assessed by a cytopathologist, unaware of the area of interest. The results were classified as either negative/benign or positive/malignant. The cytological criteria for malignancy were defined by anisokaryosis, anisocytosis, increased nuclear size, and the ratio of nucleus:cytoplasm, heterochromatic nuclei, prominent and multiple nucleoli, and raised and abnormal concentrations of mitotic cells.
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Cell line and culture conditions A human oral SCC cell line of the head and neck was established from a cervical lymph node metastasis of an invasive SCC of the soft palate. The metastasis occurred seven years after treatment of the primary tumour and metastasised to the lung and brain.19 Cells were bought from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (DSMZ no. ACC 417) and cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) with 10% (v/v) fetal calf serum (Gibco, LifeTechnologiesTM ). The positivity of head and neck cells for integrin ␣v6 has been shown previously.18 Synthesis of integrin αvβ6-selective RGD peptides A cyclic RGD-peptide was synthesised as described recently.18 It has a high metabolic susceptibility and stability in human plasma for three hours with a subnanomolar binding affinity (IC50 0.26 nmol) and a high selectivity for ␣v6, compared to other integrin subtypes (IC50 ␣v3 632 nmol, ␣51 72.9 nmol, ␣v8 23.6, ␣v5, and ␣IIb3 >1000 nmol). For medical applications, the ␣v6-selective RGDpeptide was activated with a 6-aminohexanoic acid linker, bearing a Cy 5.5 fluorescent dye. Both the linker and the dye were attached through successive amide bonds to the Lys9 side chain of the cyclic RGD-peptide. Fluorescence imaging of HN cell line with αvβ6-selective RGD peptides SCC cells from the head and neck (25 × 103 cells/well) were ® cultivated for 24 hours on micro chamber slides (Nunc Lab® TM Tek Chamber Slide system, Sigma-Aldrich), coated with fibronectin (FN) (2 g/ml) (F1141 SIGMA, Sigma-Aldrich). The Cy5.5-conjugated ␣v6-binding peptide, dissolved in phosphate-buffered saline and 5% (v/v) dimethyl sulphoxide, was applied at a final concentration of 10 M for at least 20 minutes at room temperature, followed by three washes in phosphate-buffered saline. The slides were mounted and the intensity of the fluorescence signal visualised by a laserscanning Zeiss LSM 700 microscope (Zeiss). To convert the intensity of fluorescence-staining into colours, we used the “orange-to-white” scale provided with the software ZEN (Zeiss) (low intensity = red; medium intensity = yellow; and high intensity = white). Fluorescence-imaging of infiltrating carcinoma cells with αvβ6-selective RGD peptides Cytological preparations, which fulfilled the cytological criteria for malignancy and negative controls, were investigated by fluorescence staining with ␣v6-selective peptides. The Cy5.5-conjugated compound was applied at a final concentration of 10 mol for 20 minutes at room temperature, followed by three washes in phosphate-buffered saline. Fluorescence
signals were detected by the Zeiss LSM 700 microscope as described. Statistical analysis The fluorescence-imaging results were compared to the cytological findings of the corresponding sample. Sensitivity, specificity, the positive (PPV) and negative predictive value (NPV), correct classification and false classification rates, diagnostic odds ratio (OR) and the corresponding 95% CI were analysed by the IBM SPSS Statistics for Windows (version 20). The degree of agreement between the fluorescence-cytological and cytological findings was measured with Cohen’s kappa coefficient (). Substantial agreement was defined by > 0.61, almost perfect agreement by > 0.81, and a probability of 0.01 or less was accepted as significant.
Results Demarcation of αvβ6-positive, invasive carcinoma cells by fluorescence imaging with αvβ6-selective RGD peptides Fluorescence imaging showed a distinct signal on the membranes of malignant cells. These were highly selective for ␣v6-positive malignant cells and enabled the demarcation to ␣v6-negative cells on a single cell level. No non-specific background signal was detectable. Merging of the fluorescence imaging with the differential interference contrast enabled us to gain information about the molecular characteristics - for example, ␣v6 expression - in addition to the cytological morphology of the cells (Fig. 1). The high-binding affinity of the ␣v6-targeting RGD peptide together with its selectivity against other RGD-binding integrins enabled us to discriminate the fluorescent images of ␣v6-positive cells. Cell imaging with ␣v6-selective RGD peptides therefore offers a way to demarcate ␣v6-positive, invasive carcinoma cells. Targeting αvβ6-positive, infiltrating malignant cells for a fluorescence-assisted intraoperative cytological assessment of bony resection margins (F-AICAB) Cytological preparations were obtained intraoperatively within 7-10 minutes. A thorough cytological assessment took about15 minutes. Fluorescence imaging with ␣v6-selective RGD peptides was feasible within 20 minutes. Staining with haematoxylin and eosin enabled detection of carcinoma cells among bone marrow and blood cells.2 Fluorescence imaging of cytological samples, which were defined as positive or negative for bone-infiltrating malignant cells, showed a specific signal that was restricted to carcinoma cells. Differential interference contrast imaging of specimens of cancellous bone resulted in strong reflection of light and scattering at
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Fig. 1. Fluorescence imaging of invasive carcinoma cells by Cy5.5 activated ␣v6- selective RGD peptides. Imaging of the invasive head and neck cell line showed specific demarcation of a subgroup of ␣v6-stained carcinoma cells. No background signal was detectable in ␣v6-unstained cells. The intensity levels are indicated by the largest amounts of ␣v6 at the cell membranes (left). The merge of fluorescence signal with the differential interference contrast imaging enabled us to gain information about the molecular characteristics of ␣v6 expression in addition to the cytomorphological findings (right).
Fig. 2. The ␣v6-stained, squamous carcinoma cells that were infiltrating bone were stained with Cy5.5 activated ␣v6-selective RGD peptides for a fluorescence-assisted intraoperative cytological assessment of bony margins. The intraoperative finding, based on cytomorphological assessment, was obvious in the carcinoma cells. F-AICAB enabled a specific detection of bone-infiltrating carcinoma cells with activated ␣v6-selective RGD peptides, which correlated with the cytomorphological finding (left). Cancellous bone fragments did not show any false positive fluorescent signals despite reflection and scattering of light, which further emphasises the specificity of fluorescence imaging (arrow). The differential interference contrast imaging of squamous cell carcinoma cells that are infiltrating bone are shown on the right.
the fragments of bone, with no false positive signals in the fluorescence channel (Fig. 2). This indicated specificity of the fluorescence signal by ␣v6-selective RGD peptides. Negative controls (healthy specimens of bone) showed no non-specific or false positive signals (Fig. 3). Bony resection margins, which showed malignant cells, were confirmed by ␣v6-specific F-AICAB, which showed strong fluorescent signals in all the affected cells.
In summary, F-AICAB was diagnostically valuable in the demarcation of bone-infiltrating malignant cells from bone marrow derivatives, blood cells, and cancellous bone residues with 100% sensitivity and 98.3% specificity (Table 1). Fluorescence imaging with high affinity ␣v6-selective RGD peptides enabled specific detection of invasive malignant cells that were infiltrating bone after 20 minutes incubation, which is within the time frame for intraoperative analysis
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Fig. 3. Imaging of carcinoma-free mesenchymal bone with Cy5.5 activated ␣v6- selective RGD peptides. Overview of a carcinoma-free preparation as a negative control. No fluorescence signal was detectable because of non-specific cell binding of Cy5.5 activated ␣v6-selective RGD peptides (left). Differential interference contrast imaging of a benign specimen of mesenchymal bone showed no signs of malignancy in the cytomorphological finding (right). Table 1 Results of fluorescence-assisted intraoperative cytological assessment of bone resection margins. There were no false negatives. No. (%) (n = 362)
Sensitivity (%) (95% CI)
Specificity (95% CI)
PPV (95% CI)
NPV (95% CI)
CCR
FCR
DOR
AUC (95% CI)
True positive
58 (16)
92.06% (85.8% to 100%) – –
100% (90.8% to 99.7%) – –
1.38%
∞
95% p<0.001
0.99 (98% to 100%)
299 (82) 5 (1.3)
98.3% (95.1% to 100%) – –
98.6%
True negative False positive
100% (74.9 to 99.1) – –
– –
– –
– –
– –
– –
Fluorescence-assisted intraoperative cytological assessment of bone resection margins (F-AICAB), positive predictive value (PPV), negative predictive value (NPV), correct classification rate (CCR), false classification rate (FCR), diagnostic odds ratio (DOR), Cohen kappa coefficient (), confidence interval (CI), area under the curve (AUC)
of the margin. It confirmed the diagnostic results = 95% (p < 0.001).
Discussion Invaded margins increase by 90% the risk of death within five years, and double the risk of local recurrence, which makes the status of the margins the most important predictor of outcome for patients with SCC of the head and neck.20 Consequently, intraoperative assessment of the resection site remains a crucial diagnostic tool during operative treatment. The current guidelines recommend the microscopic inspection of frozen sections to control resection sites, avoid unnecessary radical operations, and preserve function.21 In case of R1-margins, further resection of residual carcinoma is suggested.22 The resection status at bony margins has been described as an independent prognostic factor for survival, and so the management of these margins is a relevant part of a curative treatment.3 As analysis of bony margins requires tissue decalcification before histological assessment, further resection of bony margins would necessitate a second oper-
ation, which takes time, costs more, and risks the result of the previous operation. After primary reconstruction with microvascular implants, a salvage operation would risk the state of the implants and the stability of wound healing, and delay adjuvant treatments. However, the aim for initial adequate resection margins does not justify the sacrifice of healthy tissue beyond the resection site.20 To enable the intraoperative management of bony resection sites, operative cytological assessment was introduced for the microscopic analysis of the sites where bone had been resected, enabling immediate further resection.2 It gave a high diagnostic yield and its clinical use resulted in a higher disease-free and overall survival for patients with SCC of the head and neck, and a considerable reduction in R1 resections.3 Any further improvements in the detection or demarcation of bone-infiltrating carcinoma cells, therefore, could improve clinical practice. A molecular marker with diagnostic applications should have three characteristics. First, it should be expressed on the surface of the cell for easy access; secondly, it should display low expression or be absent from normal tissues; and, thirdly, it should to be expressed at considerably higher levels in the corresponding
M. Nieberler et al. / British Journal of Oral and Maxillofacial Surgery 56 (2018) 972–978
malignant tissue than in surrounding tumour. Expression of integrin ␣v6 by bone-infiltrating carcinoma cells complies with these requirements. In contrast to other cell membrane-based proteins, which often serve as markers to discriminate cells of epithelial origin from mesenchymal tissue, ␣v6 expression has a further advantage: epithelial mesynchmal transition involves processes that alter cell morphology and profiles of gene expression, and result in the downregulation of epithelial marker proteins, such as cytokeratins or E-cadherin. Consequently, these markers fail to detect undifferentiated carcinoma cells. In oral SCC cells, ␣v6 expression initiates mesenchymal transition, accompanied by the expression of the mesenchymal marker vimentin, and reduced expression of E-cadherin, which facilitate motility and invasiveness of carcinoma cells.23 The value of targeting ␣v6 is further emphasised by its diverse functions in tumour biology, that have an impact on the proliferation of carcinoma cells; their migration, invasion, cell-cell and cell-extracellular matrix interactions; and so evade apoptosis that is attributable to the lack of appropriate ligand binding to extracellular matrix proteins.24,17 Imaging of ␣v6 directly therefore provides biological information about the tumour, which is beyond cytomorphology. Considering that high ␣v6 expression was detected in bone-infiltrating carcinoma cells, it becomes particularly interesting that ␣v6 upregulates MMP2, which promotes an osteolytic response.25 In principle, an immunofluorescence cytological stain would be an alternative choice to imaging ␣v6 expression, but antibody binding to ␣v6 would demand time beyond that available for an intraoperative analysis. For the first time, therefore, we applied high affinity selective ␣v6-targeting fluorescence-labelled RGD peptides for cytological imaging of ␣v6 expression. F-AICAB was highly specific with a sensitivity of 100%. In five cases false positive fluorescence signals were detected, which resulted from insufficient washing after the incubation with ␣v6-targeting fluorescent RGD peptides. In all consecutive applications, three washes were implemented routinely, which solved this technical problem. In summary, fluorescence cytological imaging of boneinfiltrating SCC cells in the head and neck using integrin ␣v6-targeting RGD peptides can improve the intraoperative control of bony margins. Future prospective application of F-AICAB as clinical routine will clarify whether it provides the reliability and diagnostic value to support intraoperative cytological assessment of bony margins as a diagnostic tool with therapeutic consequences that may have an impact on the clinical outcome of patients with SCC of the head and neck.
Conflict of interest We have no conflicts of interest.
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Ethics statement/confirmation of patients’ permission The study was approved by the ethic committee of the Medical Faculty of the Technische Universität München (Approval number: 2435/09). All patients provided written informed consent.
References 1. Casiglia J, Woo SB. A comprehensive review of oral cancer. Gen Dent 2001;49:72–82. 2. Nieberler M, Häußler P, Drecoll E, et al. Evaluation of intraoperative cytological assessment of bone resection margins in patients with oral squamous cell carcinoma. Cancer Cytopathol 2014;122: 646–56. 3. Nieberler M, Häußler P, Kesting M, et al. Clinical impact of intraoperative cytological assessment of bone resection margins in patients with head and neck carcinoma. Ann Surg Oncol 2016;23:3579–86. 4. Nieberler M, Häußler P, Kesting MR, et al. Intraoperative cell isolation for a cytological assessment of bone resection margins in patients with head and neck cancer. Br J Oral Maxillofac Surg 2017;55:510–6. 5. Nieberler M, Reuning U, Reichart F, et al. Exploring the role of RGDrecognizing integrins in cancer. Cancers (Basel) 2017;9:e116. 6. Niu G, Chen X. Why integrin as a primary target for imaging and therapy. Theranostics 2011;1:30–47. 7. Kang Y, Massagué J. Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 2004;118:277–9. 8. Higashikawa K, Yoneda S, Taki M, et al. Gene expression profiling to identify genes associated with high-invasiveness in human squamous cell carcinoma with epithelial-to-mesenchymal transition. Cancer Lett 2008;264:256–64. 9. Ruoslahti E, Pierschbacher MD. Arg-Gly-Asp: a versatile cell recognition signal. Cell 1986;44:517–8. 10. Busk M, Pytela R, Sheppard D. Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein. J Biol Chem 1992;267:5790–6. 11. Cheresh DA, Smith JW, Cooper HM, et al. A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells. Cell 1989;57:59–69. 12. Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984;309:30–3. 13. Breuss JM, Gillett N, Lu L, et al. Restricted distribution of integrin beta 6 mRNA in primate epithelial tissues. J Histochem Cytochem 1993;41:1521–7. 14. Yamada KM, Even-Ram S. Integrin regulation of growth factor receptors. Nat Cell Biol 2002;4:e75–6. 15. Breuss JM, Gallo J, DeLisser HM, et al. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 1995;108:2241–51. 16. Ramos DM, But M, Regezi J, et al. Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma. Matrix Biol 2002;21:297–307. 17. Bandyopadhyay A, Raghavan S. Defining the role of integrin alphavbeta6 in cancer. Curr Drug Targets 2009;10:645–52. 18. Maltsev O, Marelli UK, Kapp TG, et al. Stable peptides instead of stapled peptides: highly potent alphavbeta6-selective integrin ligands. Angew Chem Int Ed Engl 2016;55:1535–9. 19. Kawamata H, Nakashiro K, Uchida D, et al. Possible contribution of active MMP2 to lymph-node metastasis and secreted cathepsin L to bone invasion of newly established human oral-squamous-cancer cell lines. Int J Cancer 1997;70:120–7. 20. Binahme A, Nason RW, Abdoh AA. The clinical significance of the positive surgical margin in oral cancer. Oral Oncol 2007;43:780–4.
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21. Wolff KD, Follmann M, Nast A. The diagnosis and treatment of oral cavity cancer. Dtsch Arztebl Int 2012;109:829–35. 22. Bailey JS, Blanchaert Jr RH, Ord RA. Management of oral squamous cell carcinoma treated with inadequate excisional biopsy. J Oral Maxillofac Surg 2001;59:1007–11. 23. Ramos DM, Dang D, Sadler S. The role of the integrin alpha v beta6 in regulating the epithelial to mesenchymal transition in oral cancer. Anticancer Res 2009;29:125–30.
24. Janes SM, Watt FM. Switch from alphavbeta5 to alphavbeta6 integrin expression protects squamous cell carcinomas from anoikis. J Cell Biol 2004;166:419–31. 25. Dutta A, Li J, Lu H, et al. Integrin alphavbeta6 promotes an osteolytic program in cancer cells by upregulating MMP2. Cancer Res 2014;74:1598–608.
ScienceDirect British Journal of Oral and Maxillofacial Surgery 56 (2018) 972–978
Fluorescence imaging of invasive head and neck carcinoma cells with integrin ␣v6-targeting RGD-peptides: an approach to a fluorescence-assisted intraoperative cytological assessment of bony resection margins M. Nieberler a,∗,1 , U. Reuning b , H. Kessler c , F. Reichart c , G. Weirich d,2 , K.-D. Wolff a,1 a
Department of Oral and Maxillofacial Surgery, University Hospital rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81679 Munich, Germany b Klinische Forschergruppe der Frauenklinik, University Hospital rechts der Isar, Technischen Universität München, Ismaninger Strasse 22, 81675 München, Germany c Institute for Advanced Study and Center for Integrated Protein Science (CIPSM), Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany d Institute of Pathology, Technische Universität München, Trogerstr. 18, 81675 Munich, Germany Accepted 5 November 2018 Available online 28 November 2018
Abstract We assessed the use of peptides containing arginylglycylaspartic acid (RGD) that target integrin ␣v6 as a potential approach for a fluorescenceassisted intraoperative cytological assessment of bony resection margins (F-AICAB) in patients who had bone-infiltrating squamous cell carcinoma (SCC) of the head and neck. This was assessed to demarcate invasive carcinoma cells that stained for ␣v6. Specimens from bony resection margins (n=362) were defined as either malignant or benign according to the results of cytological and histological examinations. Integrin ␣v6-targeting fluorescence-labelled RGD peptides were added to the cytological samples and the accuracy of the resulting signal assessed by comparing it with the cytological findings. The value of F-AICAB was evaluated to find out if it could help to improve future diagnoses, tests, and treatments. Integrin ␣v6 was strongly expressed in invasive SCC cells and qualified as a marker for bone-infiltrating carcinoma cells. It showed a high affinity to bind to invasive SCC cells and enabled swift and specific demarcation of ␣v6-stained carcinoma cells. It was also diagnostic, with a sensitivity of 100% (95% CI 81.3% to 99.3%), specificity of 98.3% (95% CI 94.4% to 99.0%), positive predictive value of 92% (95% CI 70.2% to 94.3%), and negative predictive value of 100% (95% CI 96.9% to 99.9%), compared with the cytological findings. The targeting of specific integrin subtypes with selective, synthetic ligands, adapted for multimodal imaging, is a promising new approach to diagnosis. Further studies are necessary to provide more evidence for successful clinical translation and to establish the impact on clinical procedures. ﾩ 2018 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Head and neck squamous cell carcinoma; Intraoperative margin control; Cytology; RGD peptide; Fluorescence imaging; Integrins; ␣v6 integrin
∗ Corresponding author. Tel.: +49 89 4140 5916 or +49 163 4714383, Fax: +49 89 4140 4993. E-mail address: [email protected] (M. Nieberler). 1 Tel.: +49 89 4140 2921; Fax: +49 89 4140 4993. 2 Tel.: +49 89 4140 4161; Fax. +49 89 4140 4865.
https://doi.org/10.1016/j.bjoms.2018.11.003 0266-4356/ﾩ 2018 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
M. Nieberler et al. / British Journal of Oral and Maxillofacial Surgery 56 (2018) 972–978
Introduction For patients with squamous cell carcinoma (SCC) of the head and neck, the life-limiting factors are recurrence and metastases. Prognosis correlates with incomplete resection (R1-status), undetected second primary carcinomas, and occult metastases. Despite elaborate guidelines, advances in preoperative and intraoperative diagnostics and curative therapeutic strategies, the five-year survival rate remains at about 50% .1 Consequently, additional approaches to diagnosis and treatment should be considered. The technique of the resection deserves special consideration because it is under the direct control of the surgeon. Adequacy of resection is judged from frozen sections, but because of the technical limitations of processing calcified tissue, the intraoperative histological assessment of the bony margins is not possible. To bridge this diagnostic gap, the intraoperative cytological assessment of bony resection margins (ICAB) was introduced.2–4 Although the assessment has diagnostic value, a targeted fluorescent imaging of invasive malignant cells may provide diagnostic information beyond cytomorphology. In the search for diagnostic markers, the cell adhesion and signalling receptors of the integrin superfamily are well recognised as biologically relevant targets for malignant cells.5 Integrins are heterodimeric transmembrane receptors for extracellular matrix ligands, consisting of non-covalent, associated subunits (␣ and ). The 18 ␣ subunits can interact with eight  subunits, resulting in 24 different integrin heterodimers. The combination of the subunits ␣ and  defines the integrin-binding specificity of the ligands and occurs within distinct pathophysiological units of cell-cell and cellextracellular matrix interactions that communicate essential oncological functions.6 As well as their adhesive functions when they bind to the ligands, integrins initiate bidirectional signalling across cell membranes (“outside-in” and “insideout”), and have an impact on adhesion, invasion, migration, proliferation, survival, and apoptosis of cells.7,8 As receptors within the cell membrane, integrins are easily accessible for diagnostic purposes, such as imaging with targeted fluorescence-labelled, integrin subtype-selective, synthetic ligands.6 Integrin ␣v6 is a member of the integrin subfamily, which specifically recognises the tripeptide arginylglycylaspartic acid (RGD) within extracellular matrix protein ligands, including fibronectin, osteopontin, vitronectin, tenascin-C, together with the latency-associated protein of transforming growth factor- (TGF-). This keeps TGF- in an inactive state.9–12 Within the integrin subfamily, ␣v6 exerts unique features. It is primarily expressed during the epithelialmesenchymal transition during embryonic development, but downregulated in healthy, differentiated tissues.6,13 However, it becomes upregulated again with transition-associated processes such as tissue remodelling, wound healing, and the growth of invasive tumours.14,15 Considering that ␣v6 is specifically expressed as a high-affinity receptor in malignant
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cells, it qualifies not only as a promising diagnostic, but also as a therapeutic target.16,17 For use in imaging we exploited its binding specificity to the RGD-motif of extracellular matrix proteins. A new stable and ␣v6-selective RGD-peptide with subnanomolar binding affinity IC50 (inhibitory concentration50 ) of 0.26 nmol towards ␣v6 has recently been designed and developed.18 In this study we evaluated the use of this peptide, functionalised with Cy5.5, for fluorescent cytological imaging of bone-infiltrating carcinoma cells, as an aid to the intraoperative cytological assessment of bone resection margins (ICAB).
Patients and methods Patients with infiltrating SCC of the head and neck (n=122; 39 women; 83 men; mean (range) age 62 (39-89) years) who presented between March 2009 and January 2015 at the department of oral and maxillofacial surgery, University Hospital rechts der Isar, Technische Universität, München, were recruited. The primary tumour, including suspected involved bone, was resected. The extent of resection was defined preoperatively by clinical and computed tomographic (CT)/magnetic resonance (MRI) morphological staging. We then made a cytological assessment of the margins of resected bone.2–4 All patients provided written informed consent. The study was approved by the ethics committee of the Medical Faculty of the Technische Universität München (Ethics approval number: 2435/09). Tissue samples Specimens were taken from the bony resection margins, and, for positive controls, from bone that had been histologically confirmed as infiltrated with carcinoma (n = 362). Specimens from healthy bone were used as negative controls. Cytological preparations were imaged by fluorescent cytology. In cases of complex tissues, samples were mechanically disintegrated, suspended, and filtered before assessment (Supplemental Fig. S1 in the online version at DOI: 10.1016/j.bjoms.2018.11.003). Corresponding histological slides were prepared from each site according to standard protocols. Cytological assessment Cytological preparations were assessed by a cytopathologist, unaware of the area of interest. The results were classified as either negative/benign or positive/malignant. The cytological criteria for malignancy were defined by anisokaryosis, anisocytosis, increased nuclear size, and the ratio of nucleus:cytoplasm, heterochromatic nuclei, prominent and multiple nucleoli, and raised and abnormal concentrations of mitotic cells.
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Cell line and culture conditions A human oral SCC cell line of the head and neck was established from a cervical lymph node metastasis of an invasive SCC of the soft palate. The metastasis occurred seven years after treatment of the primary tumour and metastasised to the lung and brain.19 Cells were bought from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (DSMZ no. ACC 417) and cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) with 10% (v/v) fetal calf serum (Gibco, LifeTechnologiesTM ). The positivity of head and neck cells for integrin ␣v6 has been shown previously.18 Synthesis of integrin αvβ6-selective RGD peptides A cyclic RGD-peptide was synthesised as described recently.18 It has a high metabolic susceptibility and stability in human plasma for three hours with a subnanomolar binding affinity (IC50 0.26 nmol) and a high selectivity for ␣v6, compared to other integrin subtypes (IC50 ␣v3 632 nmol, ␣51 72.9 nmol, ␣v8 23.6, ␣v5, and ␣IIb3 >1000 nmol). For medical applications, the ␣v6-selective RGDpeptide was activated with a 6-aminohexanoic acid linker, bearing a Cy 5.5 fluorescent dye. Both the linker and the dye were attached through successive amide bonds to the Lys9 side chain of the cyclic RGD-peptide. Fluorescence imaging of HN cell line with αvβ6-selective RGD peptides SCC cells from the head and neck (25 × 103 cells/well) were ® cultivated for 24 hours on micro chamber slides (Nunc Lab® TM Tek Chamber Slide system, Sigma-Aldrich), coated with fibronectin (FN) (2 g/ml) (F1141 SIGMA, Sigma-Aldrich). The Cy5.5-conjugated ␣v6-binding peptide, dissolved in phosphate-buffered saline and 5% (v/v) dimethyl sulphoxide, was applied at a final concentration of 10 M for at least 20 minutes at room temperature, followed by three washes in phosphate-buffered saline. The slides were mounted and the intensity of the fluorescence signal visualised by a laserscanning Zeiss LSM 700 microscope (Zeiss). To convert the intensity of fluorescence-staining into colours, we used the “orange-to-white” scale provided with the software ZEN (Zeiss) (low intensity = red; medium intensity = yellow; and high intensity = white). Fluorescence-imaging of infiltrating carcinoma cells with αvβ6-selective RGD peptides Cytological preparations, which fulfilled the cytological criteria for malignancy and negative controls, were investigated by fluorescence staining with ␣v6-selective peptides. The Cy5.5-conjugated compound was applied at a final concentration of 10 mol for 20 minutes at room temperature, followed by three washes in phosphate-buffered saline. Fluorescence
signals were detected by the Zeiss LSM 700 microscope as described. Statistical analysis The fluorescence-imaging results were compared to the cytological findings of the corresponding sample. Sensitivity, specificity, the positive (PPV) and negative predictive value (NPV), correct classification and false classification rates, diagnostic odds ratio (OR) and the corresponding 95% CI were analysed by the IBM SPSS Statistics for Windows (version 20). The degree of agreement between the fluorescence-cytological and cytological findings was measured with Cohen’s kappa coefficient (). Substantial agreement was defined by > 0.61, almost perfect agreement by > 0.81, and a probability of 0.01 or less was accepted as significant.
Results Demarcation of αvβ6-positive, invasive carcinoma cells by fluorescence imaging with αvβ6-selective RGD peptides Fluorescence imaging showed a distinct signal on the membranes of malignant cells. These were highly selective for ␣v6-positive malignant cells and enabled the demarcation to ␣v6-negative cells on a single cell level. No non-specific background signal was detectable. Merging of the fluorescence imaging with the differential interference contrast enabled us to gain information about the molecular characteristics - for example, ␣v6 expression - in addition to the cytological morphology of the cells (Fig. 1). The high-binding affinity of the ␣v6-targeting RGD peptide together with its selectivity against other RGD-binding integrins enabled us to discriminate the fluorescent images of ␣v6-positive cells. Cell imaging with ␣v6-selective RGD peptides therefore offers a way to demarcate ␣v6-positive, invasive carcinoma cells. Targeting αvβ6-positive, infiltrating malignant cells for a fluorescence-assisted intraoperative cytological assessment of bony resection margins (F-AICAB) Cytological preparations were obtained intraoperatively within 7-10 minutes. A thorough cytological assessment took about15 minutes. Fluorescence imaging with ␣v6-selective RGD peptides was feasible within 20 minutes. Staining with haematoxylin and eosin enabled detection of carcinoma cells among bone marrow and blood cells.2 Fluorescence imaging of cytological samples, which were defined as positive or negative for bone-infiltrating malignant cells, showed a specific signal that was restricted to carcinoma cells. Differential interference contrast imaging of specimens of cancellous bone resulted in strong reflection of light and scattering at
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Fig. 1. Fluorescence imaging of invasive carcinoma cells by Cy5.5 activated ␣v6- selective RGD peptides. Imaging of the invasive head and neck cell line showed specific demarcation of a subgroup of ␣v6-stained carcinoma cells. No background signal was detectable in ␣v6-unstained cells. The intensity levels are indicated by the largest amounts of ␣v6 at the cell membranes (left). The merge of fluorescence signal with the differential interference contrast imaging enabled us to gain information about the molecular characteristics of ␣v6 expression in addition to the cytomorphological findings (right).
Fig. 2. The ␣v6-stained, squamous carcinoma cells that were infiltrating bone were stained with Cy5.5 activated ␣v6-selective RGD peptides for a fluorescence-assisted intraoperative cytological assessment of bony margins. The intraoperative finding, based on cytomorphological assessment, was obvious in the carcinoma cells. F-AICAB enabled a specific detection of bone-infiltrating carcinoma cells with activated ␣v6-selective RGD peptides, which correlated with the cytomorphological finding (left). Cancellous bone fragments did not show any false positive fluorescent signals despite reflection and scattering of light, which further emphasises the specificity of fluorescence imaging (arrow). The differential interference contrast imaging of squamous cell carcinoma cells that are infiltrating bone are shown on the right.
the fragments of bone, with no false positive signals in the fluorescence channel (Fig. 2). This indicated specificity of the fluorescence signal by ␣v6-selective RGD peptides. Negative controls (healthy specimens of bone) showed no non-specific or false positive signals (Fig. 3). Bony resection margins, which showed malignant cells, were confirmed by ␣v6-specific F-AICAB, which showed strong fluorescent signals in all the affected cells.
In summary, F-AICAB was diagnostically valuable in the demarcation of bone-infiltrating malignant cells from bone marrow derivatives, blood cells, and cancellous bone residues with 100% sensitivity and 98.3% specificity (Table 1). Fluorescence imaging with high affinity ␣v6-selective RGD peptides enabled specific detection of invasive malignant cells that were infiltrating bone after 20 minutes incubation, which is within the time frame for intraoperative analysis
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Fig. 3. Imaging of carcinoma-free mesenchymal bone with Cy5.5 activated ␣v6- selective RGD peptides. Overview of a carcinoma-free preparation as a negative control. No fluorescence signal was detectable because of non-specific cell binding of Cy5.5 activated ␣v6-selective RGD peptides (left). Differential interference contrast imaging of a benign specimen of mesenchymal bone showed no signs of malignancy in the cytomorphological finding (right). Table 1 Results of fluorescence-assisted intraoperative cytological assessment of bone resection margins. There were no false negatives. No. (%) (n = 362)
Sensitivity (%) (95% CI)
Specificity (95% CI)
PPV (95% CI)
NPV (95% CI)
CCR
FCR
DOR
AUC (95% CI)
True positive
58 (16)
92.06% (85.8% to 100%) – –
100% (90.8% to 99.7%) – –
1.38%
∞
95% p<0.001
0.99 (98% to 100%)
299 (82) 5 (1.3)
98.3% (95.1% to 100%) – –
98.6%
True negative False positive
100% (74.9 to 99.1) – –
– –
– –
– –
– –
– –
Fluorescence-assisted intraoperative cytological assessment of bone resection margins (F-AICAB), positive predictive value (PPV), negative predictive value (NPV), correct classification rate (CCR), false classification rate (FCR), diagnostic odds ratio (DOR), Cohen kappa coefficient (), confidence interval (CI), area under the curve (AUC)
of the margin. It confirmed the diagnostic results = 95% (p < 0.001).
Discussion Invaded margins increase by 90% the risk of death within five years, and double the risk of local recurrence, which makes the status of the margins the most important predictor of outcome for patients with SCC of the head and neck.20 Consequently, intraoperative assessment of the resection site remains a crucial diagnostic tool during operative treatment. The current guidelines recommend the microscopic inspection of frozen sections to control resection sites, avoid unnecessary radical operations, and preserve function.21 In case of R1-margins, further resection of residual carcinoma is suggested.22 The resection status at bony margins has been described as an independent prognostic factor for survival, and so the management of these margins is a relevant part of a curative treatment.3 As analysis of bony margins requires tissue decalcification before histological assessment, further resection of bony margins would necessitate a second oper-
ation, which takes time, costs more, and risks the result of the previous operation. After primary reconstruction with microvascular implants, a salvage operation would risk the state of the implants and the stability of wound healing, and delay adjuvant treatments. However, the aim for initial adequate resection margins does not justify the sacrifice of healthy tissue beyond the resection site.20 To enable the intraoperative management of bony resection sites, operative cytological assessment was introduced for the microscopic analysis of the sites where bone had been resected, enabling immediate further resection.2 It gave a high diagnostic yield and its clinical use resulted in a higher disease-free and overall survival for patients with SCC of the head and neck, and a considerable reduction in R1 resections.3 Any further improvements in the detection or demarcation of bone-infiltrating carcinoma cells, therefore, could improve clinical practice. A molecular marker with diagnostic applications should have three characteristics. First, it should be expressed on the surface of the cell for easy access; secondly, it should display low expression or be absent from normal tissues; and, thirdly, it should to be expressed at considerably higher levels in the corresponding
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malignant tissue than in surrounding tumour. Expression of integrin ␣v6 by bone-infiltrating carcinoma cells complies with these requirements. In contrast to other cell membrane-based proteins, which often serve as markers to discriminate cells of epithelial origin from mesenchymal tissue, ␣v6 expression has a further advantage: epithelial mesynchmal transition involves processes that alter cell morphology and profiles of gene expression, and result in the downregulation of epithelial marker proteins, such as cytokeratins or E-cadherin. Consequently, these markers fail to detect undifferentiated carcinoma cells. In oral SCC cells, ␣v6 expression initiates mesenchymal transition, accompanied by the expression of the mesenchymal marker vimentin, and reduced expression of E-cadherin, which facilitate motility and invasiveness of carcinoma cells.23 The value of targeting ␣v6 is further emphasised by its diverse functions in tumour biology, that have an impact on the proliferation of carcinoma cells; their migration, invasion, cell-cell and cell-extracellular matrix interactions; and so evade apoptosis that is attributable to the lack of appropriate ligand binding to extracellular matrix proteins.24,17 Imaging of ␣v6 directly therefore provides biological information about the tumour, which is beyond cytomorphology. Considering that high ␣v6 expression was detected in bone-infiltrating carcinoma cells, it becomes particularly interesting that ␣v6 upregulates MMP2, which promotes an osteolytic response.25 In principle, an immunofluorescence cytological stain would be an alternative choice to imaging ␣v6 expression, but antibody binding to ␣v6 would demand time beyond that available for an intraoperative analysis. For the first time, therefore, we applied high affinity selective ␣v6-targeting fluorescence-labelled RGD peptides for cytological imaging of ␣v6 expression. F-AICAB was highly specific with a sensitivity of 100%. In five cases false positive fluorescence signals were detected, which resulted from insufficient washing after the incubation with ␣v6-targeting fluorescent RGD peptides. In all consecutive applications, three washes were implemented routinely, which solved this technical problem. In summary, fluorescence cytological imaging of boneinfiltrating SCC cells in the head and neck using integrin ␣v6-targeting RGD peptides can improve the intraoperative control of bony margins. Future prospective application of F-AICAB as clinical routine will clarify whether it provides the reliability and diagnostic value to support intraoperative cytological assessment of bony margins as a diagnostic tool with therapeutic consequences that may have an impact on the clinical outcome of patients with SCC of the head and neck.
Conflict of interest We have no conflicts of interest.
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Ethics statement/confirmation of patients’ permission The study was approved by the ethic committee of the Medical Faculty of the Technische Universität München (Approval number: 2435/09). All patients provided written informed consent.
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