Pathology of sentinel lymph nodes for melanoma

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1 Postgraduate Medial Shool, University of Surrey and Department of Histopathology, Royal Surrey County Hospital, Guildford, Surrey, UK Correspondene to: Professor M G Cook, Royal Surrey County Hospital, Egerton Road, Guildford, Surrey GU2 7XX, UK; m.ook@nhs.net Aepted 5 May 2008 Published Online First 30 May 2008 Pathology of sentinel lymph nodes for melanoma M G Cook, S Di Palma ABSTRACT As a onept sentinel lymph node biopsy seems attrative in that it attempts to identify the first lymph node, rather than the nearest node, draining a partiular anatomi area where a tumour has arisen. Pathologial assessment an then indiate whether metastases are present and the proedure is either a strong prognosti indiator or possibly therapeuti in itself. These omments apply to any tumour type, but with melanoma the pathologial proedure is more problemati and any benefits above prognosis and staging are not universally aepted. The proedure does give aurate staging without the extra morbidity of regional node dissetion and many patients gain psyhologial support from the information gained. POTENTIAL BENEFITS OF SENTINEL LYMPH NODE BIOPSY FOR MELANOMA Exision of the sentinel lymph node (SLN) enables a prognosti assessment to be made that is more powerful than thikness measurements on the primary. 1 2 This in itself is suffiient, to many, to justify the proedure sine it has low morbidity. 3 The SLN status is an important riterion for entry into linial trials and therefore until a lear effetive systemi therapy is established SLN biopsy (SLNB) will have importane and the SLN status may be the threshold to entry into a treatment programme. The initial reason for development of SLNB was to enable seletion of patients more likely to reeive benefits from regional node dissetion (RND). It had already been shown that RND was not effetive on unseleted patients but in the absene of alternatives the surgial treatment option was retained. The SLNB seemed a way of direting RND to a group of patients more likely to benefit from wide exision of nodes. This remains the usual proedure, although evolution towards offering RND to an even more restrited group of patients is proeeding. Whether SLNB by itself is of therapeuti benefit is not yet established. There is a suggestion of prolongation of disease-free survival for SLNB patients as ompared with observation followed by RND only when linial evidene of metastases develops. Prolongation of overall survival has not yet been demonstrated but longer follow-up may reveal this. 2 POTENTIAL PROBLEMS OF SLNB Any disadvantages of SLNB are also not established. The anxieties about an inrease in in transit metastases 4 5 have not been upheld. 6 7 The theoretial onern about removal of what is likely to be the first line of immunologial defene still requires further study. A suggested alternative proedure is high-grade ultrasound, 8 but 4.5 mm My approah is the smallest size that an be onsistently deteted with this tehnique and the majority of metastases in SLN for melanoma are smaller than that and therefore this alternative does not appear aeptable as a substitute for SLNB. The SLNB proedure has some assoiated morbidity but this is muh less than for RND, and at least 70% of patients with melanoma will avoid this morbidity by having the lesser proedure. The National Institute for Health and Clinial Exellene (NICE) report on skin tumours 9 has onluded that SLNB has merit for staging, and that it should ontinue to be done in existing entres as part of linial trials, but has not wholeheartedly reommended SLNB for all melanoma. This reommendation is in spite of the urrent pauity of suitable trials into whih to enter patients. The NICE advie may need modifiation in the light of the reent report of MSLT-1 sine some prolongation of disease-free survival has been demonstrated 2 and improved overall survival annot be ruled out. SURGICAL PROCEDURES EFFECTING PATHOLOGICAL ASSESSMENT The identifiation of the SLN by the surgeon should be based on preoperative lymphosintigraphy and intraoperative injetion of vital blue dye and radioolloids. The lymphosintigraphy demonstrates the diretion of the lymph drainage from any utaneous site and enables the first or sentinel lymph nodes in eah basin to be identified and marked on the overlying skin. There is some variation in definitions of SLN and so there may be variation in the numbers of SLN exised between surgeons or between entres. The SLN should be exised with a uff of fat and without trauma to the node. Examination of the subapsular zone of the SLN is most likely to reveal metastases and so it is important that this should not be damaged by the surgery. 10 Confirmation of what is a true SLN may be obtained by using arbon dye injeted at the primary site. This tends to be loalised in the area most likely to ontain metastases and its presene onfirms the node as a true SLN. Unfortunately this arbon preparation is not available in all ountries. Where the surgeon sees the afferent lymphatis with the blue dye he an use a ligature to identify this aspet of the node so that the pathologist is aware of the orientation of the node, and this therefore failitates the sliing through the median plane. HISTOPATHOLOGICAL AND PATHOBIOLOGICAL PECULIARITIES OF MELANOCYTIC TUMOURS AFFECTING INTERPRETATION OF SLN In ontrast to interpretation of SLN for other malignanies, melanoma presents extra diffiulties due to its tendeny to metastasise in small groups J Clin Pathol 2008;61: doi: /jp

2 or even single ells, whih on onventional staining are similar to other ells normally present in lymph nodes at least at sanning magnifiation. The assessment of the lymph node must be designed to detet as many as possible of these metastases. To detet all of them would involve serial setioning of the SLN to extintion resulting in thousands of setions per node. This is learly not pratiable and a balane between workload and effiieny is required and several proedures have been developed. PATHOLOGICAL PROCEDURES Dissetion Dissetion of the SLN an be by random parallel bread loaf sliing 11 or single sliing through the entral plane (bivalve tehnique). 12 Sine Cohran, in an early study, 13 showed that the majority of metastases are in a entral plane (see fig 1), the bivalve tehnique, in whih a slie is made through the onvex apsule and the hilum to reveal the maximum ut surfae area, has been adopted by us. 14 The alternative tehnique reommended by Starz et al 11 onsists of sliing the SLN at 2 mm intervals. It is attrative in that it does not depend on the slightly diffiult identifiation of a entral plane and also in that it does not require multiple setions on eah slie. However, in larger nodes, there may be a need for several bloks. On balane we have preferred to use the bivalving approah, but using some dissetion of the lymph node from the surrounding fat followed by a ombined visual assessment and palpation to identify the entral plane through whih the node is biseted. Setioning and staining Further options exist in the setioning and staining of the bivalved node. Cohran 12 reommended serial setions from the two mid-plane surfaes stained with a ombination of and melanoyte markers (, HMB45, Melan A). Starz 11 examined only two setions from eah blok disseted in a different way, and used and immunohistohemistry. Both of these proedures deteted metastases in 15 20% of patients. Sine immunohistohemistry inreases the detetion rate of metastases by at least 10% 15 we have used it from the beginning. Figure 1 Metastases of melanoma are said to our lose to the median plane of the sentinel lymph node and therefore bivalving, as shown, and embedding fae down in the assette are reommended. The partiular antibody used depends on loal preferene. We have tried HMB45, MelanA, MiTF and. Melan A and HMB45 are widely used but they an be ritiised as being less sensitive; however, on the other hand they have the advantage of being more speifi. has been hosen as the first-line antibody sine it is the most sensitive, the least demanding tehnially, and the heapest. However, it does require the pathologist to gain experiene in its interpretation, partiularly in distinguishing melanoytes from lumps of dendriti ells. We have overome this rare problem by always having unstained adjaent setions for more speifi antibodies in reserve for use in more problemati ases. We use Melan A most often for this onfirmatory purpose. We have found HMB45 positivity in only 70% of melanomas and therefore do not regard it as suffiiently sensitive. A oktail of antibodies inluding Melan A, HMB45, and tyrosinase suh as Pan Mel+ (Bioare Medial, Conord, California, USA) has high sensitivity and speifiity but is more expensive and tehnially more demanding. With experiene, has the highest sensitivity and partiularly in ombination with diaminobenzidine hromogen even single ells an be identified on sreening magnifiation and onfirmed on the adjaent. This enables distintion from other potentially positive ells suh as dendriti ells and nerves. We assess the same ells on immediately adjaent setions stained with. Unless we are onvined that the immunohistohemistry positivity orresponds with a ell with ytologial features of a melanoyte, we do not regard the SLN as positive. Melanophages may sometimes be identified without melanoytes in the SLN and it ould be speulated that these features represent intranodal regression, but the melanophages ould equally well represent drainage of melanophages from the primary melanoma. These are partiularly numerous when the primary melanoma shows features of regression. Balaning extended setioning against reverse transription-pcr In the early stages of development of SLN pathology protools, the possibility of using moleular tehniques suh as reverse transription (RT)-PCR on fresh tissue instead of histology was onsidered. Positivity in SLN for melanoma using tyrosinase and or MART-1 or gp100 was found to be muh higher than that obtained using histology, even up to 72% positive. 16 This high positivity far exeeded what would be expeted from our knowledge of the natural history of melanoma unless the seletion of ases was highly seleted in favour of extremely poor prognosis patients. However the omparison between histology and moleular tehniques was not adequately ontrolled, most obviously in that the lymph nodes were divided in an unspeified way and the separate parts assessed by only one of the two tehniques. This took no aount of the distribution of the metastases in the nodes. We deided that the role of moleular biology needed to be evaluated in a more ontrolled manner, 14 and we found that a positivity of 45% with RT-PCR on frozen setions did exeed what was obtained from immediately adjaent setions stained onventionally (18% positive), but that the disrepany ould be explained partly by the presene of naevus ells, whih would also give a positive RT-PCR result, and partly by the presene of further melanoma deeper in the node only deteted by more setions. We ahieved a detetion rate of 33.8% by extending the setioning protool to six pairs of setions at 50 mm intervals. Eah pair was stained with and. Other unstained spare setions were taken at eah 50 mm step. Further levels 898 J Clin Pathol 2008;61: doi: /jp

3 deeper than the sixth pair produed only a minimal improvement in detetion rate and therefore the six pairs at 50 mm intervals protool was adopted by us as a reasonable balane between workload and sensitivity. 14 The detetion rate of 33.8% was still below 45% deteted by RT-PCR but this is explained almost entirely by the presene of naevus ells. Others 17 have suggested that this protool is too labour intensive or expensive. More importantly the signifiane of the additional metastases needs to be established. We do not regard the extra work as exessive sine in more than 70% of SLN that are negative only seven slides need to be examined per lymph node. This is ahieved by using as a preliminary stain for sanning and one to rule out other pathology. In the 30% of ases that are positive, more detailed assessment is required. Oasional melanomas are negative (1% in our experiene), but metastases are still identified easily as a negative or lear area against the blue ounterstained bakground. The only variation in the setioning protool ours when lymph nodes are unusually rounded rather than approximately reniform. In those thiker lymph nodes we use a wider step between the pairs of stained setions, inreasing it from 50 mm to 75 or 100 mm. It may be that the protool we have adopted 14 in the long term may be shown to be exessively rigorous sine Van Akkooi et al 18 have shown that metastases,0.1 mm are not assoiated with further disease. However we onsider that all metastases however small have yet to be shown not to be apable of progressing to more aggressive disease even after many years. We have some experiene of metastases remaining small and loalised in lymph nodes for several years after exision of the primary melanoma. This is presumably the explanation of how some melanoma metastases oasionally progress 10 or more years after exision of the primary. It remains to be shown whether some melanoma metastases are not apable of progression by reason of different biology related to their geneti profile, and therefore at least until that is proved and suh ases are identifiable, detetion of as many as possible of metastases should be the aim. This is supported by finding that so-alled isolated tumour ells in groups less than 0.2 mm are assoiated with a lower overall survival. 27 At least for the foreseeable future we reommend the protool as outlined in table 1 using as a sreening stain so that the proedure is not too time onsuming. RT-PCR is not ideal at least for detetion of metastases but work of Hoon et al 19 and Chew et al (unpublished) indiate that it may be useful in providing additional prognosti information. Naevus ells in SLN One of the main diffiulties in interpretation of SLN is the presene of benign naevus ells. These are deteted by but are almost always negative with HMB45, so that marker an be used to distinguish naevus ells from metastati melanoma. 10 However, not all melanomas are positive with HMB45 (70% in our experiene) so that negativity is not a onfirmation of benign nature. More importantly, naevus ells an be distinguished from metastases by their site and ytology. We have assessed the SLN from 2005 patients with utaneous melanoma, and 14% of them ontained naevus ells mostly in the substane of the fibrous apsule but oasionally in the fibrous trabeulae. The ells are usually harateristially naevoid with small dense nulei or little ytoplasm. However, very oasionally, olletions of naevoid ells are quite large so that they appear to enroah into the adjaent lymph node. In those ases, while nulei are still highly uniform, nuleoli an beome evident. In these rare diffiult ases we use HMB45 (as reommended by Murray et al 10 ) and MIB1. The HMB45 status is an important guide but sometimes a more definite onlusion is ahieved with MIB1. Naevus ells should show no positivity with this stain, but melanoma would be expeted to show at least some positive nulei. The interpretation of MIB1 an be diffiult in lymph nodes beause of the high proportion of positivity in surrounding lymphoytes. P16 an be used in a similar way being usually positive in naevus ells and lost in melanoma. We have identified naevus ells in 279 SLN patients. Sixtytwo (22.2%) of these were assoiated with melanoma metastases but the majority (217) were not. WHICH PATIENTS SHOULD BE OFFERED SLNB The riteria of the primary melanoma that are used to justify performing SLNB are not standardised but most ommonly a minimum thikness of 1 mm has been used. Others have suggested that there is no benefit in performing SLNB on patients with melanomas thiker than 4 mm sine these are already known to have a bad prognosis. However, we would argue that a large proportion (40%) of the thik melanomas still have long-term survival and therefore the SLN an be used to attempt to identify that partiular group. We originally onsidered that any patients with a melanoma in vertial growth phase should be offered SLNB and we did identify several patients with melanomas less than 1 mm down to 0.6 mm thikness that had metastasised. Nevertheless, the number of suh ases was a small proportion of the total and therefore began to restrit offering SLNB to patients with melanoma thiker than 1 mm. Gimotty et al 20 have suggested that thin melanomas (,1 mm) an be graded in risk aording to their mitoti ounts and the gender of the patient. Males with thin and invasive mitotially ative melanomas are said to have a 31% risk of metastasis and females a 13% risk. Therefore we have begun to offer SLNB to patients with thin melanomas and dermal mitoses that by definition are in vertial growth phase. Regression has also long been onsidered as an important feature of melanomas, with impliations of an adverse effet on prognosis by some. This effet is not aepted universally and there are roughly equal numbers of studies who regard regression as having an unfavourable effet on survival as those who onsider it to have no or even a favourable effet. 21 Nevertheless prognosti tables 22 showed regression as a poor prognosti fator and for that reason we did use it as a riterion for offering SLNB. Our own studies 23 omparing melanoma patients with or without regression in terms of SLN status, as a surrogate for survival, showed as one might expet intuitively that regression appeared to onfer benefit to the patient, therefore we have disontinued aepting regression as a riterion. Morton et al 2 have identified some prolongation of diseasefree survival with SLNB on melanomas with thikness between 1.2 and 4 mm but this does not imply that SLNB should only be offered to patients with this range of thikness of melanoma sine the staging benefits also apply to those outside this group. At this stage we think it appropriate to offer SLNB to all patients with melanoma over 1.0 mm in thikness and to those patients with thinner invasive melanomas in whih mitoses are identified in the dermal omponent unless there are other linial features that override this deision. J Clin Pathol 2008;61: doi: /jp

4 Table 1 Sequene of setioning and staining for sentinel lymph node for melanoma Setion Treatment Setion 1 (first full setion) Setion 2 Setion 3 50 mm gap (+50 mm) Setion 4 Setion 5 Setion 6 Setion 7 Setion 8 Setion 9 50 mm gap (+100 mm) Setion 10 Setion 11 Setion mm gap (+150 mm) Setion 13 Setion 14 Setion mm gap (+200 mm) Setion 16 Setion 17 Setion mm gap (+250 mm) Setion 19 Setion 20 In large or round lymph nodes, the 50 mm gap may be inreased up to a maximum of 100 mm. MULTICENTRE SLN TRIALS The first multientre SLN trial (MSLT-1) has made a preliminary report. 2 In 1269 patients, this trial has shown that SLNB with immediate RND for positive ases had a 4.8% improvement in disease-free survival ompared with no SLNB and RND only on linial evidene of metastasis. The SLN status was onfirmed as a potent prognosti fator in that positive nodes onferred a 5-year survival rate of 72.3% on the patients ompared with 90.2% in patients with negative SLN. No overall survival benefit was noted at 5-year follow-up. This does not exlude the possibility of survival advantage being demonstrated after longer follow-up sine metastases from thinner melanomas tend to our later if at all. The seond multientre SLN trial (MSLT-2) proposes to evaluate whether it is neessary to proeed to RND in all patients with positive SLN irrespetive of the tumour burden, and also to further examine the role of RT-PCR positivity in histologially negative SLN. Some ases that are histologially negative but positive on RT-PCR will still enter the trial. The Sunbelt melanoma trial, whih is another multientre study, 24 emphasises the prognosti and staging value of SLNB histology and also proposes to evaluate the role of RT-PCR in assessment of SLN, but has not yet onluded its study. It has reported a definition of SLN based on the ratio of radioativity in a node to that in the bakground. The latter should be 10% or less of that in the SLN. The Sunbelt trial has not identified fators in the SLN metastases that enable predition of metastases elsewhere and therefore they advoate ompletion lymphadenetomy for every patient with a positive SLN. They have noted a lower rate of metastases to SLN in older patients despite the shorter survival in the same group, suggesting that the biology of melanomas of younger patients is often different from that of older patients, with the latter showing greater tendeny to haematogenous metastases. This is supported by the findings of Viros et al 25 in whih patients under 55 years were muh more likely to have melanomas with BRAF mutation than older patients. This supports the growing support for the idea that melanoma is not one diagnosis but a olletion of melanoyti tumours with different behaviours. A FUTURE ROLE FOR RT-PCR The MSLT-2 proposals have been stimulated in part by the observations of further RT-PCR studies 19 that indiate that RT- PCR positive, histologially negative SLN have a worse prognosis than those that are negative by both tehniques. Our reent studies (unpublished) also suggest that nodal metastases deteted by melanoma-speifi markers suh as MAGE-3 revealed by RT-PCR on paraffin-embedded tissue from SLN gives an added dimension to the prognosis beyond that whih an be obtained with histology alone. However the proess is laborious, therefore potentially expensive, and only dissets out an additional group of poorer prognosis patients from those 70% or so who are negative by histology. Rather than perform this additional assessment on the whole 70% of SLN that are negative, there is a need for a way of foussing on those within that group who are more likely to be positive. This would make this proedure more likely to be aeptable. For example, perhaps RT-PCR would be preditably negative in SLN-negative patients who were thinner than say 1.2 mm and had no mitoses in the dermis, or were of a histologial type with suspeted better prognosis suh as spindle ell melanomas, or had a diffuse and intense tumour-infiltrating lymphoyte response. Conversely SLN-negative ases that are histologially negative, but have a high mitoti ount, would be expeted to be more likely to be positive with RT-PCR. IMPROVEMENT OF PREDICTIVE ACCURACY OF HISTOLOGICAL ASSESSMENT OF SLN ACCORDING TO TUMOUR BURDEN A straightforward reord of presene or absene of metastases in SLN has great prognosti value, 1 but several authors have onsidered whether speifi features of the metastases or their distribution may give more preise prognosti information. Starz et al 11 desribed an index derived from the depth of the metastases from the apsule and the number of slies involved. This index enabled a predition of the status of the rest of the lymph nodes in the same basin to be made with onsiderable auray. Cohran 12 was able to provide inreased auray of survival predition by using a relative tumour area alulation, based on some omputer-failitated measurement and alulations. Our group 26 noted a orrelation between the miroanatomi distribution of the metastases in the SLNB and the presene of additional metastases in the RND. Those patients in whom the metastases were entirely subapsular (fig 2) were found not to have any metastases in the nodes of the subsequent RND. The subapsular pattern was seen in approximately 30% of all positive SLN. More reently Van Akkooi et al 18 showed that metastases,0.1 mm in maximum dimension, irrespetive of site, were not assoiated with further metastases or any evidene of tumour progression. Govindarajan et al 27 supported similar findings in noting that patients with metastases,0.2 mm maximum dimension did not have positivity in their RND. There have been no reurrenes in this group although 900 J Clin Pathol 2008;61: doi: /jp

5 the follow-up period was short. These findings require further study as the number of suh ases that were not subapsular was very small. In addition 28 Sheri et al have noted that metastases,0.2 mm in maximum dimension have a signifiantly higher risk of melanoma-speifi death than SLNnegative patients thereby questioning the assumption that small metastases have limited signifiane. These assessments of tumour burden all have merits, but whether one is more aurate or appropriate than another is not finally deided. All of them depend on areful examination of all available setions sine metastases are not neessarily present in all. 10 The Starz tehnique is perhaps most simple to assess being a measure from the apsule to the deepest metastasis. However one ould ask from whih part of the apsule does one measure. Logially it should be from the afferent aspet, but this is not always obvious. Van Akkooi et al reommends the maximum dimensions of the largest deposit; this sounds straightforward but it needs to be understood that they mean the largest ohesive groups of ells, even though that may be only one of many, eah of them showing only slight separation from eah other. The loation of the metastases in our hands gives more reliable information, very easily assessed, in that subapsular metastases are only very rarely assoiated with metastases in the rest of the nodes in the same basin. Even so, the definition of subapsular site needs to be onsidered arefully. Subapsular sinuses are not learly visible and metastases in that zone are not usually identifiable as in a lumen. Their subapsular zone loation is important, with the onfiguration of the deposit being reasonably smooth, that is with no irregular projetions on the parenhymal aspet. On the basis of these studies it seems reasonable to presume that prognosis based on SLN an be refined, and that low volume, small or subapsular metastases may not need to be followed by RND, although the riteria aepted may require further refinement. Whether these small or subapsular metastases are preursors to larger tumours or represent a different harater of metastases with more indolent growth properties remains to be established. It seems likely to us that Figure 2 Sreening of sentinel lymph node stained with failitates identifiation of metastati melanoma. Distintion from dendriti ells, seen lower left, is made on the basis of ytology, with the dendriti ells having a poorly defined outline and smaller nulei. The -stained adjaent setion enables onfirmation of the ytologial features and the site of the metastasis in this ase subapsular. they represent a mixture of early but potentially aggressive metastases with some more indolent ones. In the meantime the European Organization for Researh and Treatment of Caner (EORTC) has suggested a linial trial of melanoma patients with SLN metastases onfined to the subapsular zone or less than 0.1 mm in maximum dimension irrespetive of site. These will be followed by observation for 5 years in the first instane. If these metastases are not assoiated with further disease then there would be some validation of a more seletive approah to RND. It would also put more pressure on pathologists to assess the SLNB in greater detail. At the moment these assessments are performed only as part of studies or trials, but virtually all patients having SLNB are in that ategory sine the proedure is to a large extent still being evaluated. SUMMARY: PART 1 Proposals for SLN assessment On the basis of our work and other published work we suggest that SLN for melanoma should be bivalved aording to Cohran s method. The resulting paraffin-embedded bloks should be setioned in our opinion in a stepwise manner. We urrently use 50 mm steps produing six pairs of setions to an approximate total depth of mm. There is an argument to make the steps wider or less numerous, 29 but bearing in mind the small size of many metastases and our lak of ertainty on the ritial dimension of the metastases we feel this hange in protool needs to be done in a systemati way. Van Akkooi et al 18 suggest metastases,0.1 mm diameter an be ignored. The EORTC observation study, in part, is intended to validate that observation and therefore we will ontinue with the existing EORTC protool of six pairs of setions at 50 mm intervals at least until that study reports. We have strongly reommended the need for immunohistohemistry in the assessment partly beause the detetion rate is then higher and partly beause the reognition of metastases is muh failitated, making the pathologist s task less onerous. Whether or Melan A is used is a matter of personal hoie. It is wise to retain some unstained setions at eah level for problemati ases. The features that should be reorded are not only the presene of metastases but their site and size. A metastasis in a subapsular site but with a markedly irregular aspet towards the parenhyma or single ells breaking away from the main metastasis should not be regarded as being entirely subapsular in site. Other measurements on whih further refinements of management may be based are the depth of the metastasis from the apsule to its deepest point, and the maximum dimension of the largest deposit. Van Akkooi et al 18 have suggested that this last dimension should be applied only to ohesive groups of ells rather than looser aggregates. Groups of 10 ells or less are already regarded by that group as negative. Others are taking a more autious line and would prefer to await the results of more studies on this point. Sine, at least in the UK, the SLNB proedure is not regarded as standard, and in all ountries is being arefully evaluated, it is not suggested that the pathologial assessments should simply be loaded onto pathology departments without areful onsideration of the labour or ost impliations. Furthermore, it is important that ommitted pathologists are involved in the reporting of SLN to ensure the reprodutibility of the detailed assessments urrently being undertaken in multientre trials. J Clin Pathol 2008;61: doi: /jp

6 We aept that SLNB for melanoma is in a phase of assessment and therefore we believe that the riteria we have set out and any new riteria need to be evaluated ritially before disarding or adopting them formally as reommendations for general appliation. SUMMARY: PART 2 Probable further developments A lower threshold thikness of the primary melanoma for entry to SLNB ould be onsidered as well as greater reliane on mitoti ativity, partiularly in males, and perhaps in due ourse on geneti profile. Sine urrently at least 70% of patients having SLNB have negative results, a greater fous on those who are more likely to be positive is predited. This may be ahieved by hanges to the riteria of the primary melanoma used to justify performing SLNB. It is also possible that RND will be offered to only those patients with metastases involving a parenhymal site or above a ertain size. There is some uriosity that some patients (30%) with histologially negative SLNB nevertheless still develop reurrent disease and have a poor prognosis. Moleular studies may play a role at this point. Attempts will be made to predit who these patients are, using more speifi markers. If RT-PCR is adequately validated it is possible that the protool for setioning and staining ould beome less extensive. It seems likely that the need for or Melan A will persist. Other assessments may be needed sine it is lear that the status of the immunologial reation to the metastases in the lymph node may be useful information when deiding upon immunologially mediated therapy. Dendriti ells are one omponent of this reation that have been shown to orrelate with survival. 30 Tumour infiltrating lymphoytes (TIL) 22 in the primary melanoma have prognosti signifiane in some studies. TIL in metastases may have a similar signifiane. Irrespetive of the ontroversy that SLNB seems to provoke it has stimulated muh interesting analysis of the biologial proesses involved in metastasis, some of whih may result in more benefiial therapy for the patients than the SLNB itself, and therefore it is likely to remain an important part of the management of melanoma patients for many years. It provides a major opportunity for pathologists to play a pivotal role in melanoma researh and management. Competing interests: None delared. REFERENCES 1. Gershenwald JE, Thompson W, Mansfield PF, et al. Multi-institutional melanoma lymphati mapping experiene: the prognosti value of sentinel lymph node status in 612 stage I or II melanoma patients. J Clin Onol 1999;17: Morton DL., Thompson JF, Cohran AJ, et al. Sentinel-node biopsy or nodal observation in melanoma. N Engl J Med 2006;355: Morton DL, Cohran AJ, Thompson JF, et al. Sentinel node biopsy for early-stage melanoma: auray and morbidity in MSLT-I, an international multienter trial. Ann Surg 2005;242: Thomas JM, Clark MA. Seletive lymphadenetomy in sentinel node-positive patients may inrease the risk of loal/in-transit reurrene in malignant melanoma. Eur J Surg Onol 2004;30: Estourgie SH, Nieweg OE, Kroon BB. High inidene of in-transit metastases after sentinel node biopsy in patients with melanoma. Br J Surg 2004;91: Kang JC, Wanek LA, Essner R, et al. Sentinel lymphadenetomy does not inrease the inidene of in-transit metastases in primary melanoma. J Clin Onol 2005;23: Pawlik TM, Ross MI, Thompson JF, et al. The risk of in-transit melanoma metastasis depends on tumor biology and not the surgial approah to regional lymph nodes. J Clin Onol 2005;23: Take-home messages Sentinel lymph nodes merit detailed histologial assessment at several levels. Immunohistohemistry ( or MelanA) greatly failitates the reognition of metastases of melanoma. Additional prognosti information an be obtained by assessing the sites and dimensions of metastases. 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