Joint Practice Guidelines for Radionuclide Lymphoscintigraphy for Sentinel Node Localization in Oral/Oropharyngeal Squamous Cell Carcinoma

Involvement of the cervical lymph nodes is the most important prognostic factor for patients with oral/ oropharyngeal squamous cell carcinoma (OSCC), and the decision of whether to electively treat patients with clinically negative necks remains a controversial topic. Sentinel node biopsy (SNB) provides a minimally invasive method for determining the disease status of the cervical node basin, without the need for a formal neck dissection. This technique potentially improves the accuracy of histologic nodal staging and avoids overtreating three-quarters of this patient population, minimizing associated morbidity. The technique has been validated for patients with OSCC, and larger-scale studies are in progress to determine its exact role in the management of this patient population. This document is designed to outline the current best practice guidelines for the This article
provision of SNB in patients with early-stage OSCC, and to provide a framework for the currently evolving recommendations for its use. Preparation of this guideline was carried out by a multidisciplinary surgical/nuclear medicine/pathology expert panel under the joint auspices of the European Association of Nuclear Medicine (EANM) Oncology Committee and the Sentinel European Node Trial (SENT) Committee.
Keywords Sentinel lymph node biopsy A Carcinomas, squamous cell A Head and neck neoplasms A Technetium Tc-99m human serum albumin colloid A Radionuclide imaging Oral/oropharyngeal squamous cell cancer (OSCC) is one of the most common cancers worldwide, with more than 274.000 new cases annually. [bib_ref] Global cancer statistics 2002, Parkin [/bib_ref] Three-quarters of cases affect people in the developing world, while in developed countries, OSCC is the eighth most prevalent form of cancer.
Determining the presence or absence of nodal metastasis is of paramount importance for staging, treatment planning, and prognosis. The incidence of occult metastases in patients with clinically node-negative (cN0) OSCC is high, with many series reporting rates greater than 30%. [bib_ref] Effectiveness of selective neck dissection for management of the clinically negative neck, Pitman [/bib_ref] [bib_ref] The patterns of cervical lymph node metastases from squamous carcinoma of the..., Shah [/bib_ref] [bib_ref] Critical assessment of supraomohyoid dissection, Spiro [/bib_ref] [bib_ref] The effect of occult nodal metastases on survival and regional control in..., Gourin [/bib_ref] Cervical lymph node involvement is the most important prognostic factor for patients with OSCC. [bib_ref] The effect of occult nodal metastases on survival and regional control in..., Gourin [/bib_ref] [bib_ref] Evaluation of cervical lymph node metastasis in squamous carcinoma of the head..., Don [/bib_ref] [bib_ref] The efficacy of comprehensive neck dissection with or without postoperative radiotherapy in..., Leemans [/bib_ref] Elective treatment of the clinically negative neck remains a controversial topic. Over the last two decades much work has been undertaken to find reliable predictors of occult metastases, of which tumor depth appears to be the best available. [bib_ref] Micrometastases in carcinoma of the upper aerodigestive tract: detection, risk of metastasizing,..., Ambrosch [/bib_ref] [bib_ref] Depth of invasion is the most significant histological predictor of subclinical cervical..., Kane [/bib_ref] [bib_ref] Predictive factors of occult metastasis and prognosis of clinical stages I and..., Amaral [/bib_ref] [bib_ref] Mode of invasion and lymph node metastasis in squamous cell carcinoma of..., Yamamoto [/bib_ref] However, the predictive capacity of tumor depth and other primary tumor characteristics are still insufficient to negate the need for surgical staging of the cervical node basin. [bib_ref] Does tumor depth affect nodal upstaging in squamous cell carcinoma of the..., Alkureishi [/bib_ref] Elective neck dissection (END) is the current gold-standard staging procedure for the cN0 neck, providing valuable prognostic information regarding nodal status and simultaneously treating those patients found to be node positive (pN?). Previously, ENDs invariably took the form of a modified radical neck dissection, however there is increasing evidence that selective neck dissection is as efficacious as comprehensive neck dissection in treating the negative neck. [bib_ref] Effectiveness of selective neck dissection for management of the clinically negative neck, Pitman [/bib_ref] [bib_ref] Modified neck dissection: a study of 967 cases from 1970 to 1980, Byers [/bib_ref] [bib_ref] Rationale for elective modified neck dissection, Byers [/bib_ref] [bib_ref] Cervical lymph node metastases-diagnostic, therapeutic and prognostic implications, Shah [/bib_ref] [bib_ref] Evolving role of modifications in neck dissection for oral squamous carcinoma, Shah [/bib_ref] [bib_ref] Results of a prospective trial on elective modified radical classical versus supraomohyoid..., Head [/bib_ref] [bib_ref] Rationale for elective neck dissection, Pitman [/bib_ref] [bib_ref] Management of the N0 neck in oral squamous cell carcinoma, Cheng [/bib_ref] The shift toward more conservative surgical procedures has occurred primarily in the last two decades, facilitated by the work undertaken by Lindberg, Byers, and Shah to describe the common patterns of lymphatic drainage. [bib_ref] The patterns of cervical lymph node metastases from squamous carcinoma of the..., Shah [/bib_ref] [bib_ref] Distribution of cervical lymph node metastases from squamous cell carcinoma of the..., Lindberg [/bib_ref] [bib_ref] Selective neck dissections for squamous carcinoma of the upper aerodigestive tract: patterns..., Byers [/bib_ref] Knowledge of these patterns has allowed the extent of neck dissections to be progressively limited to those nodal levels at highest risk, and sentinel node biopsy (SNB) represents an extension of this philosophy.
The aim of this review is to provide an evidence-based guideline for the use of sentinel node biopsy as a staging tool for patients with early OSCC, presenting the best available evidence at the time of writing.
The existing literature was reviewed, utilizing electronic techniques (Medline, Best Evidence, the Cochrane Library, Dare) and hand-searching techniques. Where little or no data existed from randomized controlled prospective trials, emphasis was given to data from large series or reports from recognized experts in the field. It is recognized that higher-level evidence from future studies may modify the recommendations made in this manuscript.

## Definition of a sentinel node
The sentinel node concept states that the spread of a tumor is embolic in nature, via the lymphatics to the firstechelon lymph node(s) encountered in the regional draining basin. These represent the lymph nodes most likely to harbor occult metastases, and are designated the sentinel lymph nodes (SLNs). Excisional biopsy and pathological evaluation of the SLNs therefore allows for prediction of the disease status of the remaining cervical lymph node basin, potentially avoiding the need for a neck dissection. Sentinel lymph nodes need not be those closest to the tumor, and there may be multiple SLNs. [bib_ref] Technical details of intraoperative lymphatic mapping for early stage melanoma, Morton [/bib_ref] With the application of early dynamic lymphoscintigraphy, lymphatic channels are usually visualized, and nodes on a direct drainage pathway may be distinguished. The practical approach may include the combination of available detection techniques.
Lymphatic mapping and sentinel node biopsy (SNB) were first reported in 1977 by Cabanas for penile cancer. [bib_ref] An approach for the treatment of penile carcinoma, Cabanas [/bib_ref] In 1992, Morton and colleagues first described the use of intradermal isosulfan blue dye injection for lymphatic mapping and SLN localization in patients with malignant melanoma. [bib_ref] Technical details of intraoperative lymphatic mapping for early stage melanoma, Morton [/bib_ref] The following year, Alex et al. described a peritumoral intradermal injection of radioactive tracer ( 99 m technetium sulfur colloid), followed by imaging and intraoperative gamma probe radiolocalization of SLNs. [bib_ref] Gamma-probe-guided lymph node localization in malignant melanoma, Alex [/bib_ref] The sentinel node concept has since been extensively studied and validated for patients with cutaneous melanoma and breast cancer. [bib_ref] Management of earlystage melanoma by intraoperative lymphatic mapping and selective lymphadenectomy, Morton [/bib_ref] [bib_ref] Surgical resection and radiolocalization of the SLN in breast cancer using a..., Krag [/bib_ref] Studies to date have also indicated a high level of accuracy in patients with OSCC. [bib_ref] Sentinel node biopsy in head and neck cancer: preliminary results of a..., Ross [/bib_ref] [bib_ref] Sentinel lymph node evaluation in squamous cell carcinoma of the head and..., Stoeckli [/bib_ref] 

## Clinical indications

## Inclusion criteria
The most important inclusion criterion for SNB is a clinically negative neck, as defined by physical examination and clinical imaging by computed tomography (CT), contrast-enhanced magnetic resonance imaging (MRI), ultrasound-guided fine-needle aspiration cytology (USg-FNAC), and/or 18F-fluoro-deoxyglucose (FDG) positron emission tomography (PET) with or without low-dose CT (PET/CT). [bib_ref] Positron emission tomography in combination with sentinel node biopsy reduces the rate..., Kovacs [/bib_ref] There remains considerable debate on the preferred imaging modality, and to date none have the ability to detect small or micrometastatic tumor deposits, but all techniques improve on the sensitivity of palpation alone and are therefore recommended prior to SNB. Recently, the high specificity of PET has been highlighted as an important means of avoiding unnecessary neck dissections. [bib_ref] Positron emission tomography in combination with sentinel node biopsy reduces the rate..., Kovacs [/bib_ref] Gross lymphatic involvement can lead to distortion of the normal architecture, leading to aberrant drainage patterns and biopsy of false sentinel nodes. [bib_ref] Predictive factors for failure to identify sentinel nodes in head and neck..., Hornstra [/bib_ref] SNB is therefore contraindicated to stage clinically positive necks.
Following the first report of SNB for OSCC, the technique has undergone extensive validation against the gold-standard END, for tumors located in the oral cavity and accessible subsites of the oropharynx. [bib_ref] Sentinel lymph node evaluation in squamous cell carcinoma of the head and..., Stoeckli [/bib_ref] [bib_ref] The gamma-probe-guided resection of radiolabeled primary lymph nodes, Alex [/bib_ref] [bib_ref] A suggested method for sentinel node biopsy in squamous cell carcinoma of..., Shoaib [/bib_ref] It has been demonstrated to be an accurate means of staging the clinically negative neck, and more recently the potential prognostic value of SNB for these tumor sites has also been highlighted. [bib_ref] Positive sentinel lymph nodes are a negative prognostic factor for survival in..., Kovacs [/bib_ref] While SNB has been successfully reported for tumors in other locations such as the hypopharynx and supraglottic larynx, there remain significant technical barriers, and SNB for these sites should therefore still be considered investigational.Poor access to these sites requires general anesthesia and endoscopic guidance for radiotracer injection, precluding the use of preoperative lymphoscintigraphy (LSG), while the close proximity of the primary tumor to the first-echelon lymph nodes can potentially obscure the true location of SLNs. Additionally, advanced stage at presentation is common for these hidden tumors, precluding the use of SNB or indeed any surgical intervention.
There is an existing consensus that SNB for OSCC should be restricted to early tumors staged T1/2. [bib_ref] The ability of lymphoscintigraphy to direct sentinel node biopsy in the clinically..., Ross [/bib_ref] [bib_ref] The first international conference on sentinel node biopsy in mucosal head and..., Ross [/bib_ref] [bib_ref] The second international conference on sentinel node biopsy in mucosal head and..., Stoeckli [/bib_ref] Larger tumors are difficult to completely surround with the tracer injection, tend to drain to multiple lymphatic basins, and in most patients require a neck dissection for access to the primary tumor or defect reconstruction. Inclusion of T3/T4 tumors in study protocols can lead to variability in the accuracy of the technique. [bib_ref] The ability of lymphoscintigraphy to direct sentinel node biopsy in the clinically..., Ross [/bib_ref] The first and most frequent indication for SNB is to stage the ipsilateral cN0 neck in patients with a unilateral primary tumor. A second indication is for assessment of bilateral cN0 necks in primary tumors close to, or crossing, the midline. The third indication is for assessment of the contralateral cN0 neck in primary tumors close to the midline with an ipsilateral cN? neck, in order to decide whether these patients need bilateral neck dissections, or an ipsilateral neck dissection and contralateral SNB only. Patients should also be fit enough preoperatively to withstand a neck dissection.
Patients who have received prior radiation or surgical treatment to the neck are routinely excluded from SNB protocols, since the previous intervention can distort the normal lymphatic pathways and give rise to unexpected patterns of metastasis. It is possible that lymphatic mapping and SNB may yield potentially useful information in these patients. Similarly, patients with small recurrent or second primary tumors may also benefit from lymphatic mapping to guide surgical intervention. However, these applications of the SNB technique, whilst clinically attractive, remain largely unexplored.

## Exclusion criteria
In pregnant women, the urgency and the necessity of staging the neck should be discussed. Lymphoscintigraphy is specifically contraindicated in the pelvis of pregnant women, but no such recommendations are currently available for the head and neck. As discussed in the ''Dosimetry'' section, the risk of fetal damage is negligible during routine SNB procedures. However, SNB protocols should be modified in pregnant patients to minimize risks of radiation exposure and blue-dye injections. For example, the use of a 1-day protocol allows a lower injected radiation dose, and the additional radiation associated with SPECT/CT imaging may not be warranted in the pregnant patient. SNB can be performed in lactating women, but it is advised that breastfeeding be discontinued following the procedure. OSCC is rare in children, though each case should be treated individually. In the UK, Nanocolloid is approved for use in children, though licencing varies between countries. The potential benefits of sentinel node biopsy are not as well delineated in the pediatric population and, in practice, most SNB trial protocols generally exclude these patients from participation. Off-label use of radiopharmaceuticals should be considered with caution and with respect to an individual risk-benefit analysis. Other relative contraindications include known allergy to albumin colloid, and primary tumor treatment by external-beam radiotherapy.
In summary, SNB is currently indicated for cT1/2, cN0 oral, and select oropharyngeal SCC, where it may be considered a valid alternative to elective neck dissection. Other head and neck sites, histologies, and clinical situations remain under investigation.

## Radiopharmaceuticals

# Introduction
A variety of colloidal and soluble tracers have been used over the years for lymph studies. It is believed that radiocolloids are taken up by macrophages in lymph nodes whereas the transit of macromolecules through lymph nodes is delayed simply because of their large molecular size. [bib_ref] Radiopharmaceuticals in sentinel lymph-node detection-an overview, Wilhelm [/bib_ref] 

## Choice of radiopharmaceutical
The main radiopharmaceutical used in European studies of sentinel node localization in oral cancers is Tc-99mlabeled human serum albumin colloid (Nanocoll O ) (GE Healthcare). Nanocoll O has a particle size range of 5-80 nm, with a reported mean size of 8-30 nm. [bib_ref] Radiopharmaceuticals in sentinel lymph-node detection-an overview, Wilhelm [/bib_ref] Although in theory a larger particle such as Albures O (GE Healthcare) or Sentiscint O (Medi-Radiopharma) may be preferred for tumors in the floor of mouth or anterior tongue where lymphatic densities are high, Nanocoll O performs satisfactorily in all tumor types studied. [bib_ref] The ability of lymphoscintigraphy to direct sentinel node biopsy in the clinically..., Ross [/bib_ref] Nanocoll O migrates to the sentinel node within minutes, yet prolonged retention allows surgery to take place the following day.
Other radiocolloids which have been used include Tc-99 m rhenium sulfide colloid (Nanocis O , IBA), which has been shown to have a mean particle size of 23-25 nm. [bib_ref] Particle sizes of colloids to be used in sentinel lymph node radiolocalization, Jimenez [/bib_ref] Tc-99 m sulfide colloid has also been used. Standard preparations of Tc-99 m sulfide colloid result in a wide range of particle sizes, so the product is often filtered through a 100-or 200-nm membrane filter to obtain a smaller and more uniform size distribution. While there are currently no clinical studies comparing different radiopharmaceuticals, investigators have described satisfactory results with each of the available colloids. [bib_ref] The diagnostic role of radioactivity in sentinel nodes in oral and oropharyngeal..., Kovacs [/bib_ref] [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] Preparation and Quality Control Nanocoll O is labeled by incubation with Tc-99 m pertechnetate at room temperature for 30 min.Radiochemical purity can be checked by thin-layer chromatography, and labeling efficiency should be [95%. The EANM guidelines on current good radiopharmacy practice (cGRPP, www.eanm.org) recommend that labeling efficiency be checked on each preparation. The stated expiry is 6 h after preparation, although extended stability has been demonstrated. [bib_ref] Validation of an extended shelf-life for Tc-99 m albumin nanocolloid injection, Millar [/bib_ref] 

## Drug interactions and adverse effects
No interactions of drugs with radiocolloids are expected following local intradermal or subcutaneous application. Adverse effects are rare and mild following interstitial application of radiocolloids, although allergic reactions have been reported with Nanocoll and the blue dyes used at surgery. [bib_ref] Allergic reaction to nanocolloid during lymphoscintigraphy for sentinel node biopsy, Burton [/bib_ref] [bib_ref] Allergy to technetium-labeled nanocolloidal albumin for sentinel node identification, Chicken [/bib_ref] [bib_ref] Hypersensitivity reaction against patent blue during sentinel lymph node removal in three..., Keller [/bib_ref] The incidence of allergic reactions is too low to quantify, but appropriate emergency medicines should be kept available during the procedure.

## Summary
Tc-99 m-labeled Nanocoll O is easy to prepare and supply, and has suitable properties for sentinel node localization in oral cancers, with rapid migration to the sentinel node and prolonged retention.

## Dosimetry: patient

## General remarks
Presently available dosimetric data are derived from the breast cancer SNB literature, where the absorbed doses to patients are determined to be low; therefore, the radiation risk associated with this procedure is low. While no specific OSCC data exist, the radiopharmaceuticals and administered activity are identical, leading to the assumption that OSCC SNB is a safe procedure from the radiation protection point of view.

## Patient exposure
The estimated local radiation dose varies greatly, depending on the administered dose and time to surgery. As mentioned in the ''Injection'' section, there is little consensus on the optimal administered dose and timing of surgery relative to the radiocolloid injection. Most centers perform LSG within 24 h of surgery, but recommendations for administered activity range from 15 MBq (for a sameday procedure) to 120 MBq (for a 2-day procedure) in a total injection volume of 0.4-1.0 ml. The aim is to achieve an activity of at least 10 MBq at the time of surgery. [bib_ref] Sentinel node detection using Tc-99 mrhenium sulphide colloid in breast cancer patients:..., Koizumi [/bib_ref] [bib_ref] EANM-EORTC recommendations for sentinel node diagnostics in melanoma, Chakera [/bib_ref] Current EANM guidelines for SNB in breast cancer report a mean value for the effective dose of 0.048 mSv. [bib_ref] Sentinel node in breast cancer procedural guidelines, Buscombe [/bib_ref] While other authors have reported doses approximately tenfold higher, these remain low compared with other nuclear medicine procedures. [bib_ref] Radiation safety of the sentinel node technique in breast cancer, Waddington [/bib_ref] Extensive calculations performed by the Memorial Sloan-Kettering Cancer Center have confirmed the safety of SNB by reporting an effective dose around 0.2 mSv. [bib_ref] Organ and fetal absorbed dose estimates from Tc-99 m-sulfur colloid lymphoscintigraphy and..., Pandit-Tskar [/bib_ref] 

## Fetal exposure
The maximum value for fetal absorbed dose has been calculated to be 0.013 mSv following injection of 18.5 MBq. [bib_ref] Organ and fetal absorbed dose estimates from Tc-99 m-sulfur colloid lymphoscintigraphy and..., Pandit-Tskar [/bib_ref] This dose is equivalent to that received by the mother from 1 day of natural background radiation in the USA, and is orders of magnitude below the 100-200-mSv threshold for deterministic effects (malformation, growth retardation, neurodevelopmental abnormalities). [bib_ref] Radiation safety of the sentinel node technique in breast cancer, Waddington [/bib_ref] Current consensus is that noncancer health effects are not detectable below 50 mSv, while congenital malformations occur above 200 mSv. [bib_ref] Organ and fetal absorbed dose estimates from Tc-99 m-sulfur colloid lymphoscintigraphy and..., Pandit-Tskar [/bib_ref] With respect to childhood cancer induction (stochastic effect), the International Commission on Radiological Protection (ICRP) reports a threshold of 10 mSv for a 40% risk increase.In summary, the advantages of SNB for OSCC outweigh the potential risks of the absorbed radiation dose, and this is also true for pregnant patients. While SNB is not contraindicated in pregnant patients, it is preferable to use a same-day protocol, enabling a lower injected dose. [bib_ref] EANM-EORTC recommendations for sentinel node diagnostics in melanoma, Chakera [/bib_ref] 

## Lactating women
The current recommendation is that nursing mothers should suspend breast-feeding for 24 h following radiopharmaceutical injection, during which time the general anesthetic agent and radiocolloid will be excreted in breast milk. 51

## Dosimetry: staff

## Staff in operating room
Studies in breast and melanoma patients have determined the mean whole-body dose received by surgical staff to be \1 lSv per operation, with the maximum dose to the surgeon of \2 lSv. [bib_ref] Radiation safety of the sentinel node technique in breast cancer, Waddington [/bib_ref] [bib_ref] Sentinel node detection in malignant melanoma patients: radiation safety considerations, Sera [/bib_ref] [bib_ref] Radiation safety with breast sentinel node biopsy, Stratmann [/bib_ref] [bib_ref] Radiation doses to staff involved in sentinel node operations for breast cancer, Klausen [/bib_ref] The absorbed doses are further minimized when SNB is performed at 24 h after injection. [bib_ref] Sentinel node biopsy in skin melanoma patients -measurements of absorbed doses of..., Nejc [/bib_ref] Monitoring of operating room personnel for occupational exposure during the procedure is therefore unnecessary, and additional shielding is not required. While the pregnant surgeon or scrub nurse requires specific consideration, participation in fewer than 100 SNB operations during the gestation will remain below the recommended radiation exposure limits for pregnant women. [bib_ref] Radiation doses to staff involved in sentinel node operations for breast cancer, Klausen [/bib_ref] 

## Staff in pathology department
Radiation exposure to pathology staff is very low, and should not require badge monitoring. Even personnel performing unusually high numbers of procedures receive radiation doses well below established limits for members of the general public. [bib_ref] Radiation safety considerations for sentinel node techniques, Glass [/bib_ref] 

## Radiation safety precautions
Labeling specimens as radioactive for transportation to the laboratory is not required, since the surface dose rate is \5 lGy/h.

## Radioactive clinical waste
Surgical instruments and pathology slides appear to stay at background radiation levels, while measurable contamination of absorptive surgical sponges and other materials used in proximity to the injection site is observed. [bib_ref] Safety guidelines for radiolocalised sentinel node resection, Nugent [/bib_ref] It is advisable to monitor these materials for contamination, and contaminated materials should be held for an appropriate period of decay in storage before disposal. 52,59

## Injection
The lymphatic anatomy within the oral cavity and oropharynx is extremely complicated and varies significantly between subsites, emphasizing the need for precise injection technique. 39,62,63

## Patient preparation
No special preparation is needed. Patients should be fully informed about the procedure, including discussion of potential problems such as bleeding and discomfort, before consent can be obtained.

## Syringe, activity, and volume
Tuberculin syringes with minimal dead space are recommended; otherwise 0.1 mL air may be drawn into the syringe behind the radiocolloid to ensure complete administration. A 25-or 27-gauge needle should be used. The total activity to be injected may vary, depending on the size and location of the primary tumor. As described in the ''Dosimetry'' section, there is currently little consensus on the optimum activity for injection, and this varies considerably from 15 to 120 MBq between studies. [bib_ref] The second international conference on sentinel node biopsy in mucosal head and..., Stoeckli [/bib_ref] [bib_ref] Sentinel node biopsy for oral and oropharyngeal squamous cell carcinoma of the..., Stoeckli [/bib_ref] [bib_ref] Lymphatic mapping to tailor selective lymphadenectomy in cN0 tongue carcinoma: beyond the..., De Cicco [/bib_ref] The total injected activity should be adjusted according to the timing of LSG with respect to surgery. Higher doses are required for a 2-day protocol, in order to ensure that the activity exceeds 10 MBq at the time of surgery. [bib_ref] EANM-EORTC recommendations for sentinel node diagnostics in melanoma, Chakera [/bib_ref] Small volumes of 0.1-0.2 mL per aliquot are recommended to minimize contamination due to the resistance of tongue tissue. Contamination can be avoided by placement of a sheet over the injected region and a gauze swab over the needle puncture site before withdrawal. Following injection, the skin/mucosa should be checked for contamination.

## Injection site and depth, and number of injections
Tracer should be injected at 0.1-0.5 cm from the tumor or scar margin. The number of aliquots to be injected varies (two to four) according to the size and location of the lesion. The tracer should be administered on each side of the tumor/scar, keeping as a reference the orientation of the surgical scar. For lesions in sites with abundant soft tissue (i.e., soft palate or floor of the mouth) four separate submucosal injections must be given around the lesion (at 3, 6, 9, and 12 o'clock). For lesions located in muscle (i.e., tongue), injections should be performed according to the depth of the lesion. Ideally, the operating surgeon should be present for the injections to ensure consistency with injection of blue dye if used. Following injection, bleeding may be controlled with a gauze swab, and the patient should be asked to use a mouth rinse to minimize pooling of the radiotracer in the oral cavity. [bib_ref] A suggested method for sentinel node biopsy in squamous cell carcinoma of..., Shoaib [/bib_ref] 

## Imaging

# Introduction
Lymphoscintigraphy uses a gamma camera to assess the drainage of injected radiotracer via the lymphatic capillaries to the larger collector lymphatics until it either passes through, or is retained within, the regional lymph nodes. [bib_ref] Clinical assessment of human lymph flow using removal rate constants of interstitial..., Modi [/bib_ref] Accurate preoperative localization and cutaneous marking of the SLNs correlates well with the precision of the surgical procedure. [bib_ref] Role of lymphoscintigraphy for selective sentinel lymphadenectomy, Uren [/bib_ref] [bib_ref] Kinetics of three lymphoscintigraphic agents in patients with cutaneous melanoma, Glass [/bib_ref] Cameras and Camera Settings (Quality Control)
A large-field-of-view gamma camera equipped with a high-or ultrahigh-resolution low-energy collimator should be used, with a 10-20% window centered on the 140-keV energy peak of Tc-99 m. A two-headed camera allows simultaneous dynamic acquisition in the anterior and a lateral projection, and saves time for the static images and SPECT. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] Quality control for the gamma camera should be routinely performed and should follow published protocols.

## Image acquisition
Dynamic acquisition for 20-30 min (20 s per frame) with a 128 9 128 matrix or 256 9 256 matrix starting immediately after radiotracer injection will show the drainage pattern and help to differentiate between sentinel nodes, which can appear very early following injection, and second-echelon lymph nodes. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] The impact of superficial injections of radiocolloids and dynamic lymphoscintigraphy on sentinel..., Tartaglione [/bib_ref] Two (or three if a three-headed camera is used) simultaneous images in the anterior and lateral projections are recommended.
Static images in the anterior and lateral projections from one or both sides (and oblique as needed) are acquired (300 s, with a 256 9 256 matrix) to localize the nodes in three dimensions. If hot nodes are not clearly depicted, static images can be repeated at, e.g., 2 h, 4-6 h or even just before surgery. The patient is imaged in the supine position with head up. A small flat pillow under the neck may help to fixate the head and neck area.
SPECT imaging may improve the identification of SLNs, especially close to the injection site. Lesion detectability is increased by attenuation and scatter correction, which is easily accomplished with hybrid SPECT/ CT devices. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] SPECT-CT for topographic mapping of sentinel lymph nodes prior to gamma probe-guided..., Wagner [/bib_ref] The increased anatomical detail provided with CT improves localization of SLNs to the anatomical neck level. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] SPECT acquisition parameters can be 128 9 128 matrix, 180in the anterior L-mode rotation, 3angle step, with 20-25 s per projection or 60 steps per head, 30 s each, with slice thickness of 4.42 mm. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] Lymphoscintigraphy for sentinel node mapping using a hybrid single photon emission CT..., Khafif [/bib_ref] CT acquisition parameters differ depending on the CT system used. To date, most reports on SPECT/CT for SNB in oral cancer have used a slow, low-end CT scanner (GE Hawkeye) with acquisition performed over 220using 16 s for each transaxial slice, with a fixed tube current of 2.5 mA, 140 kV, and slice thickness of 10 mm. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] Lymphoscintigraphy for sentinel node mapping using a hybrid single photon emission CT..., Khafif [/bib_ref] With fast, high-end CT scanners providing higher-quality CT scans, either a low dose or a higher dose of CT can be used. Low-dose parameters can be, e.g., 20 ms per slice, slice thickness/increment 3/3 mm, and 120 kV. If a diagnostic CT scan is required, IV contrast can also be used. If CT images are used for attenuation correction, inspection of both uncorrected and attenuation-corrected SPECT images is recommended, to avoid overlooking contrast-induced artifacts on the latter.
A number of studies have reported advantages of adding SPECT/CT to planar imaging, including identification of missed SLNs, exclusion of ambiguous SLNs, and/or better anatomical localization in 30-47% of patients. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] Lymphoscintigraphy for sentinel node mapping using a hybrid single photon emission CT..., Khafif [/bib_ref] However, it has been suggested that meticulous oblique planar imaging can visualize the additional SLNs seen with SPECT imaging, and this may represent an adequate alternative. [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] Furthermore, a number of investigators have reported no advantage to SPECT imaging with respect to the number and location of visualized hotspots. [bib_ref] Is there an additional value of SPECT/CT over lymphoscintigraphy for sentinel node..., Haerle [/bib_ref] [bib_ref] Sentinel lymph node mapping using SPECT-CT fusion imaging in patients with oral..., Keski-Santti [/bib_ref] The true role of SPECT imaging for OSCC SNB has yet to be determined. If used, SPECT/CT should not be a substitute for meticulous planar imaging technique.
The location of SLNs harvested during surgery does not always correlate perfectly with the preoperative imaging, though higher-quality CT images can allow visualization of individual SLNs \1 cm, leading to improved preoperative and intraoperative SLN localization. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] Multimodal image registration for localization of sentinel nodes in head and neck..., Lopez [/bib_ref] Body Contouring
To facilitate topographic localization, a 57Co flood source (or, if available, a 153Gd source) can be used for simultaneous transmission imaging in each projection. Since there is a risk of missing faint nodes when using a transmission source, it has been suggested to repeat the scan without a transmission source. [bib_ref] Lymphoscintigraphy in high-risk melanoma of the trunk: predicting draining node groups, defining..., Uren [/bib_ref] Alternatively, a radioactive point source may be used to outline the patient's contour while recording the scan.

## Image interpretation
On dynamic imaging, SLNs are identified as one or more foci to which lymphatic drainage passes, and may be multiple, in one or several areas of the neck, ipsilateral and/ or contralateral to the primary tumor. [bib_ref] Variability of cutaneous lymphatic flow rates in melanoma patients, Uren [/bib_ref] Imaging should begin immediately, since SLNs can be seen in the first minute after injection. [bib_ref] Lymphoscintigraphic details of sentinel lymph node detection in 82 patients with squamous..., Nieuwenhuis [/bib_ref] Foci appearing only on later images are also labeled as SLNs, and most appear within 1-3 h. [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] [bib_ref] Lymphoscintigraphy for sentinel node mapping using a hybrid SPECT/CT system, Even-Sapir [/bib_ref] According to some reports, SPECT/CT may identify a median of one additional SLN compared with planar imaging. In addition, SLNs located very close to the primary tumor may be detected by SPECT/CT but not with the gamma probe during surgery. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] While the benefits of SPECT/CT have not been universally accepted, both planar and SPECT images demonstrate good or excellent inter-and intraobserver agreement for evaluation of SLNs, with kappa values of 68-89%. [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] [bib_ref] Is there an additional value of SPECT/CT over lymphoscintigraphy for sentinel node..., Haerle [/bib_ref] [bib_ref] Sentinel lymph node mapping using SPECT-CT fusion imaging in patients with oral..., Keski-Santti [/bib_ref] Nonvisualization SLNs are usually detected between 15 and 60 min after radiotracer injection. Failure to detect SLNs may be related to incorrect injection technique or close proximity of SLNs to the injection site (e.g., floor of mouth tumors). In addition, metastatic deposits may block lymphatic drainage, causing nonvisualization of SLNs. [bib_ref] Predictive factors for failure to identify sentinel nodes in head and neck..., Hornstra [/bib_ref] [bib_ref] Need for intensive histopathologic analysis to determine lymph node metastases when using..., Bilde [/bib_ref] Repeat injection and imaging may be considered, however proceeding to neck dissection is preferred in order to avoid a false-negative SLN.

## Aberrant nodes and in-transit sentinel nodes
Individual lymphatic mapping by lymphoscintigraphy is a major advantage of SNB, demonstrating occasional unexpected drainage to, e.g., level IV or contralateral metastases from well-lateralized tumors. [bib_ref] Sentinel node biopsy for oral and oropharyngeal squamous cell carcinoma of the..., Stoeckli [/bib_ref] [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] [bib_ref] Need for intensive histopathologic analysis to determine lymph node metastases when using..., Bilde [/bib_ref] [bib_ref] Functional anatomy of the lymphatic drainage system of the upper aerodigestive tract..., Werner [/bib_ref] [bib_ref] Sentinel lymph node biopsy in squamous cell carcinomas of the lips and..., Frerich [/bib_ref] [bib_ref] The nodal neck level of sentinel lymph nodes in mucosal head and..., Shoaib [/bib_ref] [bib_ref] The number of sentinel nodes identified as prognostic factor in oral epidermoid..., Gallegos-Hernandez [/bib_ref] [bib_ref] Sentinel lymph-node biopsy in head and neck cancer, Hoft [/bib_ref] [bib_ref] Sentinel lymph node biopsy in oral cavity squamous cell carcinoma without clinically..., Kontio [/bib_ref] [bib_ref] Sentinel node biopsy in head and neck cancer, Mozzillo [/bib_ref] In addition, LSG has been reported to detect ''in-transit'' lymph nodes: SLNs lying between the primary tumor and the regional lymph basin. [bib_ref] Interval nodes: the forgotten sentinel nodes in patients with melanoma, Uren [/bib_ref] These have been described in the context of malignant melanoma, but to date there have been no reports of in-transit SLNs in OSCC.

## Report and display

# Introduction
There are two main indications for careful report and display of the results from lymphoscintigraphy:
(1) unambiguous guidance for surgical biopsy, and (2) a comprehensive dataset for ongoing/future studies. [bib_ref] The ability of lymphoscintigraphy to direct sentinel node biopsy in the clinically..., Ross [/bib_ref] [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] Report
The type of radiocolloid, lot number, volume injected, and effective dose should be recorded, along with the initials and title of the nuclear medicine physician or surgeon performing the injection. The type of camera used and imaging technique should be described in detail: [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] 1. Start time for dynamic imaging; 2. Timing and location of the first-echelon node(s) that appear; 3. Timing and number of anterior, posterior, and oblique recordings; 4. Timing and location of any additional (second-echelon) nodes; these should be clearly differentiated from the first-echelon nodes; 5. If CT or hybrid imaging is used, the manufacturer, software, and protocol should be described in detail. The number and location of nodes recorded by these modalities should be described and compared with planar recordings. It should be clearly stated if the results of the tomographic images differ from the planar recordings.
Shine-through from the primary tumor or opposite side should be described and marked clearly on the images. Increased absorption in the thyroid gland can be seen due to unstable colloid solution as a result of a colloid production error, and this may lead to difficulties in interpreting the lymphoscintigraphic images. [bib_ref] Lymphoscintigraphy in tumors of the head and neck using double tracer technique, Klutmann [/bib_ref] Artifacts may also occur due to cutaneous contamination at the time of injection. [bib_ref] Contamination problem with sentinel node localization procedure: a case study, Warner [/bib_ref] Rarely, a widened lymphatic capillary may form a ''colloid lake;'' however, the associated hotspot will invariably disappear during subsequent imaging, in contrast with true SLNs.

## Display
Lymphoscintigraphic findings should be summarized by the nuclear medicine physician, providing a clear, unambiguous report for preoperative consultation. In addition, hardcopy or digital copies of the LSG images should be available to the surgeon, both prior to and during surgery. [bib_ref] The ability of lymphoscintigraphy to direct sentinel node biopsy in the clinically..., Ross [/bib_ref] [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] Skin Marking
First-echelon nodes should be marked on the skin using one color of indelible marker, guided by gamma camera and handheld gamma probe. [bib_ref] Localization of sentinel nodes in breast cancer: a novel method and device..., Laasanen [/bib_ref] Second-echelon nodes should be marked with a different color, and clearly differentiated.

## Use of dye

# Introduction
The use of blue dye in head and neck mucosal cancer sentinel node surgery is optional. However, when used it is a useful adjunct to aid SLN localization and harvest. Blue dye cannot be used alone to perform OSCC SNB, but can be used in addition to radiolocalization with preoperative LSG and intraoperative gamma probe use. [bib_ref] A suggested method for sentinel node biopsy in squamous cell carcinoma of..., Shoaib [/bib_ref] Following injection, blue dye drains to the SLNs via the same lymphatic pathways as radiocolloid, staining the channels, which can then be followed to the first-echelon nodes. Direct visualization and dissection of these channels is a natural process for the head and neck surgeon.
Rarely, nonradioactive blue nodes may contain metastases in the absence of a tumor-positive radioactive node; two such SLNs were reported in a series of 40 patients undergoing SNB with both radiocolloid and blue dye injection. [bib_ref] The accuracy of head and neck carcinoma sentinel lymph node biopsy in..., Shoaib [/bib_ref] The handheld gamma probe is more sensitive for the detection of sentinel nodes, and not all radioactive nodes will also appear blue. [bib_ref] Sentinel node biopsy in head and neck cancer: preliminary results of a..., Ross [/bib_ref] However, blue dye may aid the surgeon perform SNB, both for technical success of the procedure and for identification of subclinical nodal metastasis.

## Contraindications and special precautions
Blue dye is contraindicated in children, pregnant women, lactating women, and those who have a history of allergy to the blue dye or any of its ingredients. It can, however, be used in all mucosal sentinel node procedures, for any malignant process for which the procedure has been deemed suitable, including OSCC.

## Blue dyes
In the UK and Europe, the blue dye used is Patent Blue V (Laboratoire Guerbet, Aulnay-Sous-Bois, France; which comes in 2-ml vials at 2.5% concentration. Outside of Europe, the use of other agents such as Isosulfan Blue (Lymphazurin TM) is more common. Gloves should be worn to avoid staining, and a gauze swab used to prevent dye spillage where possible. The dye will rarely mask the edge of the tumor and, if this is a concern, the tumor edge can be marked prior to injection with staples or diathermy marks.

## Fig. 1 patent blue v dye

## Timing of injection
Patent Blue V dye is injected at the time of surgery, under general anesthetic. It takes approximately 10-15 min for a significant amount of dye to travel from the injection site to the sentinel node and this is the approximate time it takes to scrub, prepare the patient, make the initial excision, and begin to explore the neck. The patient and anesthetist should be informed that the dye will be excreted in the urine, and the urine will remain discolored for approximately 2 days.

## Injection technique
One vial of dye is injected slowly into the tissues surrounding and deep to the tumor, to minimize leakage from ulcerated tumors. Gauze swabs may be used to protect normal tissue and mop up excess dye. The number of injections is usually between two and four, but is as many as is necessary to completely surround the tumor on its deep and lateral surfaces. Occasionally it may be necessary to grab the tongue with forceps or a retracting suture during the injection. The injection site should not be massaged, in order to maintain oncologic safety of the procedure.

## Adverse effects
Anaphylaxis and allergic reactions, while rare, are a possibility, and clinicians should be mindful of this during the injection. [bib_ref] Anaphylaxis to isosulfan blue and cross-reactivity to patent blue V: case report..., Scherer [/bib_ref] In the event of a reaction, the injection should be discontinued and appropriate resuscitation performed. A decision should be made as to whether to continue or abandon the procedure based on clinical findings and discussion between the surgeon and anesthetist. The mucosa is stained after injection, however the dye tends not to diffuse more than the margins of excision of the tumor and it has not been the authors' (T.S.) experience that dye interferes with pulse oximetry or pathologic interpretation of the excised tumor specimen. [bib_ref] A suggested method for sentinel node biopsy in squamous cell carcinoma of..., Shoaib [/bib_ref] In summary, injection of blue dye is a useful adjunct to gamma probe localization of the sentinel node. It is an optional procedure, but one that offers significant advantages for OSCC SNB.

## Gamma probe

# Introduction
There are a wide variety of gamma probes available with individual feature sets, each requiring specific training and information. In many countries, effective competency training is required as part of the regulations governing the use of radioactivity.

## Probe components
The gamma probe is a radiation detector, providing a count rate from gamma rays. The handheld probe contains the radiation detector, either a crystal or a solid-state device, with surrounding metal shielding and collimation to give a restricted field of view . It is connected to a power supply and an analyzer unit which receives electrical signals from the radiation detector. The analyzer and handheld probe together form the probe system, which may be powered by mains connection or battery. The analyzer provides a response related to the detected count rate, usually by audible pitch or volume variation and by a visual display as a dial or digital count rate (counts per second, cps). The probe technology is described in a number of reference books.Probe Size and Shape
Probes typically have outer dimensions of 12-15 mm, with smaller probes producing problems related to the smaller-less sensitive-detector, and less adequate shielding of the probe housing from gamma rays. Probe tips may be angled relative to the handle. This may be viewed as an advantage for minimal-access surgery, or a disadvantage due to surgeon preference.

## Probe performance
Probe performance is described in terms of its spatial resolution and its count sensitivity. [bib_ref] Specification and performance of intraoperative gamma probes for sentinel node detection, Perkins [/bib_ref] [bib_ref] Evaluation of surgical gamma probes for radioguided sentinel node localisation, Tiourina [/bib_ref] [bib_ref] A method to evaluate intra-operative gamma probes for sentinel lymph node localisation, Britten [/bib_ref] Spatial resolution indicates how spread out the signal is from a point source;
FIG. 2 Components of the gamma probe sensitivity is the number of cps for a given strength of source. At a typical node depth of 30 mm, a point-source node will appear to be about 25 mm wide due to the imperfect spatial resolution of the probe, and resolution worsens with increasing distance. Many nodes contain well below 1% of the injected activity, and with a 6 h half-life of Tc-99 m the activity in a given node can be low, particularly if the surgery is delayed after injection of the radiopharmaceutical. A probe should be able to achieve sensitivity in the range 650-900 cps/MBq of Tc-99 m for a 3-cm-deep node. For a 3-cm-deep node with 1% uptake from a 40 MBq injection of radiocolloid, with surgery at 2 h after injection, the surgeon will see a count rate of about 220 cps. The detected count rate falls rapidly with deeper nodes, and if this arises with lower percentage uptake and longer delay from surgery there may be a much lower count rate and more difficult localization. With experience, localization at low count rates is possible, but with greater variability, longer search time, and less confidence than at higher count rates.
The probe also picks up counts from sources that are not directly in front of the probe; gamma rays can penetrate through the side of the probe, and scattered gamma rays can enter the detector. Adequate shielding of the probe is therefore important, especially for OSCC due to close proximity of the injection site. The rejection of scatter is achieved by having a probe with a good ''energy resolution,'' and with a narrow window.

## Probe controls
The probe analyzer has a number of settings affecting practical performance of the probe, and therefore ease of SLN localization. These include:
Energy Window Setting For Tc-99 m, the probe should be set to a fixed energy level of 140 keV, but the ''width'' setting is variable. The wider the window, the higher the sensitivity, but the greater the scatter detected. This is especially problematic close to the injection site, and the ''high-sensitivity'' (wider window) setting is therefore most useful for low-uptake nodes remote from the injection site.
Collimation Collimators may be removable, allowing great gain in sensitivity while sacrificing spatial resolution. Removal of the collimation can aid localization of lowuptake nodes remote from the injection site.
Additional Shielding Direct penetration of gamma rays through the side of the probe may be reduced by the use of lead plates to shield the injection site.
Integration Time Some systems allow averaging of the signal over time, reducing signal variability. Integration times [1 s must be used with caution, since the user may be misled by the delay between the probe position and the corresponding sound signal.

## Count range
The probe produces an audible change in pitch between a minimum and maximum cps range, e.g., 100-1000 cps. Counts outside the set range will all produce the same (low or high) pitch, necessitating adjustment. Inappropriate range setting can lead to failure of localization. Some probe systems can automatically adjust the pitch range for the detected counts, though this can be confusing when trying to get a sense of the absolute count at any point.

## Care of the probe and quality assurance
All radiation detectors must be checked and managed within a quality assurance (QA) program. Surgeons are advised to work closely with their nuclear medicine colleagues and medical physicists in setting up quality control (QC) procedures. Recommendations for testing are:
- On purchase, tests of performance are advised to give a reference value for sensitivity, energy resolution, and spatial resolution, and to form a baseline for day-to-day checks; - Before each use, a basic check of function and performance with determination of count rate sensitivity to a long-lived radioactive source and its energy spectrum; - Visual inspection for damage, particularly cables and connectors. All users must be advised that the probe detector is easily damaged by dropping; - In the operating room, aiming the probe at the injection site can demonstrate that the probe is functioning; however, this is not a substitute for QC checks since even a 50% loss in sensitivity would not have any effect on the general response to the injection site.

## Sterility
The probe is placed into a sterile sheath, though this makes the probe tip larger. The skin surface may be scanned before sheathing, in which case the probe must be decontaminated by wiping with 70% alcohol or other supplier-recommended agent. When removing a sheath, care must be taken not to accidentally take off any removable collimator, since these are costly to replace.

## Surgical technique and gamma probe detection
The following remarks are valid provided that (a) preoperative lymphoscintigraphy is carried out and (b) no cervical cutaneous flap will be raised.

## Procedure
At the time of lymphoscintigraphy, SLNs are marked on the skin surface under scintigraphic guidance of a Co57labeled marker pen (held 90to the skin surface) and controlled transcutaneously by the nuclear medicine physician with a collimated, handheld gamma probe. Marking the skin with the head and neck in a position as similar as possible to the positioning during surgery may facilitate harvesting of the sentinel node.
Following radiotracer injection and LSG, patients undergo general anesthesia and preparation for operation. Optionally, blue dye may be injected at this time. Transoral excision of the primary tumor is performed either before or after sentinel node biopsy. Prior excision reduces the problem of shine-through from the injection site, but potentially limits the usefulness of blue dye due to rapid transit times through the lymphatics from the injection sites.
In the operating room, the gamma probe is covered with sterile latex and applied transcutaneously to confirm the accuracy of the skin markings, which may have changed due to changes in patient positioning between LSG and surgery. [bib_ref] The application of sentinel node radiolocalization to solid tumors of the head..., Alex [/bib_ref] The theoretically optimal search pattern is to start closest to the injection site, with the probe perpendicular to the skin, using a raster pattern of 2-cm-spaced parallel lines at right angles to the direction of the injection site. A rise in activity is then confirmed by scanning in the other direction. Scanning should be no faster than a few cm/s. However, excessively slow scanning can lead to loss of information from the change in pitch as the probe passes over a hot node. The drop in counts as the probe is angulated whilst over a hot spot can confirm location.

## Location
The lymphoscintigraphy images and skin markings guide the site of incision, which is placed along the relaxed skin tension lines and positioned to facilitate excision of the scar should subsequent neck dissection be required. Sentinel nodes are reached using one or more small incisions, and removed from levels I-V according to the Robbins classification. [bib_ref] Standardizing neck dissection terminology. Official Report of the Academy's Committee for Head..., Robbins [/bib_ref] Subplatysma skin flaps are not routinely raised for biopsy-only procedures. The gamma probe is introduced into the space along the plane of dissection and angled in various directions to guide the surgeon to the sentinel node, which is then excised. Sentinel nodes in the jugular chain are found close to the internal jugular vein and those in level I will usually be found in the submandibular triangle. If blue dye is used, blue-stained lymphatics may be followed to the sentinel nodes, which may be hot, blue or both. The anatomical location of the sentinel nodes should be noted, as should its color and radioactivity ex vivo in the operating theatre because both blue dye and radioactivity will dissipate before the pathological examination. In order to avoid potential confusion, surgeons and histopathologists should agree beforehand on the exact nomenclature used for labeling sentinel nodes and drainage basins. Following excision of SLNs, repeated readings are taken of the excision bed to ensure that there are no adjacent hot nodes that also need to be removed.

## Selection of nodes
In OSCC, multiple SLNs are usually present, with reported mean numbers of 1.3 to 4 (range 1-11).Preoperative LSG may underestimate the number of SLNs, especially when multiple SLNs are in close proximity. [bib_ref] Sentinel lymph-node biopsy in head and neck cancer, Hoft [/bib_ref] [bib_ref] Staging N0 oral cancer: lymphoscintigraphy and conventional imaging, Thomsen [/bib_ref] However, the numerous other advantages of preoperative LSG counter the suggestion that it may safely be omitted from the procedure. [bib_ref] Sentinel lymph-node biopsy in head and neck cancer, Hoft [/bib_ref] Careful consideration of inactive lymph nodes in the immediate vicinity of SLNs is imperative. Although nonsentinel nodes should not be excised, there may be a scenario where closely adjacent nodes are almost completely excised while dissecting out a sentinel node. Although this is uncommon, the nonsentinel node thus labeled may be sent for pathological examination. [bib_ref] Sentinel lymph node excision. Treatment method of the N0 neck in patients..., Kovacs [/bib_ref] While preoperative imaging should detect the majority of grossly involved nodes, clinical staging remains imperfect. [bib_ref] Sentinel node in head and neck cancer: use of size criterion to..., Alkureishi [/bib_ref] Any suspicious lymph nodes observed during SNB must be excised, even in the absence of radioactivity, since gross lymphatic involvement may block the flow of radiotracer to these nodes. See also the section on ''Measured Radioactivity.'' SLNs are ranked according to their respective tracer uptake ex vivo, with the SLN with the highest activity named SLN1, that with the second highest activity SLN2, and so forth. This does not mean that SLN2 is dependent on SLN1, as metastases may be found in any of the multiple SLNs independently. [bib_ref] The accuracy of head and neck carcinoma sentinel lymph node biopsy in..., Shoaib [/bib_ref] [bib_ref] The definition of a sentinel node, Nieweg [/bib_ref] [bib_ref] Number and location of radiolabeled, intraoperatively identified sentinel nodes in 48 head..., Werner [/bib_ref] [bib_ref] A new approach to pretreatment assessment of the N0 neck in oral..., Hyde [/bib_ref] Close Spatial Relation
The problems of shine-through, whereby the high radioactivity levels from the injection site are detected from behind the tissue of interest, and scatter, in which the direction of radioactivity from the injection site is changed by the tissues and detected by the probe, are most prevalent in the submandibular and submental areas of the neck.For floor-ofmouth tumors, where the distance between primary site and SLN is smallest, this creates technical difficulty and results in lower SLN identification rates (86%, versus 96% for other OSCC subsites). [bib_ref] Sentinel node biopsy in head and neck cancer: preliminary results of a..., Ross [/bib_ref] [bib_ref] Sentinel node biopsy in oral/oropharyngeal squamous cell cancer: five year follow-up, Alkureishi [/bib_ref] [bib_ref] Sentinel node biopsy in oral squamous cell carcinoma, Civantos [/bib_ref] Careful positioning of the gamma probe, judicious use of malleable lead shields, and excision of the primary tumor before SLN localization may all help to minimize these effects. [bib_ref] A suggested method for sentinel node biopsy in squamous cell carcinoma of..., Shoaib [/bib_ref] Another option to improve identification in level I is to perform some initial dissection below the level of the marginal mandibular nerve, transecting the tissues down to the level of the mylohyoid muscle. In this manner, the lymph nodes are mobilized away from the oral cavity, and the gamma probe placed into the newly created tunnel and directed inferiorly away from the injection site. [bib_ref] Sentinel node biopsy in cancer of the oral cavity. Operative technique, Civantos [/bib_ref] 

## Activity counting
Following excision, SLN radioactivity is confirmed ex vivo using the gamma probe, and must be above background activity to be classified as hot. [bib_ref] Sentinel lymph node biopsy in oral cancer: validation of technique and clinical..., Thomsen [/bib_ref] The SLN should be placed on a surface away from the patient, or on the upturned probe tip (facing the ceiling). Anatomic location and radioactivity levels (cps) are recorded for all excised nodes. All radioactive nodes should be considered SLNs because, while there exists some confusion over the exact definition of a SLN, it is best to err on the side of patient safety. [bib_ref] The definition of a sentinel node, Nieweg [/bib_ref] The lymphatic basin should be rechecked for reduced radioactivity after SLN excision. [bib_ref] A preliminary study on sentinel lymph node biopsy: feasibility and predictive ability..., Chikamatsu [/bib_ref] Bed counts in the neck after removal of SLN almost never exceed 8-10 cps (with the head of the probe slightly turned away from the injection site).

## Risk
Sentinel node biopsy is not without risks, and injuries to the facial and spinal accessory nerves are possible through minimal exposure. Although complications rates of less than 1% are reported, the risk of injury to these nerves via minimal-access incisions is theoretically higher during SNB as compared with neck dissection. Similarly, neck dissection following positive SNB represents re-exploration in a recently operated field, and carries with it the higher risks of nerve or vessel damage. This reinforces the need for minimal tissue injury during the initial SNB procedure. [bib_ref] Sentinel node biopsy in cancer of the oral cavity. Operative technique, Civantos [/bib_ref] Experience It is clear that experience is needed before a surgeon starts performing sentinel node biopsy, as it carries a steep learning curve. This has led to the recommendation of completion of at least ten SNB-assisted elective neck dissections before the procedure is performed alone. [bib_ref] The first international conference on sentinel node biopsy in mucosal head and..., Ross [/bib_ref] In addition, it is necessary for practitioners of SNB to understand the theoretical aspects, including handling of radioactivity and optimal use of gamma probes.

## Measured radioactivity (cps)
The role of measured cps during gamma probe detection is unclear. Because of the narrow anatomic relationships, defining a lower cutoff point for SLNs is practically impossible. It has been suggested that the number of harvested SLNs may be limited to the three nodes with the highest absolute cps, or the highest ratio of ex vivo nodeto-background activity (as for early studies in melanoma), in order to reduce surgical morbidity. [bib_ref] Gamma-probe-guided lymph node localization in malignant melanoma, Alex [/bib_ref] [bib_ref] Sentinel lymph-node biopsy in head and neck cancer, Hoft [/bib_ref] [bib_ref] Sentinel node detection in N0 cancer of the pharynx and larynx, Werner [/bib_ref] [bib_ref] Intraoperative radiolympho-scintigraphy improves sentinel lymph node identification for patients with melanoma, Albertini [/bib_ref] [bib_ref] Intraoperative lymphatic mapping for early-stage melanoma of the head and neck, Bostick [/bib_ref] [bib_ref] The clinical relevance of sentinel lymph nodes identified with radiolymphoscintigraphy, Brobeil [/bib_ref] [bib_ref] The use of intraoperative radiolymphoscintigraphy for sentinel lymph node biopsy in patients..., Glass [/bib_ref] For correct staging, at least the three nodes with the highest activity should be excised as SLNs, and all positive sentinels are detected within the first five nodes of highest activity in each patient. [bib_ref] Sentinel lymph-node biopsy in head and neck cancer, Hoft [/bib_ref] [bib_ref] Sentinel lymph node excision. Treatment method of the N0 neck in patients..., Kovacs [/bib_ref] [bib_ref] Sentinel node detection in N0 cancer of the pharynx and larynx, Werner [/bib_ref] More than five SLNs are very rare, with three-quarters of patients having B3 SLNs. For safety's sake, all radioactive nodes should be excised. [bib_ref] The role of SPECT-CT in the lymphoscintigraphic identification of sentinel nodes in..., Bilde [/bib_ref] [bib_ref] Sentinel lymph node excision. Treatment method of the N0 neck in patients..., Kovacs [/bib_ref] In a study investigating the role of radioactivity in SNB, Kovacs et al. found no significant difference in absolute cps between positive and negative SLNs (medians of 157 and 235 cps, respectively), and the positive SLN need not have the highest tracer accumulation (range 13-4,716 cps). [bib_ref] Sentinel lymph node excision. Treatment method of the N0 neck in patients..., Kovacs [/bib_ref] Activity in SLNs was not found to correlate with administered dose (in MBq), and the highest activity was found in level II. The authors reported that in each patient there was one SLN with a significantly higher cps rate than the remaining active nodes, and this node could be found in all levels.
Following SLN excision, the remaining lymphatic basin is searched for residual radioactive nodes by means of an in situ survey measurement. A count rate less than one-tenth of the excised node with the lowest radioactivity is considered indicative that all SLNs have been identified and removed. In some centers, lymph nodes with count rates of less than one-tenth of the ''hottest'' excised node are not removed. This practice is based on the results of the large Sunbelt Melanoma trial, demonstrating a low failure rate of 2%. [bib_ref] Sunbelt Melanoma Trial. Lessons learned from the Sunbelt Melanoma Trial, Mcmasters [/bib_ref] However, no similar data have yet been reported for SCC.
The time from injection has also been found not to affect the relative counts between SLNs, or if it does, it affects the results only in case of very large time spans of [14 h, depending on the half-life of the tracer used. [bib_ref] The definition of the sentinel lymph node in melanoma based on radioactive..., Carlson [/bib_ref] [bib_ref] Standardized probedirected sentinel node dissection in melanoma, Essner [/bib_ref] It is important, however, that the time span between injection and surgery is consistent for a given study population.
As a result, the absolute radioactivity counts are less important than relative levels between the excised nodes in the context of SNB. Similarly, absolute radioactivity counts cannot be compared between centers due to differences in protocol.

## Pathology evaluation of sentinel node
In OSCC, SNB has been examined only in the context of relatively small observational cohort studies, and the pathology protocols have typically been designed to detect micrometastases (MMs) and isolated tumor cells (ITCs) with high sensitivity. At present, the significance of finding MMs and ITCs is unknown in OSCC. The grade of evidence is currently at level III, as described in the SIGN methodology for clinical guidelines. 122 Several large-scale validation studies (ACOSOG and the University of Miami, SENT, the Danish national group, and the Brazilian head and neck group) are currently underway. [bib_ref] Sentinel node biopsy in cancer of the oral cavity. Operative technique, Civantos [/bib_ref] [bib_ref] Sentinel node biopsy in cancer of the oral cavity, Civantos [/bib_ref] [bib_ref] Sentinel node biopsy for squamous cell carcinoma of the oral cavity: preliminary..., Ross [/bib_ref] [bib_ref] Need for intensive histopathologic analysis to determine lymph node metastases when using..., Bilde [/bib_ref] [bib_ref] Predictive value of sentinel node biopsy in head and neck cancer, Chone [/bib_ref] 

## Histological definitions
The present International Union against Cancer (UICC) definitions are shown in [fig_ref] TABLE 1 UICC: No contact with vessel wall before a further six numbered serial sections... [/fig_ref].Whilst these definitions have been largely accepted, little information is available regarding the evidence on which they were based. The relationship of these definitions to tumor-node-metastasis (TNM) coding is demonstrated in [fig_ref] TABLE 2: Comparison of UICC and TNM classifications Generic TNM coding for sentinel nodes... [/fig_ref].
One element which is clearly subjective in nature is the assessment of cell viability, as the significance of individual cytokeratin-positive ''nonviable cells'' is difficult to establish. Various terms have been employed for these cells, including ''mummified cells'' or thanatosomes. [bib_ref] Thanatosomes'': a unifying morphogenetic concept for tumor hyaline globules related to apoptosis, Papadimitriou [/bib_ref] The present authors' practice (K.H., P.S.) is to include such elements in the morphological description, with careful correlation to the adjacent hematoxylin and eosin (H&E)stained section. Features which may be useful include the lack of a nucleus, but the biological potential of these cells is as yet unknown.
In addition, the presence of cells in the lymphatic plexus should always be recorded. Whilst their significance is unknown, it appears that they do not represent extracapsular extension of tumor.

## Protocol
A well-defined, written, standard operating procedure should be established between the surgical team and the reporting pathology laboratory. This should include how the specimen should be delivered to the laboratory, and outline the supporting documentation which accompanies it and appropriate elements of radiological protection practice. Other important elements may include agreed turnaround times and the manner in which the results are to be reported. An overview of the proposed pathologic evaluation protocol is presented in [fig_ref] FIG. 3: Pathology evaluation of sentinel lymph nodes FIG [/fig_ref].

## Gross sectioning
The node or nodal basin should be fixed in 10% neutral buffered formalin (or equivalent) for 24 h, as per standard laboratory practice. The nodes are described macroscopically, including dimensions, and excess fat is carefully trimmed away. Nodes less than 2 mm (longest dimension) should be processed whole, while nodes 2-5 mm should be cut through the hilum or longest pole-to-pole diameter, and both halves processed en face. Nodes greater than 5 mm should be cut into 2-mm slices, longest pole to pole, with processing of all slices en face.
Step Sectioning A routine H&E section is prepared, and metastatic disease reported if present. If negative, six exact serial sections are mounted, and separately numbered 1-6. Next, 150 lm material is discarded, or retained for research, Much of the published literature has utilized AE1/AE3 pancytokeratin antibody. However, concern has been expressed regarding the specificity of this anticytokeratin cocktail. Cross-reactivity is a problem seen with a number of pan-cytokeratin antibodies, and may mandate the use of more than one antibody in SNB protocols to clearly delineate MMs and ITCs from other elements in the node, such as dendritic cells and macrophages. However, presently it is reasonable to assume that any reputable commercially available pancytokeratin antibody (such as AE1/AE3 or MNF116) may be used. It is important to recognize artifacts, and any cytokeratin-positive components should always be compared with adjacent sections stained by hematoxylin and eosin [bib_ref] The effect of occult nodal metastases on survival and regional control in..., Gourin [/bib_ref] [bib_ref] Evaluation of cervical lymph node metastasis in squamous carcinoma of the head..., Don [/bib_ref] [bib_ref] The efficacy of comprehensive neck dissection with or without postoperative radiotherapy in..., Leemans [/bib_ref] [bib_ref] Micrometastases in carcinoma of the upper aerodigestive tract: detection, risk of metastasizing,..., Ambrosch [/bib_ref] [bib_ref] Depth of invasion is the most significant histological predictor of subclinical cervical..., Kane [/bib_ref].

## Microscopy
Sections should be examined using a good-quality bright-field microscope and equivocal findings discussed with an experienced colleague. Where pancytokeratinpositive cells are present it is essential that adjacent sections are stained to allow morphological comparison.

## Report
The side, number, and level of each node basin in the neck must be recorded. A diagram provided by the surgical team should be incorporated into the pathology record where possible.
The report must include details of the numbers of nodes found in each individual basin and which nodes were hot, blue, both or unlabeled. The dimensions of each node must be included and the macroscopic appearance of the gross and cut surfaces stated.

## - macrometastasis
- Note the largest dimension of the metastatic deposit in each node, and whether extracapsular spread is present or not.
- Micrometastasis
- Should be recorded, even in the presence of macrometastasis - The largest dimension should be recorded - Anatomical location within the node: capsular, subcapsular, parenchymal - Unifocal or multifocal: it is often not possible to be confident of the exact numbers - Presence of extracapsular spread. This is permitted if the deposit is peripherally located and is associated with a reactive stromal response

## - isolated tumor cells (itc)
- If evident, should be recorded, even in the presence of macro-or micrometastasis - If cohesive: the size of the largest deposit should be stated - If dispersed: note the anatomical distribution - Benign inclusions including nevus cells, salivary inclusions, and false-positive cytokeratin artifacts (e.g., dendritic cells or scattered nonviable anucleate cells) should be recorded. The histopathological features of ITCs need careful description, as the UICC size cutoff by necessity encompasses small metastases which vary greatly in size and presumably in biological potential. Positive findings using nonmorphological methods in the absence of histologically proven metastasis are generally considered as ITCs.

# Other methodologies
Frozen Sections The use of frozen-section evaluation for SLNs has been described in a number of recent reports, and the results appear promising, with negative predictive values ranging from 83% to 99%. [bib_ref] Sentinel node biopsy for oral and oropharyngeal squamous cell carcinoma of the..., Stoeckli [/bib_ref] [bib_ref] Intraoperative diagnosis of cancer metastasis in sentinel lymph node of oral cancer..., Terada [/bib_ref] [bib_ref] The value of frozen section analysis of the sentinel lymph node in..., Tschopp [/bib_ref] These results are in contrast to an earlier report by Civantos et al., who described poor sensitivity of frozen section compared with step-serial sectioning in a series of 43 oral cancer patients. [bib_ref] Lymphatic mapping and sentinel lymphadenectomy for 106 head and neck lesions: contrasts..., Civantos [/bib_ref] The main advantage of the technique is that it may allow a majority of patients to undergo a single-stage procedure. While the use of frozen section is advocated by some authors, it has not yet gained universal acceptance.

## Imprint cytology
The use of imprint cytology in conjunction with frozen sections in the assessment of SLNs has been described in other tumors such as breast adenocarcinoma. One study in OSCC based on 30 cases demonstrated high sensitivity and specificity, though a recent study showed frozen section to FIG. 5 A micrometastasis found in the seventh level of a sentinel node that was clear on initial sectioning. Viable nucleated squamous cells are present in a cohesive group. Section quality is suboptimal and recutting is not possible be a more accurate method of intraoperative diagnosis. [bib_ref] Intraoperative diagnosis of cancer metastasis in sentinel lymph node of oral cancer..., Terada [/bib_ref] [bib_ref] Intraoperative neck staging using sentinel node biopsy and imprint cytology in oral..., Asthana [/bib_ref] Imprint cytology has an advantage over frozen section in that no tissue is lost in the generation of the sample, however much larger studies are required before considering adoption into the protocol.
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) Methods with increased sensitivity, such as cytokeratin RT-PCR, have been suggested. The small number of studies published demonstrate expression of cytokeratins in nodes which were metastasis negative on initial assessment. However, only a proportion of the nodes demonstrated metastases on serial sectioning. [bib_ref] The diagnostic accuracy of reverse transcription-PCR quantification of cytokeratin mRNA in the..., Garrel [/bib_ref] [bib_ref] Clinical evaluation of a new molecular method for detection of micrometastases in..., Shores [/bib_ref] The clinical role of these methods in the future remains uncertain given the ongoing concerns regarding their specificity together with the associated medicolegal problems, given that significant tissue must be used which has not been assessed histopathologically.

## Burden of work
The authors recognize that, on average, 2.5 sentinel node basins are yielded per neck side. The protocol above may produce up to 12 levels per node. If three nodes are present in each basin there could be 180 slides to examine. On the other hand, the majority of sentinel nodes are small and the nodes from a single basin can often be grouped into one cassette (using ink to identify ''hot'' and ''blue'' nodes), saving laboratory time and effort. In addition, the described protocol has the ability to detect all MMs. According to Cochran's principle, metastatic deposits tend to cluster in the plane of the hilum, and some authors argue the case for examination of bisected sentinel nodes at three or six levels only. [bib_ref] Pathobiology of the sentinel node, Cochran [/bib_ref] Although such a protocol can theoretically miss a MM, it may turn out that no useful information is added by leveling through the block beyond six levels. [bib_ref] Sentinel lymph nodes in cancer of the oral cavity: is central stepsectioning..., Thomsen [/bib_ref] However, several studies report that additional sentinel nodes are upstaged by step-serial sectioning with cytokeratin immunohistochemistry, compared with H&E only. [bib_ref] Need for intensive histopathologic analysis to determine lymph node metastases when using..., Bilde [/bib_ref] 

# Outcome analysis
The success of this multidisciplinary staging technique depends on good communication between all of the individual components; imaging, surgery, and histopathology. The multidisciplinary team (MDT) setting should be utilized for the discussion of every patient, and regular audit of patient outcomes against published data should be carried out. SLNs should be successfully located and harvested in [90% patients. [bib_ref] Sentinel node biopsy in head and neck cancer: preliminary results of a..., Ross [/bib_ref] The accuracy of the technique can be assessed by the proportions of patients whose SLN contain metastases, which should match that of END (20-30%, depending on patient population and tumor size). [bib_ref] Micrometastasis in oral/oropharyngeal squamous cell carcinoma: Incidence, histopathological features and clinical implications, Woolgar [/bib_ref] Lastly, the rate of false negatives (SLN-negative patients who develop early recurrent disease) should be \5%. [bib_ref] Sentinel node biopsy: the technique and the feasibility in head and neck..., Von Buchwald [/bib_ref] Further studies are required to determine the full clinical significance of micrometastasis and individual tumor cells in OSCC. The biological potential of the tumor cells may vary with differing types of tumors, and clinical decisions currently have to be made on the basis of grade, stage, and margin status of the primary lesion as well as on microscopic findings in the sentinel node. [bib_ref] Sentinel node biopsy: interpretation and management of patients with immunohistochemistry-positive sentinel nodes..., Rutgers [/bib_ref] [bib_ref] Correlation of histopathologic findings with clinical and radiologic assessments of cervical lymph-node..., Woolgar [/bib_ref] The optimum protocol will hopefully emerge from the large-scale trials and studies currently in progress. [bib_ref] Sentinel node biopsy in oral/oropharyngeal squamous cell cancer: five year follow-up, Alkureishi [/bib_ref] [bib_ref] Sentinel node biopsy in cancer of the oral cavity, Civantos [/bib_ref] [bib_ref] Sentinel node biopsy for squamous cell carcinoma of the oral cavity: preliminary..., Ross [/bib_ref] [bib_ref] Need for intensive histopathologic analysis to determine lymph node metastases when using..., Bilde [/bib_ref] [bib_ref] Predictive value of sentinel node biopsy in head and neck cancer, Chone [/bib_ref] One of the major aims of SNB in OSCC is to achieve better staging, and thorough pathological examination of SLNs remains the standard.

## Summary
Successful application of the SNB technique is dependent on good communication between all members of the multidisciplinary team, and this joint guideline represents an extension of that approach. Sentinel node biopsy provides an additional tool for staging patients with early OSCC. However, it is not without limitations, and all practitioners of SNB must be aware of these. It is hoped that this document will serve as a reference outlining the optimal practice for the provision of SNB in patients with OSCC, based on the best currently available evidence. As such, the use of the above protocol is recommended until further data, in the form of results from currently underway large prospective studies, become available.
ACKNOWLEDGEMENT The authors would like to thank the European Association of Nuclear Medicine's Physics, Dosimetry, and Radiopharmacy Committees for their assistance in production of this manuscript.
OPEN ACCESS This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
[fig] FIG. 3: Pathology evaluation of sentinel lymph nodes FIG. 4 Isolated tumor cells stained by AE1/AE3 in a sentinel lymph node [/fig]
[fig] FIG. 6: Cytokeratin-positive cells in a sentinel node stained by CKC pan cytokeratin. The white arrow shows a contaminant squame (this can be ascertained by the geometric outline, lack of nucleus, and by focusing at high power). The black arrows show nonnucleated individual tumor cells, and dendritic cells can be seen in the background FIG. 7 A group of nucleated isolated tumor cells stained by AE1/ AE3 in a sentinel node FIG. 8 Multinucleated and mononuclear macrophages revealed by the detailed protocol in a sentinel node that was clear in the first sections. These were suspicious for ITCs within the island FIG. 9 Adjacent field to Fig. 5 stained by AE1/AE3, showing absence of tumor cells [/fig]
[table] TABLE 2: Comparison of UICC and TNM classifications Generic TNM coding for sentinel nodes pNX (sn) Sentinel lymph node could not be assessed pN0 (sn) No sentinel node metastasis pN1 (sn) Sentinel node metastasis Sentinel nodes with micrometastasis only are identified by (mi) pN1 (sn) (mi) single ipsilateral node with micrometastasis pN2 (sn) (mi) multiple ipsilateral nodes with micrometastasis Sentinel nodes with isolated tumor cells are coded separately for morphological and nonmorphological techniques such as PCR or flow cytometry pN0 (i-)(sn) No sentinel lymph node metastasis histologically, negative morphological findings for isolated tumor cells (ITC) pN0 (i?)(sn) No sentinel lymph node metastasis histologically, positive morphological findings for isolated tumor cells (ITC) pN0 (mol-)(sn) No sentinel lymph node metastasis histologically, negative nonmorphological findings for isolated tumor cells (ITC) pN0 (mol?)(sn) No sentinel lymph node metastasis histologically, positive nonmorphological findings for isolated tumor cells (ITC) [/table]