Sentinel Lymph Node Biopsy A new approach in the management of head and neck cancers

Cervical lymph node metastasis affects the prognosis and overall survival rate of and therapeutic planning for patients with head and neck squamous cell carcinomas (HNSCCs). However, advanced diagnostic modalities still lack accuracy in detecting occult neck metastasis. A sentinel lymph node biopsy is a minimally invasive auxiliary method for assessing the presence of occult metastatic disease in a patient with a clinically negative neck. This technique increases the specificity of neck dissection and thus reduces morbidity among oral cancer patients. The removal of sentinel nodes and dissection of the levels between the primary tumour and the sentinel node or the irradiation of target nodal basins is favoured as a selective treatment approach; this technique has the potential to become the new standard of care for patients with HNSCCs. This article presents an update on clinical applications and novel developments in this field.

A s opposed to primary tumours, metastasis is responsible for the high mortality rate of most cancer patients; moreover, cancer cells primarily invade the regional lymph nodes before spreading to other parts of the body. 1 Genetic instability results in tumour cell heterogeneity, leading to the emergence of metastatic clones and dissemination of the cancer from the primary tumour site. 2 Malignant cells metastasise due to an interaction between the host factors and tumour cells.Genes related to the extracellular matrix, adhesion, motility and protease inhibition constitute a significant part of the metastatic process. 3

Nodal Metastasis n o d a l m e ta s tat i c c a s c a d e
The migration and invasion of cancer cells into the lymphatic system is governed by a variety of intricate genotypic, phenotypic and microenvironmental processes.After entering the lymphatic draining channels, the tumour cells metastasise to the regional lymph nodes in the neck and form the metastatic foci. 4Macrometastases refer to lymph nodes that appear suspicious on clinical or radiographical examinations; in contrast, nodal metastases-which are not detectable by imaging methods or physical examination-indicate occult or subclinical metastasis.Hermanek microscopically differentiated occult metastases into macrometastases (metastatic deposits of >2 mm), micrometastases (metastatic deposits of <2 mm) and isolated or small clusters of tumour cells (metastatic deposits of <0.2 mm). 5 Isolated tumour cells (ITCs) can also be defined as a cluster of ≤200 tumour cells visible on one histology slide; these cells can further be categorised into those detectable by light microscopy, immunohistochemistry or molecular methods. 6Chemoradiation or elective neck dissection (END) should be considered in patients The microenvironment of the lymph node is initially hostile to cancer cells due to a predominance of immune effector cells and cytokines; immunoresistant clones in the hostile lymph node milieu subsequently proliferate, spread and invade the rest of the lymphatic system to establish metastasis. 8alignant cells follow an orderly sequence, spreading from one nodal basin to the next as the disease progresses down the neck.In some situations, lymph node groups can be bypassed, which can result in a process known as skip metastasis. 9However, controversial reports exist as to whether lymphatic tumours spread through new lymphatic vessels (i.e.lymphangiogenesis) or pre-existing peritumoural lymphatic vessels. 10Different molecular components are also involved in the metastatic cascade, including prospero homeobox 1, lymphatic vessel endothelial hyaluronan receptor 1, podoplanin, vascular endothelial growth factor receptor-3, epithelial cadherin, catenins, syndecans, focal adhesion kinase, matrix metalloproteinases-2 and -9, metallothioneins and laminins. 11These markers have recently been explored as a result of increased research interest in tumour lymphatics. 12s i g n i f i c a n t p r o g n o s t i c fa c t o r Lymph nodes in the head and neck constitute approximately 30% of the 800 lymph nodes in the human body.13 Neck node metastasis is a major determinant in the prognosis of oral, oropharyngeal and other head and neck cancers; the disease-free survival rate decreases to approximately 50% with the presence of a single cancer-positive lymph node.14 The extent of lymph node involvement should be considered as an indirect index of the systemic tumour load and is an important aspect of tumour staging; thus, the size, level and presence of a metastatic neck node determines overall survival and treatment planning.15 In patients with oral cancer, the lymph node levels I to III are most commonly involved.16 The frequency of skip metastasis to levels IV or V, bypassing the upper nodal levels, is approximately 4-5% in oral cancer, although this can increase to 16% in the tongue.17 A clinically negative neck indicates a primary tumour of either ≤2 cm or 2-4 cm with no regional lymph node metastasis (N0).18 The incidence of occult neck metastasis in stage I/II disease for patients with clinically negative necks is 30-34%.12,19 The traditional approach to treatment has been to proceed with wide-margin radical neck surgery.However, prophylactic END for all patients with carcinomas of the oral cavity and a clinically negative neck results in an overtreatment rate of 65-70%; nevertheless, overall survival may be jeopardised if the carcinoma is not treated. 20Predictive factors associated with occult metastasis of the cervical nodes include primary tumour site, size, degree of differentiation, perineural and vascular invasion, inflammatory response and tumour ploidy status.21 Advanced diagnostic approaches with greater accuracy are required, since these factors alone or along with conventional methods are insufficient to assess neck metastasis.13 Advanced imaging modalities such as computed tomography, magnetic resonance imaging, ultrasonography, positron emission tomography (PET), lymphoscintigraphy and ultrasoundguided fine-needle aspiration cytology are often used for neck evaluation and screening in patients with oral cancer.Nonetheless, these approaches can fail to detect occult neck metastasis and subcentimetric or microscopic metastatic foci.22 For patients presenting clinically without regional disease, treatment approaches for oral squamous cell carcinoma (OSCC), and head and neck cancers in general, have been debated.23 In cases of more advanced oral cancer, clinically evident and diagnostically proven locoregional metastasis give a clearcut indication for treatment of the neck, based on the treatment approach for the primary tumour.24 However, the management of patients with stage I/II disease and a N0 neck remains unclear; patients can be treated either with prophylactic END, irradiation or observation with regular follow-ups.14 A surveillance approach can result in poor survival and increased risk of occult metastasis.25 However, performing ENDs on all patients with N0 necks would lead to undue surgeries with large incisions, skin flaps and scars, the sacrifice of the spinal accessory nerve and the involvement or sacrifice of the sternocleidomastoid muscle and internal jugular vein, particularly if the surgery is bilateral.26 Neck dissection could also lead to other sequelae, including shoulder and neck dysfunction, pain, contour changes, haemorrhage, nerve injury, lower lip paresis, lymphoedema, an increased need for postoperative radiotherapy, poor cosmetic outcomes and greater expenses.27

Sentinel Lymph Node Biopsy a n o v e l a p p r o a c h
Recently, the concept of a sentinel lymph node (SLN) was introduced in head and neck squamous cell carcinomas (HNSCCs) in order to more precisely detect and evaluate neck metastasis and unpredictable lymphatic drainage patterns, following its successful application in melanomas and breast cancer. 22,28Several validation studies involving ENDs have resulted in SLN cancer detection rates of >95%. 12Following the orderly and sequential drainage in the lymphatic stream from the tumour site, the first node reached is the SLN, which could help predict the nodal stage of metastasis [Figure 1].In theory, if the SLN is free from cancer cells, then distal node involvement is assumed to be rare. 29Thus, the lymphatic basin status can be ascertained along with a reduction in poor prognosis and morbidity rates. 28,30SLN biopsy is an ancillary diagnostic method for assessing the presence of occult metastatic disease in a N0 neck.This minimally invasive technique eliminates the need for a neck dissection, which until recently was thought to be the only means of neck staging. 31A SLN biopsy is a selective procedure based on the identification and evaluation of echelon nodes (i.e.first station or levels I and II) for metastatic spread; hence, the first drainage node or group of nodes, known as the SLN, are chosen for dissection as the location at which a primary tumour first metastasises. 14In 1996, the first successful SLN biopsy was performed by Alex et al. on a patient with a laryngeal supraglottic carcinoma; Koch et al. subsequently proved the feasibility of this procedure in 1998 for selected patients with head and neck mucosal lesions. 27Morton et al. found that selective lymphatic dissection performed after a SLN biopsy was therapeutically equivalent to a comprehensive elective lymphatic dissection among patients with skin melanomas. 32

r e at m e n t p r o t o c o l s
In recent decades, improved understanding of lymphatic drainage patterns in the head and neck region have simplified the assessment of higher risk nodal levels. 27There has been a gradual shift towards a more conservative/selective surgical approach for patients with clinically negative necks, progressing from radical neck dissection to modified radical neck dissection and subsequently selective neck dissection. 330][31][34][35][36][37][38][39][40][41][42][43][44][45][46] Shoaib et al. suggested a protocol involving preoperative lymphoscintigraphy, intraoperative blue dye and gamma probe localisation [Figure 2]. 30This technique is based on observing the route of lymphatic flow via imaging after the injection of a radioactive contrast agent near the primary tumour. 42The flow and direction of the lymph, comparable to the possibly metastatic flow from the tumour, can be visualised preoperatively by means of lymphoscintigraphy or single-photon emission computed tomography (SPECT).Lymphoscintigraphy reveals SLNs associated with the primary tumour, unexpected lymphatic drainage patterns and lymphatic vessels associated with different lymphatic drainage basins. 14To enhance the detection rate, blue dye is often used in combination with radioisotopes. 47During the surgery, a handheld gamma probe is used for radionuclide detection to trace the SLN perioperatively and a gamma camera is used for dynamic monitoring of lymphatic drainage.The site of the radioactive lymph nodes, which are important anatomical landmarks, are marked using a gamma camera and the ex vivo radioactivity of the nodes and surgical bed is checked after removing the nodes. 31istopathological evaluation, immunohistochemistry and molecular markers have been suggested for a small number of harvested SLNs to help detect occult metastasis in serial lymph node sections, including molecular techniques such as polymerase chain reaction and immunohistochemistry using cytokeratin markers. 43These could potentially lead to more accurate nodal staging and the detection of nodal micrometastatic deposits and ITCs. 48It has been suggested that step serial sectioning at 150 μm intervals with pan-cytokeratin enhances nodal detection by approximately 20% in comparison to the initial routinely stained section. 15Murer et al. reported lower postoperative morbidity rates and better shoulder function following a SLN biopsy in comparison to an END. 49Hernando et al. observed  and SLN biopsies were associated with lower rates of postoperative morbidity. 44lkureishi et al. reported that the pathological review of a SLN or neck dissection specimen can affect the staging of a lymph node if it reveals occult or additional positive lymph nodes which might have been missed on a routine physical examination or radiographical evaluation. 43This alteration in lymph node staging could increase the risk for distant metastasis and change the patient's prognosis and treatment plan. 50,51In HNSCC cases, a SLN biopsy has been suggested as a valid method to improve the accuracy of pathological staging of lymph nodes and subsequently allow treatments to be tailored. 32In a meta-analysis, Thompson et al. found that a positive SLN biopsy confirmed occult metastasis in 31% of patients; this correlates with a previously reported occult metastatic rate of 33%. 45,52valuation of a positive lymph node status is critical as it is a major indicator for adjuvant radiation and chemotherapy.The use of a SLN biopsy allows adjuvant or elective chemoradiation to be avoided, as well as the associated morbidities of these treatment options. 53For example, HNSCC patients receiving chemoradiation are more likely to develop acute mucositis, oral pain, dysphagia and xerostomia; in addition, they are more frequently hospitalised. 45,54atients with a negative SLN biopsy can therefore   *A clinically negative neck indicates a primary tumour of either ≤2 cm or 2-4 cm with no regional lymph node metastasis. 18SG = lymphoscintigraphy SN = sentinel node; T/t = treatment.
avoid adjuvant therapy, which would lessen patient morbidity; this option can be reserved for a later time in the event of a second primary diagnosis or tumour recurrence. 46While a SLN biopsy is not yet considered the standard of care for cancers of the oral cavity, many single-and multi-centre studies have successfully demonstrated its feasibility in oral cancers with high detection rates (approximately 95%) and negative predictive values (88-100%), thus substantiating its significance and use in the staging and treatment of early-stage head and neck cancers. 43,55,56

Advances and Future Developments
A variety of soluble tracers and radiocolloids have been used in lymphoscintigraphy, including technetium-99m ( 99m Tc)-labelled colloidal human serum albumin, colloidal sulphur, 99m Tc colloidal rhenium sulphide and a dextran-based product modified to allow 99 Tc-labelling. 13,21,44,57Tsuchimochi et al. proposed performing a SLN biopsy using multimodality imaging and polyamidoamine-coated silica nanoparticles loaded with 99m Tc and indocyanine green dye. 47Deeply situated SLNs can be detected more accurately with the use of near-infrared dyes; recently, hybrid tracers combined with radiotracers and fluorescence dyes have resulted in high sensitivity for preoperative SLN mapping. 57Bluemel et al. evaluated the feasibility and potential advantages of freehand SPECT in oral cancer in comparison with conventional intraoperative localisation techniques for a SLN biopsy; they found that one of the most important limitations of a SLN biopsy-the 'shine-through' phenomenon-was overcome by freehand SPECT. 58cording to Denoth et al., metastatic deposits are not randomly distributed within SLNs but are predominantly found in the central planes, closer to the lymphatic inlet; analysis of the distribution pattern of metastatic spread within SLNs with a virtual microscope resulted in a detection rate of 90% and 80% for micrometastasis and ITCs, respectively. 59an Den Berg et al. introduced the concept of hybrid tracers containing both radioactive and fluorescent labels which allowed for the direct integration of preand intraoperative guidance technologies when used in combination with new surgical imaging modalities and navigation tools in SLN detection. 57Using ultrasound-guided spectroscopic photoacoustic imaging of molecularly-activated plasmonic nanosensors in an OSCC murine model, Luke et al. demonstrated that lymph node metastases as small as 50 μm could be detected in vivo at a depth of 1 cm with high sensitivity and specificity; this new approach could potentially be a sensitive alternative to a SLN biopsy. 1 In various animal studies, attempts to combine several techniques have been reported, including the addition of 99m Tc, iodine-125 or iodine-111 to phthalocyanine tetrasulfonate, dextran and Evans blue, methylene blue or blue Ficoll dyes. 47,60Tsopelas et al. found 99m Tc-Evans blue to be useful in differentiating the initial draining lymph node from higher-tier nodes in linked chains. 61The clinical applications of a SLN biopsy could be enhanced through on-going developments and innovations.These might include the preoperative use of PET, the biological staging of primary site biopsies, the discovery of more radionuclide-avid lymph nodes or ultrasound-detectable injectable contrast agents as potential second tracers, the application of intraoperative reverse transcriptase polymerase chain reaction analysis of the sentinel node and the use of endoscopic SLN biopsies. 14,26,27,62,63

Conclusion
A SLN biopsy can prevent the unnecessary removal of functional lymph nodes and limit the extent of neck dissection surgery.However, surgical precision and experience as well as specific technical devices are required for its successful application and implementation in the head and neck region.Although a review of the current literature demonstrated the reliability and worldwide acceptance of this approach, the role of SLN biopsies in HNSCCs is still under investigation.Using the sentinel node concept helps to define the surgical approach to a clinically negative neck and identify skip metastasis and unpredictable lymphatic drainage patterns, resulting in a more favourable prognosis for patients with head and neck cancers.

Figure 1 :
Figure 1: Flowchart depicting the process of primary tumour spread to a sentinel lymph node.
physical examination in comparison to an END •Saves time and expense •Shortens the recovery period •For medically fit patients •May be difficult as the approach is technique-sensitive •The use of blue dye may elicit an allergic response •False-negatives can occur due to uneven radionuclide injection, the obscuring of the SLN by the radioactive signal of the primary tumour and the obstruction of the lymphatic vessels by the gross tumour, resulting in a redirection of lymphatic flow •The accuracy of this technique in identifying true SLNs is inadequate in patients with tumours of the floor of the mouth •In cases of multiple SLNs and SLNs at different levels, the number of SLNs to be removed is still unknown, which may lead to an extensive procedure similar to that of an END •The 'shine through' phenomenon and scatter radiation due to the primary tumour can obscure identification •Variability in head and neck lymphatic drainage may result in collateral channels leading to skip metastasis T1 = primary tumour of ≤2 cm; T2 = primary tumour of 2-4 cm; N0 = without regional lymph node metastasis; SLN = sentinel lymph node; END = elective neck dissection.

Figure 2 :
Figure 2: Proposed treatment protocol for a sentinel lymph node biopsy.30