Defining secure surgical bone margins in head and neck squamous cell carcinomas: The diagnostic impact of intraoperative cytological assessment of bone resection margins compared with preoperative imaging

Oral Oncology 102 (2020) 104579

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Defining secure surgical bone margins in head and neck squamous cell carcinomas: The diagnostic impact of intraoperative cytological assessment of bone resection margins compared with preoperative imaging

T

Markus Nieberlera,⁎, Herbert Stimmerb, Daniela Rasthofera, Katharina Nentwiga, Gregor Weirichc, Klaus-Dietrich Wolffa a b c

Department of Oral and Maxillofacial Surgery, Hospital rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81679 Munich, Germany Department of Radiology, Hospital rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81679 Munich, Germany Institute of Pathology, Technische Universität München, Trogerstr. 18, 81675 Munich, Germany

ARTICLE INFO

ABSTRACT

Keywords: Head and neck squamous cell carcinoma Cytology Intraoperative surgical margin control Bone margins Imaging

Background: Imaging provides crucial staging information for treatment planning of head and neck squamous cell carcinomas (HNSCCs). Despite technical progress in imaging techniques, defining the extent of bone involvement preoperatively remains challenging and requires intraoperative information to control for adequate resection. The intraoperative cytological assessment of the bone resection margins (ICAB) provides information whether bone is infiltrated by carcinoma. The aim of this study was to assess the diagnostic value of preoperative imaging compared with ICAB in order to achieve carcinoma-free bone margins. Materials and Methods: 108 HNSCC patients underwent preoperative computed tomography (CT), magnetic resonance imaging (MRI) and orthopantomogram (OPG) for staging and surgical planning. Curative resection was planned based on imaging. Intraoperatively, the resection margins were controlled by ICAB. The diagnostic value of preoperative imaging and ICAB was assessed with reference to the histological findings. Results: CT showed a sensitivity of 89.7%, specificity of 63.0%, positive predictive value (PPV) of 85.9%, and negative predictive value (NPV) of 70.8%. MRI revealed a sensitivity of 45.5%, specificity of 66.7%, PPV of 71.4% and NPV of 40.0%. OPG-imaging had a sensitivity of 64.7%, specificity of 76.2%, PPV of 81.5%, NPV 57.1%. In comparison, ICAB provided a sensitivity of 78.6%, specificity of 95.7%, PPV 73.3%, and NPV 96.7%. The accuracy was 82.1%, 52.9%, 69.0%, and 93.5% for CT, MRI, OPG, and ICAB, respectively. Conclusion: Preoperative imaging lacks accuracy in defining adequate bone resection margins, compared with ICAB. ICAB supports preoperative imaging and intraoperative frozen sections to improve bone margin control.

Introduction Head and neck squamous cell carcinomas (HNSCC) represent 4–5% of all cancers. Among them, oral squamous cell carcinomas (OSCCs) are the most common and are localized in the oral cavity (88%), near the lips (8%), and the oropharynx (4%), with more than 300.000 new cases each year. As the 5-years survival rate remains at about 55% with no major progress having been achieved during the last years, an improvement in interdisciplinary diagnostic and therapeutic strategies is urgently needed [1–4]. Patients’ outcome depends on tumour size, localization, bone infiltration, local and distant metastasis, and margin status [5–7]. In particular, positive margins are associated with local recurrencewith a reduced 5-year survival rate [8–10]. In contrast to

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other prognostic factors, adequate resection margins are under the direct control of the surgeon. Thus, progress in intraoperative assessment of the surgical resection margins in curative intended surgery offers an approach for improving patient outcome. However, margin control may be difficult, because surgical treatment of invasive carcinomas often results in complex margins, including bone margins. As intraoperative histological information on soft tissue margins is available by frozen sections, but microscopic assessment of bone margin status is usually not available within the time-frame of the surgical treatment, the planning of the surgical treatment of bone-infiltrating carcinomas remains mainly based on preoperative imaging information. The most common imaging techniques for preoperative staging and surgical planning include MRI and CT imaging. But despite technical progress in

Corresponding author. E-mail address: [email protected] (M. Nieberler).

https://doi.org/10.1016/j.oraloncology.2020.104579 Received 2 July 2019; Received in revised form 9 January 2020; Accepted 20 January 2020 1368-8375/ © 2020 Elsevier Ltd. All rights reserved.

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imaging techniques, the definition of the extent of bone involvement preoperatively remains challenging. Further, the preoperative imaging information can not be transferred into the intraoperative setting to delineate the malignant from the healthy tissue border in real-time. This demands an improvement of direct intraoperative information in order to demarcate bone involvement by carcinoma growth for adequate resection. Curative surgical treatment aims at carcinoma-free margins with primary reconstruction of soft and hard tissue structures for functional and aesthetic rehabilitation [11–13]. Primary reconstruction demands a maximum of certainty with respect to surgical margin status and the recipient area for the microvascular transplant, because re-resection after reconstruction is frequently technically impossible [14]. Staging for surgical planning includes clinical examination, imaging and histological analysis. The demarcation of bone infiltration by carcinoma during staging is vital, as the infiltration of adjacent bone tissue may not be clinically apparent but decreases the 5-year survival rate to 50% or less [2,15]. Imaging should provide information about carcinoma localization, size and the involvement of adjacent structures, if a decision is to be made about the most effective therapy and in order to plan the surgical, but also the adjuvant treatment regimes [1,3,16]. Imaging for staging of HNSCC patients encompasses orthopantogram (OPG), computed tomography (CT) and/or magnetic resonance imaging (MRI). CT and MRI are recommended as the standard conventional imaging modality [17]. MRI is suggested for soft tissues, perineural and neurovascular involvement, and bone marrow infiltration [18–22]. CT imaging is thought to detect cortical erosion and bone medullar invasion, because of its high bone contrast and is often used for the planning of bone resection with primary reconstruction [18,22,23]. MRI should be the preferred method when metal artefacts of dental restorations are expected [22,24]. However, CT is more frequently applied because of its faster production, range of indication, and patient compliance [17]. Despite the technical progress, no imaging modality provides enough information to demarcate diseased from healthy tissue precisely, nor can the preoperative imaging information be transferred to the intraoperative situation without loss of information. Thus, uncertainty remains with respect to bone infiltration, which can lead to false assessments of the extent of bone affection by carcinoma growth and accordingly to over- or undertreatment. Because of the preoperative limitations of the assessment of bone infiltration and the intraoperative restriction of frozen sections to soft tissue, we have introduced the intraoperative cytological assessment of bone resection margins (ICAB) [25,26]. ICAB supports intraoperative margin analysis with a clinical impact on bone margin control and finally, on patient outcome [27]. Until now, the diagnostic value of ICAB and preoperative imaging modalities have not been critically compared regarding their potential to define an adequate site for resection in order to achieve carcinomafree bone margins. In this study, we have assessed the diagnostic value of ICAB compared with the information provided by preoperative imaging to determine secure surgical bone margins.

Table 1 Summary of interdisciplinary therapeutic procedures.

Type of resection

Primary reconstruction procedures of soft- and hard tissue structures

Neck dissection

Adjuvant therapy

Total number of patients: 108

n

%

Segmental mandibulectomy Horizontal marginal mandibulectomy Partial maxillectomy Lingual rim resection of the mandible Partial maxilla- and mandibulectomy Partial resection of temporal bone Resection of cortical mandible bone RFF

43 40

39.5 36.8

15 5

13.8 4.6

2

1.8

2

1.8

1

0.9

47

43.2

31 20 1 1 1

28.5 18.4 0.9 0.9 0.9

1 1 1 2 2

0.9 0.9 0.9 1.8 1.8

47 39 2 20

43.2 35.8 1.8 18.4

57 16

52.4 14.7

1 0 34

0.92 0.00 31.2

ALT flap Fibula transplant Iliac crest flap Existing perforator flap Perforator flap from lower leg Primary wound healing Iliac crest flap and RFF flap RFF and ALT flap Nasolabial flap Reconstruction without tissue transplant Both sides Ipsilateral side Lymph node biopsy No ND at the primary surgery Radiation Radiation and chemotherapy Radiation recommended Refused by patient No treatment

RFF = radial forearm flap, ALT = anterolateral thigh, ND = neck dissection.

intervention was performed in accordance with the Declaration of Helsinki and has been approved from an ethical and legal point of view by the ethical committee of the medical faculty of the Technische Universität München (registration number: 455/15 s). Preoperative CT imaging was carried out in 105 patients. MRI and OPG was part of the routine clinical staging in 34 patients and in 58 patients, respectively. Thirty-one patients underwent all three imagine modalities. Exclusion criteria, based on the histopathological results included benign odontogenic tumours, bony defects due to osteomyelitis or osteoradionecrosis and resections without bone structures. In addition, claustrophobic patients, pregnant patients, patients with a history of preoperative chemo- or radiation therapy, medically compromised patients or patients allergic to the radio contrast agent were excluded from the study. The histological findings served as a diagnostic reference. The clinical data of the selected patients are summarized in the supplementary Table 1, according to the UICC TNM Classification of Malignant Tumours (8th edition).

Materials and methods Patient cohorts The retrospective study includes 108 OSCC patients (73 men and 35 women; age range 27–89 years; mean age 63,4 at the day of surgery), who were referred to our clinic between February 2010 and April 2014 and underwent preoperative imaging. Surgical treatment was performed according to our interdisciplinary head and neck tumour board decision at the department for Oral and Maxillofacial Surgery of the Technische Universität München, Germany. Treatment included radical resection of primary tumour and radical or selective neck dissection. The study was conducted without aberration from the standard of care. Prior to primary reconstruction, soft tissue resection sites were controlled by frozen section analysis and bone margin by ICAB. The

Diagnostic and therapeutic procedures The therapeutic procedures were determined preoperatively on the basis of staging results by an interdisciplinary head and neck tumour board. The indication and extent of bone resection was defined by cancerous bone involvement and estimated risk of osseous infiltration, as suggested by clinical examination and imaging results. Eighteen images from referring hospitals or other private practice imaging centers were accepted and checked by our radiologist with regard to image quality, parameters of the CT, MRI or OPG and time interval between 2

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Table 2 Diagnostic value of ICAB and preoperative imaging to define bone resection margins in HNSCC patients. n (%) Patients CT

108 (100%) 95 (100%)

True positive True negative False positive False negative MRI

61 (64.2%) 17 (17.9%) 10 (10.5%) 7 (7.4%) 34 (100%)

True positive True negative False positive False negative OPG

10 (30.6%) 8 (22.2%) 4 (11.1%) 12 (36.1%) 55 (100%)

True positive True negative False positive False negative ICAB

22 (40.0%) 16 (29.1%) 5 (9.1%) 12 (21.8%) 107 (100%)

True positive True negative False positive False negative

11 (10.3%) 89 (83.2%) 4 (3.7%) 3 (2.8%)

Sensitivity (CI 95%)

Specificity (CI 95%)

PLHR

NLHR

PPV (CI 95%)

NPV (CI 95%)

CCR

FCR

DOR

Accuracy

κ

89.7% (79.9–95.8)

63.0% (42.4–80.6)

2.4

0.16

85.9% (78.7–90.9)

70.8% (48.9–87.4)

82.1%

17.9%

14.8

82.1%

0.55 p < 001

45.5% (25.6–67.2)

66.7% (34.9–90.1)

1.4

0.82

71.4% (44.9–99.2)

40.0% (18.1–61.6)

52.9%

47.1%

1.7

52.9%

0.11 p < 0.473

64.7% (42.1–77.1)

76.2% (52.8–91.8)

2.72

0.46

81.5% (66.3–90.7)

57.1% (44.4–69.0)

69.1%

30.9%

5.9

69.0%

0.38 p < 0.008

78.6% (49.2–95.3)

95.7% (89.4–98.8)

18.3

0.22

73.3% (44.9–92.2)

96.7% (90.8–99.3)

93.5%

6.5%

81.6

93.5%

0.72 p < 0.001

n = number; ICABs = intraoperative cytological assessments of bone resection margins; CT = computer tomography; MRI = magnet resonance imaging; OPG = orthopantomogramm; CI 95% = confidence interval; PLHR = positive likelihood ratio; NLHR = negative likelihood ratio; CCR = correct classification rate; DOR = diagnostic odds ratio; FCR = false classification rate; κ = Cohen kappa coefficient; NPV = negative predictive value; PPV = positive predictive value.

image and surgery. The time intervals between imaging and surgery are summarized in supplementary Table 2.

6856-1. OPGs were obtained during the staging process to assess the continuity of bone structures of the maxilla and mandibular bone. Postoperative OPG-morphological assessment provided information regarding the extent, localisation, and borders of resected bone.

CT imaging Preoperative multi-slice CT examination was performed with 16slice, 64-slice, 128-slice and 256-slice CT scanner. CT scans were performed 70 s after the administration of 70 ml i.v.-contrast agent. Multiplanar reconstructions were performed in all three planes perpendicular to the site of the primary tumor with both a soft-tissue postprocessing kernel and a soft-tissue window (slice thickness of 3 mm, no interslice gap).

Pre-and postoperative definition of the resections site Pre-and postoperative OPG-imaging in combination with CT/MRImorphology was used to define the location of the resection sites. Preoperatively, the resections site with presumably secure margins were defined by radiographic and MRI-morphological sings of bone infiltration by carcinoma growth and compared with the postoperative situation after bone resection. The radiographic images were assessed by a radiologist with no knowledge of the histological results and differentiated into three groups: non-infiltrated, erosive and infiltrated. The localization, tumour invasion, soft tissue extent and lymph node metastasis were evaluated. Comparison of the pre-operative images, which indicated the extent of the bone resection required for free bone margins, with the postoperative extent of bone resections was used to assess the diagnostic value of the pre-operative imaging information for defining adequate bone resection. The final margin status was defined by histology.

MR imaging MRI was performed on a 1.5 or 3 Tesla MRI Scanner, a Siemens Aera Scanner, or Siemens Avanto Scanner (Siemens Medical Solutions, Erlangen, Germany). A combined head-and-neck coil was used. The acquisition protocol consisted of the following sequences: T2w fat-saturated sequences (e.g., STIR) in all three (axial, coronal, sagittal) planes, axial T1 weighted turbo spin-echo (TSE) sequence before and after intravenous administration of gadolinium-DTPA (0.1 mmol/kg body weight), coronal T1 weighted TSE fat sat sequence after contrast. Slice thickness was 4 mm for all sequences.

Intraoperative cytological assessment of bone margins (ICAB)

OPG imaging

ICAB was performed as described previously [25,27]. Briefly, bone specimens were obtained from the bone resection margins with a cytobrush (Cytobrush Plus GT, Medscand Medical, Malmö, Sweden) at the Institute of Pathology, Technische Universität München and subjected to intraoperative cytological assessment. The specimens were fixed with Delaunay‘s solution for 1 min and stained with Hematoxylin-Solution (Merck, Darmstadt, Germany). Two cytopathologists evaluated the cytological samples independently and conveyed the results to the surgeons within 20 min. For diagnostic reference, the ICAB results were compared with the final histological findings and the preoperative

Conventional OPG examinations were obtained with orthopantomogram OraliX Multiscan (Philips GmbH, Health Systems, Hamburg, Germany). Imaging parameters (kV, mA, mAs) dependened on the constitution of each patient. On average, 70 kV were used. Attention was paid to a proper position of the patient, an aspect that can affect image quality. Other imaging-influencing factors include acquisition time, presence of metallic restorations or reconstruction plates, as well as contrast, sharpness, or density of the image. Afterwards, OPG images were evaluated systematically by our radiologist using a light box DIN 3

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imaging results.

procedures is shown in Table 1. The preoperative findings, considering the location and extent of carcinoma infiltration were compared to the final histological results. The imaging techniques revealed a sensitivity of 89.7% (CT), 45.5% (MRI) and 64.7% (OPG), and the specificity was 63.0% (CT) 66.7% (MRI) and 76.2% (OPG) to define adequate resection borders by preoperative imaging. In comparison, the cytological diagnostic by ICAB provided a sensitivity of 78.6% and a specificity of 95.7%. When the histological results were used as a reference, the accuracy of CT was 82.1% with κ = 0.55 (p < 0.001). MRI provided an accuracy of 52.9% with κ = 0.11 (p < 0.473), and standard radiographic imaging with OPG gave an accuracy of 69.0% with κ = 0.38 (p < 0.008). ICAB provided a 93.5% accuracy compared with the final histological results with κ = 0.72 (p < 0.001) Table 2. CT imaging was the most reliable preoperative source of information for assessing bone involvement by carcinoma growth. Thus, the diagnostic potential of CT imaging to differentiate between healthy, eroded and infiltrated bone by HNSCC was analyzed separately. Here, CT showed a sensitivity of 25% and a specificity of 89.5% with an accuracy of 78.3% (k = 0.16 (p < 0.001)). Infiltration in the cancellous bone was demarcated with a sensitivity of 92% and a specificity of 68%, and an accuracy of 84% (k = 0.63 (p < 0.001)). However, without the presence of cortical erosion, CT imaging revealed a high sensitivity of 92%, but a low specificity of 9.1% with an accuracy of 77.1% (k = 0.01 (p < 0.001)) Table 3. In summary, preoperative imaging lacks accuracy in defining adequate bone resection margins, compared with ICAB. ICAB supports preoperative imaging and intraoperative frozen sections to improve bone margin control Fig. 1.

Histopathological assessment Histopathological findings served as a standard of reference. The histopathological evaluation of the resected bone was provided by the Institute of Pathology, Technische Universität München. Hematoxylineosin staining was performed according the manufacturer’s instructions (Hematoxylin-Solution, Merck, Darmstadt, Germany). The histopatological findings for each patient were classified by two independent pathologists into three groups: non-infiltrated, cortical erosion, and cancellous bone infiltration. Bone infiltration was defined by carcinoma growth beyond cortical structures into the cancellous bone. Erosive carcinoma growth was defined as being osteolysis of the surface of the cortical bone. The carcinoma was classified according to the UICC TNM Classification of Malignant Tumours (7th edition December 2009). Bone resection margins were evaluated and defined according to the R-classification: carcinoma free (R0), microscopic (R1) or a macroscopic (R2) residual carcinoma. Statistical analysis. Statistical analysis Data were abstracted from the patients file records and the results of the histopathological evaluation were correlated with the preoperative formal written findings following MRI and CT. Cohen’s Kappa (κ) was used to assess the level of interobserver agreement between preoperative imaging and postoperative histopathology, whereby agreement attributable to chance was factored out. Values ranged between −1 and 1, with 1 indicating perfect agreement, −1 indicating perfect disagreement, and 0 indicating agreement attributable to chance [28]. Quality criteria such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy (DA) were calculated for both MRI and CT, by using the histopathological result as a reference. The statistical calculation was provided for all cases in which bone resection was performed based on preoperative staging examinations, and thus provided histological findings of the bone as a reference for the statistical calculation. The findings have been differentiated between non-infiltrated, cortical erosion, cancellous bone infiltration and no statement possible. With data of patients who were examined by both MRI and CT, a McNemar test was performed to compare the correctness of diagnosis of both imaging methods. A pvalue of <0.05 was considered to indicathttp://statpages.info/confint. htmle statistical significance. The data were analyzed with Excel and SPSS 20.0 (SPSS, Chicago, IL, USA) , www.alanfielding.co.uk/multivar/ accuracy.ht.

Discussion Our work has addressed the urgent issue how to control for adequate resection at bone margins. The underlying problem is defined by the lack of diagnostic value of pre- and intraoperative clinical and imaging assessment aimed at defining the presence and extent of carcinoma growth in bone. The clinical problem is not limited to HNSCCs. However, in HNSCCs functional consequences of the overtreatment or of failed resection are crucial. Overtreatment, including preventable resection of bony parts of the head region must be avoided, because loss of bone tissue can have a direct impact on vital functions, including speaking, chewing, swallowing and breathing. On the other hand, undertreatment with a missed resection, resulting in bone margins with microscopically present cancer cells (=R1 resections), reduces patient outcome, because R1status at bone margins has been recognized as an independent risk factor for HNSCC patients [27]. For staging and surgical planning, preoperative imaging by CT, MRI and functional imaging are used in clinical routine diagnostics to evaluate carcinoma infiltration [18,2931]. However, all imaging information is subjected to technical confounders, which has to be taken into account: With MRI, low image quality can be caused by motion artefacts, resulting from tongue movements or swallowing during the imaging process [32,33]. Falsepositive results can occur because of inflammatory conditions, osteoradionecrosis, dental extraction defects, or chemical shift artefacts by bone marrow fat, all of which may be interpreted as the malignant involvement of the bone and might lead to overtreatment [34-36]. On the other hand, metal artefacts can induce false positive or false negative CT results and can be limiting for the estimation of perineural involvement, bone marrow infiltration without cortical defect, or alveolar bone invasion [32]. To date, no significant differences between CT and MRI had been observed with respect to bone infiltration [18,32]. In our study, the sensitivity of CT was higher (89.7%) than that of MRI (45.5%). Thus, the risk of unnecessary resection of healthy tissue is higher, if surgical concepts would merely rely in CT imaging information.

Results The average interval between imaging and surgery was 18 days for CT and MRI and 33 days for OPG. Six patients were excluded from the study, because they underwent surgery more than 50 days after the CT/ MRI. With the exclusion of these patients, the mean range between CT and surgery was 15.6 days (minimum 1 day, maximum 47 days), MRI and surgery 17.2 days (minimum 1 day, maximum 42 days) and OPG and surgery 13.2 days (minimum 0 days, maximum 40 days). Further, patients lacing postoperative imaging control were excluded (n = 13). After being preoperatively staged, all patients were treated by primary surgical resection, based on clinical and CT/MRImorphological assessment, intraoperative cytology, and frozen sections. Forty-three patients underwent segmental mandibulectomies, 40 horizontal marginal resections of the mandible, 5 lingual rim resections and 15 partial maxillectomies. Main sites of OSCC included floor of the mouth, the tongue, and the mandible region. The interdisciplinary therapeutic procedures included primary reconstructions with microvascular fibula transplants, radial forearm flaps and anterior lateral thigh flaps. The detailed description of the interdisciplinary therapeutic 4

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Table 3 Diagnostic value of CT imaging to define bone erosion and infiltration in HNSCC patients. n (%) Patients Erosion CI 95% True positive True negative False positive False negative Infiltration CI 95% True positive True negative False positive False negative Infiltration/-Erosion CI 95% True positive True negative False positive False negative

108 (100%) 23 (100%) 1 (4.4%) 17 (73.9%) 2 (8.7%) 3 (13.0%) 75 (100%) 46 (61.3%) 17 (22.7%) 8 (10.7%) 4 (5.3%) 61 (100%) 46 (75.4%) 1 (1.6%) 10 (16.4%) 4 (6.6%)

Sensitivity

Specificity

PLHR

NLHR

PPV

NPV

CCR

FCR

DOR

Accuracy

κ

25% 0.6–80.6

89.5% 66.9–98.7

2.4

0.84

33.3% 0.8–90.6

85% 72.1–96.8

78.3%

21.7%

2.8

78.3%

0.16 p < 0.001

92% 80.8–97.8

68% 46.5–85.1

2.9

0.12

85.2% 72.9–93.4

81% 58.1–94.6

84%

16%

24.4

84%

0.63 p < 0.001

92.0% 80.8–97.8

9.1% 0.3–41.3

1.0

0.88

82.1% 69.6–91.1

20% 0.5–71.6

77.1%

22.9%

1.2

77.1%

0.01 p < 0.001

n = number; CT = computer tomography; CI 95% =confidence interval; PLHR = positive likelihood ratio; NLHR = negative likelihood ratio; CCR = correct classification rate; DOR = diagnostic odds ratio; FCR = false classification rate; κ = Cohen kappa coefficient; NPV = negative predictive value; PPV = positive predictive value.

conclusion, frozen sections and ICAB can support margin control at complex resection sites with bone and soft tissue surgical margins. In order to widen the application of ICAB, a protocol for the intraoperative cell isolation has been developed, which allows cytological assessment of complex surgical margins of bone and soft tissue [26]. Other sophisticated approaches have been described to enabling immediate microscopic bone assessment [37–44]. However, so far, none has been implemented in the clinical routine or tested to evaluate the clinical impact of intraoperative bone margin assessment. ICAB proved to be feasible in the clinical routine to supplement intraoperative frozen section analysis [25-27]. Nevertheless, the clinical establishment of ICAB disclosed technical and logistic limitations: These included desiccation of the bone resection margin, insufficient cellular or too much trabecular bone material, but also interfering blood cells, which resulted in low-quality cytological preparations. Therefore, we rinsed the bone margins to eliminate excess blood and surface contaminants, such as bone dust or floating carcinoma cells, which do not represent bone infiltration. Contamination of the surgical bone margins or the cytological preparations with carcinoma cells by instruments, gloves or the adherent carcinoma tissue during the transport to the pathological institute must be avoided to prevent false-positive findings. The diagnostic quality of ICAB further strongly depends on the cytological assessment, which should be reserved for an experienced cytologist, as previous radiotherapy may altered cell morphologies and immature hematopoietic precursor cells may even be mistaken for malignant cells. Further, complex margins, an infiltrative carcinoma growth, and preceding radiotherapy might challenge the techniques of both, frozen sections and ICAB. For these cases, a protocol for intraoperative cell isolation during cytological assessment of bone margins (ICI CAB) has been developed, which allows the analysis of isolated cells from complex surgical margins [26]. The time for the cytological preparation with subsequent intraoperative staining amounts to a maximum of seven minutes. The overall time, including the cytological assessment, takes about thirty minutes and does not exceed the time for standard frozen section. The ICAB and ICI CAB procedure do not demand specific surgical planning or logistics on the day of surgery. The cytological assessment of a larger amount of isolated cells can be supported by a further development of ICAB involving the application of fluorescently labelled synthetic integrin ligands that specifically bind to the integrin subtype αvβ6, which is expressed by invasive HNSCC cells [45]. These efforts aim to achieve a

Although unspecific inflammatory reaction of the bone marrow might result in an enhanced MRI signal that might be interpreted as carcinoma infiltration, MRI revealed a lower sensitivity compared to CT in our cohort. The reason might be that CT imaging has a high sensitivity for contrast-enhanced signals, derived from the cancellous bone. The specificity of both imaging techniques was comparable with 63.0% (CT) and 66.7% (MRI). Although CT imaging had a high sensitivity in cases of HNSCC infiltration of the cancellous bone, it revealed low sensitivity when used to assess cortical bone erosion by carcinoma growth. This brings the risk of false-positive findings and resection of healthy cancellous bone structures. In contrast, the low sensitivity to detect cortical erosion preoperatively can lead to inadequate bone margins. Further, the time between the preoperative imaging and the time of surgery has to be considered, because surgical treatment may be delayed for weeks to finish the staging process and to complete medical examinations. As carcinoma growth continues in the mean time, the imaging information may under-represent the extent of carcinoma infiltration. Consequently, preoperative staging cannot provide the information needed to define the extent of bone resection precisely, and thus each surgical concept with curative intent involves the risk of inadequate bone resection margins [25]. A further drawback is, that the imaging information cannot be transferred into the intraoperative setting to support the surgeon in real-time. Because of this lack of diagnostic information, ICAB was introduced to offer intraoperative information, regarding the resection status, with a microscopic standard to enable a controlled and guided bone resection. In this study, ICAB provided a sensitivity of 88.0% and a specificity of 78.6% and a specificity of 95.7% for the determination of adequate resection. Prior to the presented study, ICAB proved to be a valid diagnostic means for the assessment of carcinoma cell infiltration of bony tissue and a support for intraoperative margin analysis [25]. The clinical impact of ICAB on margin control and patient outcome is of relevance: Resection in case of positive ICAB finding can reduced osseous R1 resections, resulting in a significant reduction of overall R1 resections from 17 to 7.8 % (p = 0.026), compared to a control group. Patients, where ICAB was applied, revealed a higher disease-free survival [p(log-rank) = 0.045] and overall survival [p(log-rank) = 0.014] [27]. The exclusion of cases with advanced local (pN1) and distant metastasis (M = 1) further revealed that the resection status at bone margins is an independent prognostic factor for survival [27]. As a 5

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Fig. 1. CT-and MRI-morphological illustration of bone infiltration of HNSCC and the corresponding histological and cytological findings. A: CT imaging with signs of bone infiltration by an oral squamous cell carcinoma (OSCC) of the alveolar ridge. The borders are defined by osseous defect, as indicated by arrows. The soft tissue contrast does not allow a defined demarcation of carcinoma tissue. Bone-infiltrating carcinoma tissue cannot be specifically illustrated. B: MRI-morphological demonstration of the same patient reveals enhanced contrast in the region of prospective carcinoma infiltration. In contrast to the CT-morphological illustration, the region of enhanced contrast suggests bone infiltration beyond the osseous defect within the cancellous bone, as indicated by arrows. MRT-imaging information did not allow to distinguish between carcinoma tissue and edema or inflammation of the cancellous bone. C: Cytological finding of ICAB. HNSCC cells showed cytomorphological criteria for malignancy, which was correlating to the corresponding histological finding shown in D. (Magnification 1500×) D: Histological finding of an infiltrating pattern of HNSCC carcinoma (arrows) in the mandible at the bone resection margin *. (Magnification 150×, Hematoxylin and Eosin (H&E) staining is illustrated).

carcinoma-free resection status during a single surgical intervention. Primary reconstruction then gains importance, considering the advances that have been made in free-flap reconstructive techniques, which allow surgical concepts that were previously impossible. Large HNSCCs demand complex resections, which often result in compromised breathing, swallowing, and speech. To enable functional and morphological rehabilitation, the primary reconstruction of soft and hard tissue structures with microvascular osteo-(myo-)cutaneous transplants is often inevitable. However, if positive margins are confirmed by final histology, re-resection in a second operation after complex primary reconstructions might be technically impossible or compromise the microvascular transplant. ICAB might improve the surgical treatment of HNSCC patients, as the control of the resection status remains an important predictor of outcome and is under the direct influence of the surgeon [9].

of intraoperative information for achieving carcinoma-free margins to improve patient outcome and to secure an adequate recipient area for microvascular transplants. Consequently, the application of ICAB could especially be valuable to support the concept of the primary reconstruction of soft and hard tissue structures to improve functional rehabilitation and quality of life.

Conclusion

References

Declaration of Competing Interest None declared. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.oraloncology.2020.104579.

[1] Howaldt HP, Vorast H, Blecher JC, Reicherts M, Kainz M. The DÖSAK tumor register results. Mund-, Kiefer- und Gesichtschirurgie 2000;4(1):S216–25. [2] Uribe S, Rojas LA, Rosas CF. Accuracy of imaging methods for detection of bone tissue invasion in patients with oral squamous cell carcinoma. Dentomaxillofac Radiol 2013;42(6):20120346.

In head and neck surgery, ICAB provides valuable intraoperative information, ifurther to that from preoperative imaging, and can be considered to complement frozen sections analysis. For a maximum of certainty with respect to adequate margins, ICAB provides a novel level 6

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[25] Nieberler M, Hausler 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(9):646–56. [26] Nieberler M, Haussler 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(5):510–6. [27] Nieberler M, Haussler P, Kesting MR, et al. Clinical impact of intraoperative cytological assessment of bone resection margins in patients with head and neck carcinoma. Ann Surg Oncol 2016. [28] Altman DG. Practical statistics for medical research. Boca Raton, Fla: Chapman & Hall/CRC; 1999. [29] Close LG, Merkel M, Burns DK, Schaefer SD. Computed tomography in the assessment of mandibular invasion by intraoral carcinoma. Ann Otol Rhinol Laryngol 1986;95(4 Pt 1):383–8. [30] Brown JS, Griffith JF, Phelps PD, Browne RM. A comparison of different imaging modalities and direct inspection after periosteal stripping in predicting the invasion of the mandible by oral squamous cell carcinoma. Br J Oral Maxillofac Surg 1994;32(6):347–59. [31] van den Brekel MW, Runne RW, Smeele LE, Tiwari RM, Snow GB, Castelijns JA. Assessment of tumour invasion into the mandible: the value of different imaging techniques. Eur Radiol 1998;8(9):1552–7. [32] Gu DH, Yoon DY, Park CH, et al. CT, MR, 18F-FDG PET/CT, and their combined use for the assessment of mandibular invasion by squamous cell carcinomas of the oral cavity. Acta Radiol 2010;51(10):1111–9. [33] Imaizumi A, Yoshino N, Yamada I, et al. A potential pitfall of MR imaging for assessing mandibular invasion of squamous cell carcinoma in the oral cavity. AJNR Am J Neuroradiol 2006;27(1):114–22. [34] Chung TS, Yousem DM, Seigerman HM, Schlakman BN, Weinstein GS, Hayden RE. MR of mandibular invasion in patients with oral and oropharyngeal malignant neoplasms. AJNR Am J Neuroradiol 1994;15(10):1949–55. [35] Beyer H-K. Artefakte bei MRT-Untersuchungen. MRT der Gelenke und der Wirbelsäule: Radiologisch-orthopädische Diagnostik. Berlin, Heidelberg: Springer Berlin Heidelberg; 2003. p. 41–50. [36] Crecco M, Vidiri A, Angelone ML, Palma O, Morello R. Retromolar trigone tumors: evaluation by magnetic resonance imaging and correlation with pathological data. Eur J Radiol 1999;32(3):182–8. [37] Sangeetha R, Uma K, Chandavarkar V. Comparison of routine decalcification methods with microwave decalcification of bone and teeth. J Oral Maxillofac Pathol: JOMFP 2013;17(3):386–91. [38] Weisberger EC, Hilburn M, Johnson B, Nguyen C. Intraoperative microwave processing of bone margins during resection of head and neck cancer. Arch Otolaryngol Head Neck Surg 2001;127(7):790–3. [39] Bilodeau EA, Chiosea S. Oral squamous cell carcinoma with mandibular bone invasion: intraoperative evaluation of bone margins by routine frozen section. Head Neck Pathol 2011;5(3):216–20. [40] Forrest LA, Schuller DE, Lucas JG, Sullivan MJ. Rapid analysis of mandibular margins. The Laryngoscope 1995;105(5 Pt 1):475–7. [41] Forrest LA, Schuller DE, Karanfilov B, Lucas JG. Update on intraoperative analysis of mandibular margins. Am J Otolaryngol 1997;18(6):396–9. [42] Oxford LE, Ducic Y. Intraoperative evaluation of cortical bony margins with frozensection analysis. Otolaryngol–Head Neck Surg: Off J Am Acad Otolaryngol-Head Neck Surg 2006;134(1):138–41. [43] Hinni ML, Ferlito A, Brandwein-Gensler MS, et al. Surgical margins in head and neck cancer: a contemporary review. Head Neck 2013;35(9):1362–70. [44] Mahmood S, Conway D, Ramesar KC. Use of intraoperative cytologic assessment of mandibular marrow scrapings to predict resection margin status in patients with squamous cell carcinoma. J Oral Maxillofac Surg: Off J Am Assoc Oral Maxillofac Surg 2001;59(10):1138–41. [45] Nieberler M, Reuning U, Kessler H, Reichart F, Weirich G, Wolff KD. Fluorescence imaging of invasive head and neck carcinoma cells with integrin alphavbeta6-targeting RGD-peptides: an approach to a fluorescence-assisted intraoperative cytological assessment of bony resection margins. Br J Oral Maxillofac Surg 2018;56(10):972–8.

[3] Loeffelbein DJ, Kesting MR, Mielke E, Jonas M, Holzle F, Wolff KD. Image fusion of bone SPECT and CT - a specific diagnostic method in oral and maxillofacial surgery. Mund Kiefer Gesichtschir 2007;11(1):33–41. [4] Li HX, Zheng JH, Fan HX, Li HP, Gao ZX, Chen D. Expression of alphavbeta6 integrin and collagen fibre in oral squamous cell carcinoma: association with clinical outcomes and prognostic implications. J Oral Pathol Med 2013;42(7):547–56. [5] Sadick M, Schoenberg SO, Hoermann K, Sadick H. Current oncologic concepts and emerging techniques for imaging of head and neck squamous cell cancer. GMS Curr Top Otorhinolaryngol, Head Neck Surg 2012;11:Doc08. [6] Roh JL, Yeo NK, Kim JS, et al. Utility of 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography and positron emission tomography/computed tomography imaging in the preoperative staging of head and neck squamous cell carcinoma. Oral Oncol 2007;43(9):887–93. [7] Brandwein-Gensler M, Teixeira MS, Lewis CM, et al. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival. Am J Surg Pathol 2005;29(2):167–78. [8] Loree TR, Strong EW. Significance of positive margins in oral cavity squamous carcinoma. Am J Surg 1990;160(4):410–4. [9] Binahmed A, Nason RW, Abdoh AA. The clinical significance of the positive surgical margin in oral cancer. Oral Oncol 2007;43(8):780–4. [10] Rogers SN, Brown JS, Woolgar JA, et al. Survival following primary surgery for oral cancer. Oral Oncol 2009;45(3):201–11. [11] Markowitz BL, Calcaterra TC. Preoperative assessment and surgical planning for patients undergoing immediate composite reconstruction of oromandibular defects. Clin Plast Surg 1994;21(1):9–14. [12] Maciejewski A, Szymczyk C. Fibula free flap for mandible reconstruction: analysis of 30 consecutive cases and quality of life evaluation. J Reconstr Microsurg 2007;23(1):1–10. [13] Holzle F, Kesting MR, Holzle G, et al. Clinical outcome and patient satisfaction after mandibular reconstruction with free fibula flaps. Int J Oral Maxillofac Surg 2007;36(9):802–6. [14] Ord RA, Aisner S. Accuracy of frozen sections in assessing margins in oral cancer resection. J Oral Maxillofac Surg 1997;55(7):663–9. discussion 669–671. [15] Schaarschmidt BM, Heusch P, Buchbender C, et al. Locoregional tumour evaluation of squamous cell carcinoma in the head and neck area: a comparison between MRI, PET/CT and integrated PET/MRI. Eur J Nucl Med Mol Imaging 2016;43(1):92–102. [16] Shum JW, Dierks EJ. Evaluation and staging of the neck in patients with malignant disease. Oral Maxillof Surg Clinics North America 2014;26(2):209–21. [17] Sarrión Pérez MG, Bagán JV, Jiménez Y, Margaix M, Marzal C. Utility of imaging techniques in the diagnosis of oral cancer. J Cranio-Maxillof Surg 2015;43(9):1880–94. [18] Vidiri A, Guerrisi A, Pellini R, et al. Multi-detector row computed tomography (MDCT) and magnetic resonance imaging (MRI) in the evaluation of the mandibular invasion by squamous cell carcinomas (SCC) of the oral cavity. Correlation with pathological data. J Exp Clin Cancer Res 2010;29(1):73. [19] Kanda T, Kitajima K, Suenaga Y, et al. Value of retrospective image fusion of 18FFDG PET and MRI for preoperative staging of head and neck cancer: comparison with PET/CT and contrast-enhanced neck MRI. Eur J Radiol 2013;82(11):2005–10. [20] Wiener E, Pautke C, Link TM, Neff A, Kolk A. Comparison of 16-slice MSCT and MRI in the assessment of squamous cell carcinoma of the oral cavity. Eur J Radiol 2006;58(1):113–8. [21] Palasz P, Adamski L, Gorska-Chrzastek M, Starzynska A, Studniarek M. Contemporary diagnostic imaging of oral squamous cell carcinoma - a review of literature. Pol J Radiol 2017;82:193–202. [22] Handschel Jr, Naujoks C, Depprich RA, et al. CT-scan is a valuable tool to detect mandibular involvement in oral cancer patients. Oral Oncol 2012;48(4):361–6. [23] Li C, Men Y, Yang W, Pan J, Sun J, Li L. Computed tomography for the diagnosis of mandibular invasion caused by head and neck cancer: a systematic review comparing contrast-enhanced and plain computed tomography. J Oral Maxillofac Surg 2014;72(8):1601–15. [24] Kolk A, Schuster T, Chlebowski A, et al. Combined SPECT/CT improves detection of initial bone invasion and determination of resection margins in squamous cell carcinoma of the head and neck compared to conventional imaging modalities. Eur J Nucl Med Mol Imaging 2014;41(7):1363–74.

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