Biomarkers in melanoma: staging, prognosis and detection of early metastases

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1 Review CONTENTS Staging of melanoma Prognostic biomarkers for Stage I & II melanoma Individualized prognosis for Stage I & II melanoma patients Detection of metastasis in Stage I III melanoma Expert opinion Five-year view Key issues Information resources References Affiliations f Author for correspondence Division of Dermatopathology, Albany Medical College MC-81, Albany, NY 12208, USA Tel.: Fax: carlsoa@mail.amc.edu KEYWORDS: biomarkers, melanoma, metastases, prognosis, staging Biomarkers in melanoma: staging, prognosis and detection of early metastases J Andrew Carlson, Andrzej Slominski, Gerald P Linette, Martin C Mihm Jr and Jeffrey S Ross Currently, melanoma remains a surgical disease since early detection and excision of thin melanomas offers the best chance of a cure. Despite intensive clinical investigation, no effective systemic therapies exist for metastatic melanoma. Sentinel lymph node biopsy has greatly aided the staging and prognostic evaluation of primary cutaneous melanoma, however, approximately a third of patients diagnosed with metastatic melanomas present without prior regional lymph node involvement. Additional prognostic biomarkers exist which help determine the risk of advanced melanoma but the accuracy for each current marker is less than 100%. A greater understanding of the biology of melanomas and the development of new methods to identify patients with early (subclinical) metastatic disease may allow for selective and more effective therapy for patients at-risk for advanced disease. In this paper, current and novel potentially more accurate biomarkers for the staging and prognostic evaluation of melanoma patients, and for the detection of subclinical metastases are reviewed. Expert Rev. Mol. Diagn. 3(3), (2003) Early primary cutaneous melanoma, characterized by a radial growth phase confined to the epidermis and/or to the superficial dermis, can evolve and exhibit a vertical growth phase capable of invading the deep dermis, ultimately giving rise to metastasis [1,2]. In general, thickness (Breslow s depth) of the primary lesion directly correlates with prognosis. For most in situ or thin melanomas without evidence of regression or expansive junctional nests, complete excision is curative [2 4]. In contrast, the prognosis for individuals with advanced or metastatic melanoma is poor with a 5-year survival of 5 10% [5]. This poor survival rate is, in part, the consequence of the lack of effective treatment for metastatic disease [5,6]. Therefore, most clinical experts contend that prevention and early detection of thin cutaneous melanoma is currently the best means of reducing melanoma mortality [7 10]. A search for biomarkers that can predict which patients are most likely to develop metastasis is an approach that would allow earlier therapeutic intervention for patients deemed at high risk for relapse. Novel biologic and improved conventional therapy protocols have raised hopes that metastatic melanoma may eventually become more manageable or, ultimately, curable [11 25]. Indeed, the current challenge for melanoma is to determine which of the new prognostic biomarkers should become part of a standard pathology report and in what context should they be included. This supplementary information could be added to the standard pathology report and has the potential to significantly increase the accuracy of predicting melanoma prognosis for the individual. This review will cover conventional and novel biomarkers for the staging, prognosis and detection of early metastatic disease. Staging of melanoma Overall, the single best indicator of prognosis of melanoma patients is the stage at first clinical presentation. The staging of melanoma, specifically for Stages I, II and III, is heavily reliant on histologic findings. Specifically, tumor depth, the presence or absence of ulceration, the Future Drugs Ltd. All rights reserved. ISSN

2 Carlson, Slominski, Linette, Mihm & Ross presence or absence of microscopic metastases either peritumoral microsatellites or regional sentinel lymph node and the number of regional lymph nodes involved are key to the correct staging of early and intermediate stage melanoma [26]. The American Joint Committee on Cancer (AJCC) staging system for melanoma has recently been revised and is based on followup of 17,600 patients at 13 institutions entered into the database [27,28]. This revised staging system has been approved by the AJCC, the International Union Against Cancer (UICC) Tumor-Node-Mestatasis (TNM) Committee, the World Health Organization (WHO) Melanoma Program and European Organization for Research and Treatment of Cancer (EORTC) Melanoma Co-operative Group [27,29]. This revision fulfills the criteria for staging [27]: Practical, reproducible and applicable to all medical disciplines, it adequately reflects the biology of melanoma Evidence-based and utilizes the dominant prognostic factors identified by Cox multivariate regression analyses Relevant to and regularly incorporated into clinical trials and yields data that is easily culled from medical records for tumor registry use. The revised AJCC melanoma staging scheme is a substantially modified and more complex staging system for cutaneous melanoma and considered a marked improvement of past staging systems. The following modifications are the most important: The primary determinant of tumor (T) staging is tumor thickness as measured in millimeters and the Clark level of invasion is now used only for defining T1 ( 1 mm) melanomas The cut-off points for tumor thickness are less than or equal to 1, 1 2, 2 4 and greater than 4 mm Ulceration has been added in describing the primary tumor and is defined as an absence of an intact epidermis overlying a major portion of the primary melanoma based on microscopic examination and, moreover, ulceration is an independent factor that has successfully predicted the presence of a sentinel lymph node (SLN) metastasis [30] Clinical satellites, microsatellites and in-transit metastases have similar prognostic implications, are categorized nodal (N) disease and are placed in the regional Stage III disease classification Size of lymph node as prognostic factor has been eliminated and replaced with the number of positive nodes and, in addition, presence of so-called micrometastases identified by SLN biopsy is factored into pathologic staging. Although controversy still exists as to whether or not the SLN biopsy procedure alters a patient s survival, it has been demonstrated to be a powerful prognostic indicator [31] The presence of an elevated serum lactic dehydrogenase (LDH) level is used in the metastasis (M) category This revised staging system more precisely defines prognosis and will improve the stratification of patients in future clinical trials. The importance of this data lies in pathologic reporting of melanoma where the above information should be documented. In addition, this revised staging scheme has been validated by different melanoma databases showing both correlation between increasing stage and decreasing survival with incremental differences between subcategories (TABLE 1A&B) [32 34]. Clinical evaluation & investigations in the staging of melanoma patients At first presentation, clinical Stage I and II melanoma patients require a complete clinical examination to detect a second primary melanoma and/or metastases, particularly regional nodal disease [35,36]. In addition, some authorities recommend baseline chest x-ray and blood testing for serum alkaline phosphatase and LDH [37,38]. In contrast, other investigators do not Table 1A. Melanoma staging and survival by updated AJCC 2000 classification [27]. Tumor-node-metastasis (TNM) classification T classification T1a 1.00 mm, no ulcer T1b T2a T2b T3a T3b T4a T4b 1.00 mm, ulcerated or level IV/V mm, no ulcer mm, ulcerated mm, no ulcer mm, ulcerated >4.00 mm, no ulcer >4.00 mm, ulcerated N classification N1a 1 lymph node, micrometastasis N1b 1 lymph node, macrometastasis N2a 2-3 lymph nodes, micrometastasis N2b 2-3 lymph nodes, macrometastasis N2c N3 In-transit/satellite metastasis 4 metastatic lymph nodes/matted lymph nodes/ combinations of in-transit/satellite metastasis or ulcerated melanoma with metastatic lymph nodes M classification M1 Distant skin, subcutis or lymph node metastasis, normal LDH M2 M3 Lung metastasis, normal LDH All other visceral metastases, normal LDH/any distant metastasis, elevated LDH Micrometastasis diagnosed after sentinel or elective lymph node dissection. Macrometastasis are defined as clinically detectable metastases confirmed by therapeutic lymphadenectomy or when any lymph node exhibits extracapsular extension. AJCC: American Joint Commitee on Cancer; LDH: Lactic dehydrogenase. 304 Expert Rev. Mol. Diagn. 3(3), (2003)

3 Biomarkers in melanoma Table 1B. Melanoma staging and survival by updated American Joint Commitee on Cancer 2000 classification [27]. Stage T (Tumor) N (Node) M (Metastasis) 5-year survival (%) [28] 10-year survival (%) [28] IA T1a N0 M (94.4) 87.9 (88.5) IB T1b N0 M (92.8) 83.1 (84.6) T2a N0 M IIA T2b N0 M (76.7) 64.4 (65) T3a N0 M IIB T3b N0 M (74.9) 50.8 (61.8) T4a N0 M IIC T4b N0 M (46.0) 32.3 (30.1) IIIA T1-4a N1a M (62.5) 63.0 (54.7) T1-4a N2a M IIIB T1-4a N1a M T1-4b N2a M T1-4a N1b M T1-4a N2b M IIIC T1-4b N1b M T1-4b N2b M any T N3 M (42.6) 18.4 (25.7) IV any T any N any M (14.3) (7.1) M1a M1b M1c Survival data from [34]. recommend additional testing due to low yield in the absence of clinically symptomatic regional or systemic disease [36,39,40]. Most clinical experts believe that elective lymph node dissections (ELND) are not routinely indicated [36,39,40]. However, the randomized Intergroup Melanoma Surgical Trial published in 1996 defines two subgroups that benefit have improved 5-year survival with ELND: patients less than 60 years of age and patients with a primary lesion between 1.1 and 2.0 mm in depth [41]. More recently, SLN biopsy for melanomas larger than 1 mm thickness designed to detect micrometastases has become the de facto standard of care in many regions of the world for the staging of primary cutaneous melanoma [37,42,43]. According to the hypothesis proposed by Morton and colleagues, the SLN accurately reflects the status of the regional nodes based upon the lymphatic patterns of spread from a primary cutaneous melanoma and allows the pathologist an opportunity to examine in detail one or several nodes [42]. For patients presenting with regional, in transit, and nodal metastases (clinical Stage III melanoma), more extensive clinical studies, such as CT scans, are indicated to complete the staging evaluation [36,37]. For example, CT imaging of the regional area is suggested for patients with locally advanced melanoma of the head/neck or inguinal-femoral area to better assess deep nodal involvement that is difficult to monitor with physical examination. Additional imaging studies (such as brain MRI, bone scan and CT scan of the chest/abdomen/pelvis) are usually performed only if symptoms and physical signs warrant. In approximately 10% of these cases, distant asymptomatic metastases will be found [36]. Finally, for patients presenting with metastatic melanoma (clinical Stage IV), a careful search for the primary tumor, which includes a history of all cutaneous procedures, biopsies or treatments, is warranted. In addition, proper staging for newly diagnosed Stage IV melanoma includes CT imaging of the chest/abdomen/pelvis, brain imaging (MRI with gadolinium is recommended if available), chest x-ray and complete blood work including LDH [36,37]. Confirmation of the metastasis should be achieved by cytologic (fine needle aspiration), or histologic examination, which may include immunohistochemical testing to confirm the melanocytic lineage [36]

4 Carlson, Slominski, Linette, Mihm & Ross Prognostic biomarkers for Stage I & II melanoma Data from the 2001 AJCC staging committee conclusively demonstrates that thin melanomas (<1 mm) have an excellent prognosis with greater than 90% survival at 5 years. In contrast, thick melanomas (>4 mm with ulceration) are associated with a relatively poor prognosis, with approximately 45% survival at 5years [28,33]. However, it has become clear that a minority of patients with thin melanomas will succumb to metastatic disease, while a subset of patients with thick ulcerated primary lesions never recur. In an effort to better predict survival for Stage I and II melanomas, numerous individual prognostic factors and multifactor models have been used to predict disease outcome in both small and large melanoma cohorts. Amongst all of these potentially useful prognostic biomarkers, the new AJCC staging system incorporates only tumor thickness, ulceration, microsatellites and nodal status [28]. The elimination of routine use of Clark s level of invasion has created some debate. Marghoob and coworkers from the New York Melanoma Cooperative Group published evidence that for any given thickness, the 10-year survival is worse in patients with deeper Clark s levels of invasion [44]. Omission of this biomarker and many of the other clinicopathologic prognostic factors from staging/prognostic assessment is likely due, in part, to an absence of reproducible definitions, easy applicability and lack of agreement amongst the world s experts as to their relative importance. Apart from tumor or Breslow thickness, most of these other putative biomarkers have not been systematically studied, rigorously defined and tested for reproducibility, nor used for prognosis assessment in large groups of melanoma patients with 5 years or more follow-up (TABLE 2) [45]. For example, potentially useful biomarkers, such as mitotic count or vascular invasion, both found to be strong predictors of survival in small collectives of melanoma patients [46,47], have not been evaluated in large cohorts of melanoma patients [32 34]. Moreover, molecular prognostic biomarkers, such as growth fraction as measured by Ki-67 monoclonal antibody (mab) immunolabeling [48 50], are not considered. Molecular markers that correlate with melanoma progression may help in patient selection for adjuvant therapies or represent novel targets for treatment as they are related to the pathogenesis and progression of melanoma. TABLES 2 & 3 list clinicopathologic and molecular biomarkers, respectively, that are suggested as part of clinicopathologic report (TABLE 2) or have been identified by multivariate analysis as having a significant (e.g., independent of tumor thickness) association with outcome (TABLE 3). Clinicopathologic biomarkers predictive of prognosis The concept of melanoma tumor progression and multiple factor analysis of melanoma was first developed by Day and coworkers in the Melanoma Co-operative Group [51 53]. These ideas were later more fully delineated and tested by Clark and coworkers, who demonstrated that metastasis was associated with a specific pattern of tumor growth, termed the vertical growth phase [1,2]. Most melanomas are suspected to evolve through an initial stage termed the radial growth phase (in situ and microinvasive melanomas), which, if excised, has an almost 100% probability of cure, although by the time of diagnosis, the majority of melanomas are in the vertical growth phase with a risk of metastasis that is directly related to its depth of invasion. However, the presence of the vertical growth phase and tumor thickness, by themselves, do not accurately predict outcome [4,54] and should, therefore, not be used as a sole criterion for adjuvant therapy [54]. For example, high mitotic counts and regression are more common in thin metastasizing melanomas [4,55], whereas thick melanomas with prolonged survival have low mitotic counts and spindle cell populations significantly more often [56]. Ulceration superimposed on primary melanoma is also an independent biomarker of survival that upstages melanomas of equal depth [33]. However, ulceration may ultimately be a clinicopathologic sign of the presence of SLN metastasis [30] because the ulceration significance drops to borderline level when SLN status is factored into survival analysis [57]. Similarly, vascular invasion and tumor angiogenesis are independent risk factors for decreased survival and likely histologic markers of the metastatic process [47,58]. Both of these histologic features are highly predictive of SLN metastasis [59] and tumor ulceration [58], respectively. In addition to ulceration and tumor thickness, clinical features of site, age and sex, and the histologic characteristics of level of invasion, mitotic rate, tumor-infiltrating lymphocytes, histologic regression, microscopic satellites, histologic subtype, vascular invasion, tumor vascularity and tumor cell type have all been found to have independent prognostic value in small and/ or large melanoma cohorts [2,3,44,47,58,60 62]. Apart from anatomic site, ulceration and tumor thickness, most of these other clinicopathologic prognostic factors have been found to be independently significant in less than 75% of studies with greater than 450 patients and 5 years or longer mean or median follow-up (TABLE 2). Nonetheless, recent guidelines for the reporting of melanoma include most of these clinicopathologic observations for reasons of patient stratification, research use and the belief that multiple-factor is more accurate than singlefactor prognosis assessment [29,36,38,63 66]. Small melanoma cohorts, inadequate follow-up and the use of varying criteria or techniques to measure the specific histologic finding have hampered acceptance of most of these pathologic biomarkers in risk-assessment. Therefore, a need exists for the development of well-defined criteria for all of these pathologic biomarkers and for education sessions to disseminate the criteria and standardize the reporting of melanoma [66]. For example, in a review of melanoma patients referred to a major cancer center, only a minority of routine melanoma pathology reports commented on the presence or absence of ulceration, and approximately half did not report ulceration when present [67]. Consequently, these melanoma patients would not have been correctly staged by AJCC standards if not for this independent review. Many clinicopathologic biomarkers could play an important role in refining the prognosis and determining subsequent treatment of melanoma patients. Before their relative worth can be tested in large melanoma collectives, thus accepted in melanoma 306 Expert Rev. Mol. Diagn. 3(3), (2003)

5 Biomarkers in melanoma staging, specific criteria and standardized reporting of their presence must be universally accepted and rigorously applied. Novel protein, molecular & chromosomal prognostic biomarkers Numerous molecular and immunohistochemical studies have found biomarkers that identify patients at risk for advanced melanoma, either locoregional or distant metastases by Cox proportional hazards univariate and multivariate analyses [26,60,68 72]. Most of these biomarkers simply highlight the pathogenic mechanisms of melanoma progression and, in multivariate analyses, do not improve upon morphologic phenotypic markers used for staging melanoma on routinely processed tissue sections [60,66,70,73]. Nonetheless, these molecular biomarkers improve our basic understanding of the biology of cutaneous melanoma, potentially offering new targets for novel treatment Table 2. Clinicopathologic prognostic biomarkers that are standard or suggested as part of the pathologic report [36,38,63 65]. Biomarker Categorization Survival (%) Ref. Studies with an impact on prognosis Ref. Clinical variables Age (years) (10-year) [33] 14/21 (67%) [32,33,45,90, ] Sex Male Female 57 (8-year) 83 [2] 13/20 (65%) [32,33,45,90,93,264, 265, ,274] Anatomic site Extremities (arms & legs) Trunk, head & neck Volar or subungal 87 (8-year) [2] 16/20 (80%) [33,45,60,89,90,93,94, ,272,273] Margins of excision Wide local excision (standard) Narrow local excision 84 OS 84 NS [275] Surgical interval between biopsy and excision <14 days >92 days 72 (10-year) 75.9 NS [276] Clinical stage Localized melanoma Regional lymph nodes (gross clinical disease) Distant metastases 79 (10-year) 14 2 [277] Histologic variables Pattern of melanoma growth Radial growth phase Vertical growth phase (dermal tumor nodule formation) 100 (8-year) 71 [2] Thin 1 mm melanomas Junctional expansion nodule Adnexal involvement [4] Tumor thickness 1 mm mm mm >4.0 mm 91 (10-year) [278] 22/22 (100%) [32,33,44,45,60, 89 94, ] Clark level of invasion Mitotic count 0/mm 2 6/mm 2 >6/mm 2 II (papillary dermis) II (expansion of the papillary dermis) IV (reticular dermis) V (subcutis) 98 (10-year) (8-year) [44] 6/12 (50%) [33,44,89,90,264,265] [2] 5/8 (62%) [32,60,89 91] 307

6 Carlson, Slominski, Linette, Mihm & Ross Table 2. Clinicopathologic prognostic biomarkers that are standard or suggested as part of the pathologic report [36,38,63 65]. Ulceration Thin 1 mm melanomas Lymphoid response Absent Present Absent Present (histologic absence of intact epidermis over tumor) Brisk (diffuse lymphocytic infiltrate) Nonbrisk (foci of lymphocytic infiltrates) Absent (no lymphocytes infiltrating tumor) (10-year) [2] 10/12 (83%) [32,33,45,89,90,93, 264,267,271,273] [33] [279] 3/5 (60%) [45,89,93] Regression Absent or incomplete 77 (8-year) [2] 1/6 (17%) [89] Present 60 Thin 1.5 mm melanoma Absent Present (tumor thickness plus regression thickness in mm) 93 (5-year) 64 [280] Vascular involvement/invasion (predictor of sentinel lymph node metastasis) [59] Tumor vascularity (correlates with ulceration) Microsatellites (microscopic equivalent of clinical in-transit metastasis) [33] Presence of a benign nevus Histologic classification Melanoma cell morphology (phenotype) Spindle cell vs. conventional melanoma Sentinel (regional) lymph node metastasis Absent Present (melanoma cells within or fixed to the walls of vessels) Absent (no apparent change in normal vascular plexus) Sparse/moderate (increased small and dilated vessels) Prominent (easily apparent widely dilated and small vessels) Absent Present (nests of tumor 0.05 mm distant from main tumor) Absent Nevus present Acral melanoma Superficial spreading melanoma Conventional Stage I and II melanoma Desmoplastic and neurotropic melanoma Conventional melanoma >4 mm (Stage II) Spindle cell melanoma, mean 3.9 mm (Stage II) 54 OS OS (10-year) (5-year) 87 NS 75 (8-year) (5-year) 90 (5-year) 34 (12%) 3% (8%) [47] [58] [281] [91] 0/4 (0%) [282] 2/14 (14%) [90,273] Studies evaluating prognosis of melanoma with more than 450 cases with 5 years or longer median or mean follow-up. Found to be significantly associated with metastasis 41 versus 20% and 18 vs. 4% for junctional expansion nodules and melanomatous adnexal involvement, respectively, for thin metastasizing melanomas versus thin melanomas without metastasis and 5 years follow-up. Frequency of positive lymph nodes in these forms of cutaneous melanoma (% of clinically and pathologically positive lymph nodes reported in [283]). OS: Overall survival; NS: No significant differences found for survival. [283] [31] [169] strategies. These molecules can be broadly categorized according to their mechanisms of action and are related to the histologic prognostic markers tumor thickness, ulceration, mitotic rate, histologic regression, tumor vascularity and presence of tumor-infiltrating lymphocytes. For example, tumor thickness and mitotic rate reflect the degree of cellular proliferation, a process representing the balance between cell birth and death (apoptosis) influenced by cell cycle regulatory proteins such as p16 INK4a. Tumor thickness is also influenced by the degree to which tumor cells can invade the dermis and maintain an 308 Expert Rev. Mol. Diagn. 3(3), (2003)

7 Biomarkers in melanoma adequate blood supply, processes governed by proteolytic enzymes, adhesion molecules and angiogenic growth factors. Regression and tumor-infiltrating lymphocytes are indicative of the host immune response and the melanoma s putative escape from immune surveillance with progression a process intricately controlled by cytokines and other immunoregulatory molecules and denoted by less immunogenic, more malignant tumor cells [68]. Some of the most promising molecular markers are discussed and include cell cycle biomarkers [46,50,74,75], p16 INK4a /CDK2NA [48,76], MLSN-1 (Melastatin, Millennium Pharmaceuticals Inc., Cambridge, MA, USA) [77], transcription factor activator protein (AP)-2 [78], matrix metalloprotein (MMP)-2 [79], DNA content (ploidy) and fractional allelic loss analysis [80,81]. Other independent molecular biomarkers have prognostic significance in subgroups of melanoma patients, such as c-myc gene amplification in acral melanomas [82,83] or β3 integrin expression in intermediate thickness melanomas [84]. In addition, melanoma cells possess a remarkable repertoire of biosynthetic capacities represented by the production of hormones, growth factors and their receptors that may sustain and accelerate tumor development and progression (TABLE 3) [11,85]. Table 3. Independent prognostic biomarkers in Stage I and II melanoma identified by multivariate analysis or subgroup analysis. Biomarkers Specific finding (subgroup) Survival (%) Ref. Cell cycle markers/regulators Ki-67 (MIB-1) PCNA >20% (VGP) >20% (>4 mm) >median >35% (VGP)! index 9 (5-year) OS 10 (10-year) OS 0 (5-year) OS 9/10 (5-year) DFS/OS Worse (5-year) OS Mitotic index >1% 50 (10-year) OS [46] S-phase fraction >4% 50 OS [46] Nuclear organizing regions! count 50 (10-year) DFS [75] Cyclin A 5% (SSM) 40 (10-year) DFS [97] Cyclin D3 >5% (SSM) 40 (10-year) OS, DFS [96] p16 INK4a Absent p16 37 (10-year) OS [48] CDK2NA 540 C>T polymorphism 70 (10-year) OS [76] p27 kip1 <5% (NM/p21Waf1 >5%) 35 (5-year) DFS [98] [48] [49] [50] [284] [74] p53 + expression (VGP) >10% (>1.5 mm) - expression 50 (10-year) OS 68 (5-year) OS 16 (10-year) OS [48] [285] [286] Melanocyte differentiation-related antigens Microphthalmia-associated transcription factor <50% ( mm) 60 (5-year) DFS [287] HMB-45 (gp100) >91% 41 (5-year) DFS [284] Other gene markers Activator protein (AP)-2 transcription factor 0 75 index 35 (10-year) DFS [78] N-ras No codon 18 mutation DFS 288] c-myc High expression (ALM) 0 (10-year) OS [82] Metallothionein + expression 60 (10-year) OS [289] MLSN-1 Loss of mrna 51 (8-year) DFS [77] Cell adhesion and motility markers Integrins- β3 + expression ( mm) 40 (10-year) OS [84] CD % 45 (10-year) DFS [290] 309

8 Carlson, Slominski, Linette, Mihm & Ross Table 3. Independent prognostic biomarkers in Stage I and II melanoma identified by multivariate analysis or subgroup analysis. Immunoregulators/immune markers Human leucocyte antigen (HLA)-ABC <90% (group 3 )! OS [60] Growth factors Osteonectin >50% ( 0.75 mm) 30 (10-year) OS, DFS [291] Extracellular degrading/proteolytic enzymes Plasminogen activating system 6 50% ( mm) 50 (10-year) OS, DFS [292] Matrix metalloprotein (MMP)-2 >34% 55 (5-year) OS [79] Chromosome aberrations/dna content Anuesomy- fractional allelic loss (FAL) High FAL 20 (10-year) OS, DFS [80] DNA ploidy! DNA index Aneuploid (VM) Aneuploid " DFS 20 (10-year) OS 20 (10-year) DFS Group 3 as defined in [293]; mm with or without ulceration and >1.5 mm, nonulcerated on extremities. Log rank test used. Thickness and vascular invasion were also significant predictors of metastasis. Multivariate logistic regression analysis for risk of metastasis. ALM: Acral lentiginous melanoma; DFS: Disease-free survival; ER: Early relapse; NM: Nodular melanoma; OS: Overall survival; SSM: Superficial spreading melanoma; VGP: Vertical growth phase melanoma; VM: Vulvar melanoma. [81] [123] [124] Cell cycle markers Proliferative activity correlates with metastatic potential and has significant prognostic value in several cancers [86 88]. For melanoma, five out of eight (62%) studies on large melanoma collectives (>450 patients with 5 years median or mean followup) have demonstrated a significant, independent relationship between increasing mitotic count and decreasing survival [32,60,89 94]. Ki-67/MIB-1 index, proliferating nuclear antigen (PCNA) index, S-phase fraction and silver-stained nuclear organizing region (AgNOR) counts all are reliable and widely used methods of measuring proliferative index (growth fraction) [95] and have been demonstrated to be independent prognostic factors in small melanoma cohorts [46,50,74,75]. Moreover, cell cycle regulators, such as cyclins A and D3, p27 Kip1 and p16 INK4, correlate with proliferative index markers and independently predict for prognosis in subsets of melanoma patients (e.g., cyclin D3 index >5% in superficial spreading melanoma) [96 98]. Thus, detection of cell cycle biomarkers is feasible for prognostic purposes in melanoma staging. Ki-67, for instance, may well prove to be the most effective biomarker as it is a widely available immunohistochemical test that busy practitioners can reasonably and reproducibly use to supplement the pathologic analysis (FIGURE 1). This antigen can be detected in the nucleus of cells during late G1, S, G2 and M phases, which effectively measures most cells in the cell cycle [95]. Most studies in melanoma have demonstrated that the Ki-67 index is a useful prognostic biomarker for melanoma progression with decreased overall survival (OS) and risk for metastatic disease [48 50,99 102]. However, this correlation appears to be related to melanoma thickness because high Ki-67 index in melanomas thicker than 1.5 mm are at risk for metastases or death from melanoma [48,49,99 101] but this is not the case for thin melanomas [99,100]. Highlighting this relationship is a novel proliferation-based prognostic index (tumor thickness x Ki-67 index) which improves upon Ki-67 index s prognostic capability [101]. Contrarily, two studies failed to demonstrate the prognostic value of Ki-67 index [103,104]. Nonetheless, most of the evidence points towards a significant relationship of proliferative activity with outcome warranting, in our opinion, evaluation in large melanoma collectives to ultimately test the usefulness of proliferative index in prognostic assessment. p16 INK4a P16 INK4a, also known as CDK2NA, inhibits the ability of cyclin-dependent kinases (CDK)-4 and -6 to activate critical substrates necessary for progression from G1 to the S-phase of the cell cycle [ ]. As a tumor suppressor gene, p16 INK4a appears to play a fundamental role in melanoma progression [70,76], whereas aberrations (mutation, homozygous deletion) involving other products of CDK2NA locus, such as p14, p15 and p18, are extremely rare in melanoma cell lines and sporadic melanomas [105]. Germline mutations of the p16 INK4a gene are found in approximately 25% of families with inheritable melanoma [9]. In addition to mutations, promoter methylation and loss of heterozygosity are common mechanisms of silencing p16 gene activity in sporadic melanomas [76]. Benign melanocytic nevi uniformly express the p16 protein [ ], while increasing loss of p16 INK4a expression is seen with melanoma progression [48, ]. This loss of p16 INK4a protein expression correlates with increased cell proliferation (Ki-67 index), advanced stage and metastases [48]. However, loss of p16 INK4a appears to be a late rather than initiating event in melanoma as 310 Expert Rev. Mol. Diagn. 3(3), (2003)

9 Biomarkers in melanoma melanoma in situ indicates no alterations, or only a decrease, but not loss of p16 INK4a expression [ ]. For invasive, vertical growth phase melanomas, loss of nuclear p16 INK4a immunoreactivity predicts for poor 10-year survival (37%) [48], whereas the presence of p16 INK4a gene polymorphisms 540 C > T has a positive effect on 10-year survival (70%) [76]. Transcription factor AP-2 The transcription factor AP-2 is a 52 kda DNA-binding protein that is thought to inhibit tumor growth through the activation of p21. In a consecutive series of 369 clinical Stage I cutaneous malignant melanoma patients, loss of AP-2 expression is significantly associated with low p21 expression, increased tumor thickness, high TNM category and recurrent disease [78]. In this study, low AP-2 index was an important predictor of both recurrence-free survival (RFS) and OS. Based on these results, loss of AP-2 expression can be associated with malignant transformation and tumor progression, with the tumor-suppressive action of AP-2 mediated through p21 regulation and where decreased AP-2 expression independently associates with elevated risk of subsequent metastatic behavior. MLSN-1 MLSN-1 is a member of a novel family of at least three distinct putative calcium channel proteins that are distantly related to the transient receptor potential (Trp) calcium channel family [113]. The regulation of intracellular calcium is widely used in biologic signaling and appears to be a particularly important component of signaling pathways that affects cell cycle regulation and cell survival. By in situ hybridization techniques with 35 S-labeled probes on 150 patients with localized melanoma, loss of MLSN-1 mrna independently predicted poor survival (77 ± 15 and 51% ± 8 8-year OS for Stage I and Stage II disease, respectively) (FIGURE 2) [77]. However, it has not yet been demonstrated that loss of MLSN-1 expression is necessary or sufficient for the acquisition of metastatic potential during the progression of melanoma [77]. The authors recommend that future studies should explore MLSN-1 together with other immunohistochemical and molecular markers to determine whether there is added prognostic utility in the combined marker set for predicting metastatic potential and/or the presence of micrometastases in the SLN. This knowledge could be used to direct staging investigations, such as SLN biopsy and therapy. MMP-2 Proteolytic enzymes, such as MMPs, are instrumental in the degradation of extracellular matrix components and are crucial in the first step of invasion of normal tissues by cancer cells. MMP-2, also known as Type 4 collagenase, is suspected to augment the metastatic capability of melanoma cells by degrading the basement membrane zone and surrounding tissues [114,115]. Specifically, MMP-2 expression correlates with hematogeneous metastases [115] but not tumor thickness [79] and in male patients particularly, high MMP-2 expression independently predicts for poor 5-year survival [79]. Chromosome analysis (fractional allelic loss) & DNA content (aneuploidy) Gross chromosomal losses and gains characterize most human tumors including melanoma [116,117]. Moreover, aneuploidy is believed to be the ultimate driving force in carcinogenesis, rather than mutations in tumor suppressor genes and oncogenes [118]. Indeed, melanoma exhibits nonrandom chromosomal break points, specific aneusomies or specific allelic imbalances, and metastasis is suppressed by specific chromosomes implicating chromosomal instability in melanoma pathogenesis [83,117, ]. Underscoring the importance of chromosomal instability in the natural history of melanoma is the finding that increasing fractional allelic loss correlates with poor survival and shortened disease-free survival [80]. Similarly, increasing DNA index (aneuploidy) predicts for both decreased disease-free-survival and poor OS (FIGURE 3) [81,123,124]. Figure 1. Ki-67 (MIB-1) expression in primary melanoma directly correlates with outcome [48 50,99 102]. Example of nodular melanoma where most of the nuclei are labeled by Ki-67. Individualized prognosis for Stage I & II melanoma patients The current approaches to prognostication allow for assignment of patients to risk categories but they do not permit accurate assessment of predicted outcome for any one individual with Stage I or II melanoma. Most melanoma patients are cured by local excision and therefore do 311

10 Carlson, Slominski, Linette, Mihm & Ross Figure 2A. in situ hybridization image using a radiolabeled MLSN-1 probe and dark-field illumination. MLSN-1 expression loss (arrows) can be seen in this thick nodular melanoma [302]. not require additional therapy more extensive surgery or new adjuvant therapies that could lead to significant and unacceptable morbidity. However, a minority of patients, mostly those with thick primaries, have an intermediate or high risk for recurrent melanoma and comprise a unique subset of patients with surgically treatable melanoma for whom cure is possible but relapse and distant metastases likely. Strategies to improve the prognosis for the latter group of patients with effective adjuvant therapies are critical and require accurate, individual-based staging. For example, in recent randomized trials on patients at high risk for recurrence (patients with thick primary melanomas, Stage II and those with regional lymph node metastases, Stage III), those patients administered adjuvant therapy with high-dose interferon (IFN)-α2b had significantly improved RFS and OS rates [125]. Clark and coworkers at the University of Pennsylvania, USA, demonstrated that multifactorial prognostic assessment is superior to that based on thickness alone [2] and their multifactorial model is approximately 85 89% accurate [2,126]. In a more recent attempt to improve risk assessment, Cochran and coworkers developed a simple, two-step multifactorial formula based on the five variables: gender, age, anatomical site, thickness and ulceration to determine likelihood for recurrence of melanoma using readily available demographic information that is likely to be more accurate than single-factor analysis [127]. Similarly, Thomé and coworkers applied mathematical formula to determine the potential benefit of adjuvant interferon therapy for individual patients based on revised AJCC melanoma staging system and results of meta-analysis of the Eastern Cooperative Oncology Group (ECOG) studies [21]. Based on their assumptions, survival improvements ranged up to 13%. Information such as from this latter study, would allow patients to make more informed decisions regarding the management of their melanoma. Therefore, the current question to answer is whether additional clinical and histologic, or novel immunohistochemical, molecular or chromosomal biomarkers in primary melanoma have any supplemental role in the prediction of prognosis and selection of therapeutic protocols in addition to standard AJCC staging. Ultimately, as the presence or absence of minimal residual cancer cells is at question, detection of melanoma cells at secondary sites may be more significant than presence or absence of specific biomarkers of systemic disease in the primary tumor [128]. Highlighting this point is the finding that tumor thickness does not carry prognostic significance in thick ( 4 mm) melanomas after multivariate analysis [54] and ulceration has borderline significance when SLN status is included in prognosis assessment [57]. Detection of metastasis in Stage I III melanoma The most important factor affecting melanoma patients is whether the tumor has spread before curative excision. Patients who develop recurrent disease clearly had occult systemic spread that was undetectable by the methods routinely employed for staging. A palpable node is the first and most frequent sign of melanoma recurrence. In fact, approximately two-thirds of patients who develop clinical metastases following treatment of a primary cutaneous melanoma initially present with locoregional metastases and the remaining third present with distant metastases [129,130]. SLN biopsy may detect the majority of patients with early regional lymph node involvement as well as predict for the probability of systemic disease [31]. However, the time course of the development of distant metastasis has been found to be more or less the same, irrespective of the metastatic pathway [129]. Furthermore, the SLN procedure alters the pattern of first recurrence with negligible regional lymph node basin and increased locoregional cutaneous and distant recurrences [131]. These findings suggest that for patients with in-transit or satellite metastasis and/or regional lymph node metastasis, hematogenic metastatic spread has already taken place. Thus, the diagnostic value of SLN biopsy and the therapeutic benefit of elective lymph node dissection may be limited, as satellite and in-transit metastases or direct distant metastases will not be detected and blood-borne spread may have already taken place when this intervention is performed. Nonetheless, at this time, it is unclear what the impact 312 Expert Rev. Mol. Diagn. 3(3), (2003)

11 Biomarkers in melanoma Melastatin expression correlates with melanoma thickness Tumor thickness Melastatin mrna diffusely present Focal or diffuse loss of melastatin mrna >2.0 mm 20% expression in thick melanoma Risk of metastsis mm mm 50% expression in intermediatethick melanoma 75% expression in intermediatethin melanoma <0.5 mm 100% expression in thin melanoma Figure 2B. MLSN-1 downregulation is more often encountered in thick versus thin melanomas and independently associated with survival. Image courtesy of Millennium Pharmaceuticals, Inc., Cambridge, MA, USA on survival is for the early detection of asymptomatic metastases, such as those found by SLN biopsy. In addition, the success of adjuvant chemotherapy is based on the assumption that it eradicates occult metastases before they are clinically evident. Therefore, the development of more sensitive and specific techniques to detect occult systemic spread, such as SLN biopsy, are crucial to spare patients with local disease unwarranted therapy and to more effectively treat patients who are at risk of dying of metastatic disease. Current follow-up recommendations for Stage I III melanoma The goals of follow-up are to detect disease recurrence and additional primary melanomas independent of the initial primary lesion at an early stage, with subsequent reduction in morbidity and mortality. Follow-up interventions can include patient education, patient self-examination of skin and lymph nodes, physician interval examination and laboratory/imaging studies. Controversy exists as to the frequency of clinical surveillance and also whether laboratory monitoring is costeffective [35,38 40, ]. Generally, follow-up of melanoma patients to detect melanoma recurrence is based on clinical examination with the encouragement of patient self-surveillance as the majority of all recurrences are patient detected [133,135,140,141,144]. Moreover, physician and patient surveillance should be life-long as melanoma can demonstrate late (>10 years after initial diagnosis) or ultra-late (>15 years) recurrence [ ]. In fact, recent parametric statistical analysis and past conventional nonparametric methods have demonstrated that late recurrence is to be expected, particularly for patients with thin melanomas even though the probability of cure increases with progressively long disease-free survival [146,150]. Therefore, the interval of physician examination is based on the individual: tumor thickness, risk for multiple melanomas (multiple or atypical nevi, family history of melanoma and past melanoma), and patient s anxiety and/or ability to recognize signs and symptoms of melanoma recurrence. As most melanoma recurrences occur within the first 2 years of diagnosis, intervals of 1 to 4 per year for the first 2 years and 1 to 2 per year thereafter are recommended [38,39,145]. In the clinical practices at the Albany Medical College (NY, USA), Siteman Cancer Center (Washington University, St Louis, MO, USA) and the Harvard Medical School (Massachusetts General Hospital, Boston, MA, USA), the current recommendations for patients with Stage I/II melanoma are office visits with full skin examination every 3 months for the first 2 years and then tapered over the following 3 years with a yearly follow-up after the fifth year. Physical examination for lymph node inspection and other anatomic sites clinically relevant are also documented during each visit. Chest x-ray (A/P only) and liver function tests, including LDH, are performed yearly. At this time, there is no evidence to support routine blood tests (such as blood counts or electrolytes) or imaging studies in the absence of clinical signs or symptoms as they rarely reveal asymptomatic metastases [38,39,133,135,136,140,145]. New techniques and laboratory tests that are more sensitive and specific in their ability to detect early, subclinical locoregional and distant metastases are currently being developed and validated. These studies could potentially be added to the initial work-up and follow-up care of melanoma patients (TABLE 4)

12 Carlson, Slominski, Linette, Mihm & Ross 1.1 mm level 3 60 Distribution of DNA mass (DNA index) Number of cells pg DNA Figure 3. Increasing DNA content (aneuploidy) and fractional allelic loss predicts for worse survival and decreased disease-free survival [80,81,123,124]. Example of an intermediate thickness melanoma exhibiting aneuploid DNA content from a patient who subsequently developed local in-transit metastases. Imaging studies As greater than 90% of disease recurrences are detected by history and/or physical examination [39,140,144,145], extensive imaging examinations are not indicated in the initial staging evaluation or follow-up of patients with Stage I or II melanoma unless suggested by review of systems or physical examination. Currently, it is not feasible nor cost-effective to perform whole body scans of asymptomatic patients with Stage I, II, or III melanoma [39,151,152]. Limitations of conventional CT imaging include an inability to detect normal-sized (<1 cm) tumor bearing lymph nodes or differentiate nontumor bearing enlarged lymph nodes from enlarged malignant ones, or postoperative/postradiotherapy scarring from actual tumor recurrence. Small metastases, less than 1 cm, are also often missed using conventional imaging techniques. Nonetheless, evidence exists that demonstrates the usefulness of sophisticated imaging techniques for the detection of subclinical metastatic melanoma, such as B-scan ultrasonography of local and regional lymph nodes and whole body 2-[153]fluoro-2-deoxy- D-glucose positron emission tomography (PET) [154,155]. Both techniques hold promise for increasing detection rates during follow-up of clinically silent (early asymptomatic) Stage III and IV melanoma patients, respectively. For detection of locoregional melanoma recurrence, ultrasound examination of the regional lymphatics in patients at significant risk for metastases (thick, ulcerated melanomas) was found to be more sensitive than palpation for detecting in-transit and nodal metastases and increased metastasis detection by 30% [156]. PET, due to a low sensitivity of 17%, should not be used for initial regional staging of clinical Stage I-II patients [157]. However, for distant metastases, PET shows promise in the earlier detection of recurrent melanoma as it appears to be more sensitive than CT scans and can thus alter surgical management of some patients [151,158]. PET can distinguish between same-sized benign and malignant lymph nodes, between surgical/postradiation scarring and viable tumor, and detect metastases smaller than 1 cm [155]. Recent meta-analyses of PET suggest that it has a high diagnostic accuracy, particularly for systemic staging, with a diagnostic odds ratio of 36.4 [159,160]. With respect to the evaluation of clinical Stage III patients, PET is cost-effective, relatively quick, easy to perform and delivers less radiation than a total body CT scan [161,162]. However, at this time, Mijnhout and coworkers believe it is not yet possible to develop guidelines for the effective use of PET in patients with melanoma due to poor methodological quality of available studies, specifically because of problems with verification, review and selection bias [159]. SLN The role of the ELND for patients with cutaneous melanoma remains one of the most debated topics of surgical oncology. ELND or SLN biopsy are the only known ways of confidently detecting microscopic nodal disease. Lymphatic mapping and SLN biopsy are supported as the standard of surgical care of melanoma by the WHO and the Sunbelt Melanoma Clinical Trial [42,163]. SLNs are the first set of nodes to receive drainage 314 Expert Rev. Mol. Diagn. 3(3), (2003)

13 Biomarkers in melanoma and cancer cells from a primary tumor site, thus this procedure is the optimal approach to detect microscopic nodal metastasis. Typically, a single SLN is identified by the surgeon, however, often multiple (two or more) SLNs are identified during the procedure and removed for pathologic analysis. The development of lymphatic mapping over the last decade has led to dramatic changes in the surgical approach to regional nodes draining solid tumors [164,165]. Regional lymph nodes are a common site of melanoma metastases and the presence or absence of melanoma in regional lymph nodes is the single most important prognostic factor for predicting survival [31]. Furthermore, identification of metastatic melanoma in lymph nodes and excision of these nodes may enhance survival in a subgroup of patients whose melanoma has metastasized only to their regional lymph nodes and not to distant sites [166]. SLN biopsy was developed as a low morbidity technique to stage the lymphatic basin without the potential morbidity of lymphedema and nerve injury. The presence or absence of metastatic melanoma in the SLN accurately predicts the presence or absence of metastatic melanoma in that lymph node basin. When performed by experienced centers, the false-negative rate of SLN biopsy is very low. As such, the nodal basin that contains a negative SLN will usually be free of microscopic disease. Since occult micrometastatic disease affects approximately 20% (range 9 to 42% [167]) of patients with Stage I and II melanoma, selective SLN dissection allows those patients with negative SLN biopsies to be spared a formal lymph node dissection, thus avoiding the complications usually associated with that procedure. Despite the fact that patients with primary tumors less than 1 mm thick or spindle cell melanoma, of any thickness, have a low incidence of positive SLN [168,169], SLN mapping is offered to certain patients with thin melanomas (< 1 mm) at some medical centers. While standard pathologic evaluation of lymph nodes may miss metastatic melanoma cells, more sensitive techniques are being developed which may identify micrometastases more accurately (TABLE 4) [170,171]. Immunohistochemistry & RT-PCR in SLN evaluation Since routine histologic examination of lymph nodes underestimates the presence of micrometastatic disease, more sensitive assays for detecting tumor cells have been developed [171,172]. Immunohistochemistry (IHC) is more sensitive than hematoxylin and eosin (HE) alone in the identification of micrometastases [173]. In this setting, HMB-45 (mab specific for gp100 lineage-restricted antigen) as well as A103 (mab specific for MART-1/Melan-A antigen) are relatively specific but lack sensitivity since approximately 20% of melanomas do not express either marker. In addition, the highly sensitive S-100 is often used in conjunction with gp100 and MART-1/Melan-A to assist in the pathologic evaluation [ ]. Using RT-PCR, it is possible to define a population of patients at higher risk for both recurrence, compared with routine HE histology [172, ]. Recently, Shivers and coworkers have compared molecular staging of patients by RT-PCR with conventional S-100 IHC staining of the SLNs [172]. In these studies, SLN specimens were bivalved and half of each specimen was examined by routine histology, including both HE and S-100 IHC. The other half of each specimen was analyzed by a nested RT-PCR assay. HE examination alone detected metastases in 16% of patients tested. Serial sectioning and IHC increased micrometastasis detection to 22%. In the remaining HE and IHC negative SLN, RT-PCR detected micrometastatic disease in 62%, further increasing the proportion of patients with evidence of nodal disease to 70% overall. This subset of patients with RT-PCR positive, IHC and HE negative SLN are at increased risk for recurrence (range 10.2 to 25%) compared with RT-PCR, IHC and HE negative SLN (range 1.6 to 6%) but at lower risk compared with RT-PCR, IHC and HE positive SLN (range 36 to 67%) [178,181,182]. This important study highlights the potential impact of well-controlled and validated molecular staging, however, independent confirmation is required prior to widespread adoption in the clinical laboratory. Approximately 30% of patients with invasive melanoma develop recurrence, while the average detection rate for RT-PCR is 50% [183]. This 20% difference may represent false-positives due to lymph node melanocytic nevi, melanophages and S100 protein positive Schwann cells or melanoma cells incapable of tumor progression [66,177,182, ]. Lymph node melanocytic nevi have an incidence of between 4 and 22% and can be differentiated from metastatic melanoma cells by HMB-45 and Ki-67/MIB-1 IHC negativity [174,189,193]. Alternative explanations for false-positives include mechanisms related to tumor dormancy and immune surveillance, both of which are frequent hypotheses related to the biologic heterogeneity of melanoma. With extended follow-up, it is plausible that a subset of the false-positive subgroup will recur, however, at present, this issue of the accuracy of molecular staging remains a lingering concern. False-negative results obtained using RT-PCR include presence of tyrosinase negative amelanotic melanoma cells or phenomenon of alternative splicing, such as tyrosinase and other melanogenesis-related proteins (MRPs) that are coded by genes containing several exons, that after transcription may generate alternatively spliced products [194]. Additional melanocyte specific genes, such as tyrosinase related proteins (TRP) Type 1 and 2, and use of primers at different exon locations may lessen the problem of false-negatives [194]. Another potential marker in the molecular evaluation of SLN is the apoptosis-related gene survivin. Survivin protein expression has been correlated with melanoma progression [195] and by RT-PCR of SLN, survivin gene expression significantly correlates with death or disease progression (38.5/100% survivin +/- disease-free patients) [196]. In addition, it is unknown whether complete lymph node dissection and/or adjuvant therapy with IFN-α2b improves outcome for these RT-PCR positive, IHC and HE negative cases. This question of the benefit of additional therapy for patients who are positive only by the molecular method is currently being investigated by the national multicenter Sunbelt Melanoma Trial [172]. Another potential benefit of RT-PCR in SLN evaluation is labor and cost effectiveness. SLN can be preselected based on RT-PCR positivity for further analysis by IHC [197]

14 Carlson, Slominski, Linette, Mihm & Ross Table 4. Techniques and biomarkers used for the detection of regional and distant melanoma metastases. Method and biomarker Sensitivity (+) (%) Specificity (-) (%) Detection rate a (%) Outcome Regional lymph node metastasis Ref. Clinical examination [156] Imaging Ultrasound [156] Ultrasound B-scan with fine needle aspirate cytology [154] PET with FDG [157] SLN biopsy Histology & IHC ( ) b 18 (12 26) b [157] HE [173] HE & IHC: By thickness (+) 17 (total) <0.76 mm 0 [294] mm mm 25 + >4.00 mm 39 + Tyrosinase gene expression (RT-PCR) 96 c 81 d [172] HE & immunoperoxiadase negative lymph nodes in Stage I & II melanoma 49 [69] Path-/RTPCR- 49 (37 56) 0 (deaths) [172] Path-/RTPCR+ 33 (31 41) 9% (deaths) [172] Path+/RTPCR+ 18 (13 22) 39% (deaths) [172] Survivin gene expression (RT-PCR) 72 e Unknown e 100% (survival - cases) 38.5% (survival + cases) Distant metastasis [196] Imaging CT scan [155] PET with FDG 79 (66 93) 86 (78 95) [159] Serology Melanoma associated proteins: -S-100-β protein 86 (CI 68 91) 91 (CI 87 94) 54% mets/" OS [226,228] Stage I/II (review) 4 (0 9) + 99 (96 100) - [202] Stage III (review) 29 (5 98) + 99 (96 100) - [202] Stage III (recent study) NR [230] -MIA 80 (CI 56 94) 62 (CI 50 70) Predicts metastasis [226] Stage I [295] 316 Expert Rev. Mol. Diagn. 3(3), (2003)

15 Biomarkers in melanoma Table 4. Techniques and biomarkers used for the detection of regional and distant melanoma metastases. Stage II 23 + Stage III/IV Predicts metastasis -TA90 antigen: 78 (92) f 77 (86) f 36% (5-year) OS [234] 4 mm thickness " DFS [235] NSE (all Stages) No significance [229] LASA-P Recurrence 50% [296] Cytokines, adhesion molecules, other proteins: -SIL-2R (Stages I III) " OS [297] -VEGF (all Stages) Predicts progression [298] -VCAM (Stage IV) 21 " OS [299] -ICAM (Stages I III) Not significant [297] -IL-10 (Stages I III) " OS [297] -Albumin (all Stages 15 (CI 4 35) 99 (96 100) Predicts metastasis [226] -LDH (all Stages) 48 (CI 30 68) 98 (CI: 96 99) Predicts metastasis [226] Melanogenesis related factors: -L-dopa/L-tyrosine ratio (all Stages) Predicts progression [230] -5-S-CD (Stages I III) Predicts metastasis [247] -6H5MI2C (all Stages) Rarely + End-stage finding [239] -Tyrosinase mrna RT-PCR 30/72% (2-year) DFS/OS [300] Stage I 18 (3-22) 100 (99 100) NR [207] Stage II 28 (23-34) 100 (99 100) StageI/II 19 (16-21) 100 (99 100) Stage III 30 (26-34) 100 (99 100) -Multimarker RT-PCR 40% (4-year) DFS g [208] Stage I/II 64 ± ± 11.8 Stage III 70.8 ± ± 13.3 Urine analysis -6H5MI2C (Stages I-III) 52 " levels,! OS [245] -5SCD (Stages I-III) 83 " OS [245] a For this column, + signifies detection rate percentage of melanoma patients is positive and - signifies percentage of controls is negative. b Range from [301] c Sensitivity for RT-PCR assay for tyrosinase in histologically positive lymph nodes. d Specificity for RT-PCR assay for tyrosinase in histologically negative, nonmelanoma lymph nodes. e Sensitivity calculated based on tyrosinase + mrna SLN (sensitivity = 26 + survivin/ 36 + tyrosinase SLN patients); no tyrosinase negative patients were tested for survivin mrna expression to determine specificity. f Sensitivity and specificity when patients receiving adjuvant immunotherapy with CancerVax excluded. g Worst overall survival for tyrosinase positive, Mart-1 negative patients. 5-S-CD: 5-S-cysteinyldopa; 6H5MI2C: 6-hydroxy-5-methoxyindole-2-carboxylic acid; CI: 95% confidence interval; DFS: Disease-free survival; DOR: Diagnostic odds ratio for a positive test result (true-positives/false-positives); FDG: Fludeoxyglucoselasa; HE: Hematoxylin eosin; IHC: Immunohistochemistry; LASA-P: Lipid-associated sialic acid; MIA: Melanoma inhibitory activity; NR: Not reported; NSE: Neuron specific enolase; OS: Overall survival; Path: Pathologic examination positive with or without immunohistochemistry; PET: Positron emission tomography; SLN: Sentinel lymoh node VCAM: vascular cell adhesion molecule; VEGF: Vascular endothelial growth factor

16 Carlson, Slominski, Linette, Mihm & Ross Should regional lymph node dissection follow positive SLN? The goals of regional lymph node surgery consist of establishing prognosis and obtaining regional control with the hope of improving survival. The method of SLN evaluation affects the overall detection rate of metastasis (TABLE 4) [66,165]. In the past, a positive regional node, macroscopic or microscopic, was followed by a default regional node resection. In light of these new SLN techniques and new knowledge of natural history of micrometastases, it has been recommended that the principles guiding the extent of nodal surgery be re-examined [165,198]. For example, if only % of subsequent regional lymph node dissections for micrometastases yield extra-lymph node metastases [57,164,199], then the majority of these patients may not have benefited from the subsequent complete lymph node dissection. Currently, the methods for SLN evaluation to date vary considerably as few SLN studies examine the entire lymph node and diverse patient populations have been analyzed by SLN biopsy. Moreover, sufficient follow-up has not elapsed to record definitive outcomes after SLN biopsy. Due to these aforementioned limitations, Krag and Weaver recommend that pathologists and clinicians conduct definitive research toward better defining the role and impact of SLN surgery on the goals of regional lymph node management [165]. Refinement of SLN evaluation to prognosticate about additional regional lymph node metastases based on the extent and depth from the capsule of the metastasis could be one improvement on present day management [200]. For example, melanoma micrometastases confined to the subcapsular sinus are not associated with additional metastases in the regional nodes whereas parenchymal and partial-extensive nodal replacement may be associated with additional regional lymph node disease (FIGURE 4) [66]. Serologic evaluation for detection of metastatic disease & circulating melanoma cells Numerous molecular tumor biomarkers are synthesized, secreted or shed into the blood directly by melanoma cells, or indirectly due to destruction of melanoma cells by the host immune response or chemotherapy [69, ]. Moreover, viable melanoma cells can be found circulating in the blood of all stages of melanoma patients [ ]. These serologic biomarkers of melanoma metastasis and disease progression consist of melanoma associated antigens (e.g., tyrosinase, TRP-1, TRP-2 and gp100), intermediate products of melanogenesis (e.g., L-dopa, 5-S-cysteinyldopa [5-S-CD] or dihydroxyindole [DHI] derivatives), cytokines (e.g., soluble interleukin-2 receptor [sil-2r]), immunomodulatory neuropeptides (e.g., α-msh) and adhesion molecules (e.g., vascular adhesion molecule [VCAM]-1) [24,25,69,194,202,203]. For circulating melanoma cells (CMCs), primers for melanogenesis-related mrna, such as tyrosinase, are often used with RT-PCR in molecular staging for clinical investigations (TABLE 4) [69,128,202]. Currently, tests for these melanoma tumor markers, or for CMCs, do not have a place in the routine staging and monitoring of patients as their sensitivity and specificity are insufficient to accurately predict disease recurrence and progression [7,203,208]. Although their sensitivity increases with advanced melanoma, it is less than 100% as the serum levels of many patients are affected by other conditions, such as infectious diseases, autoimmune disorders, other neoplastic disorders or immunotherapy [69,202,203,209]. Furthermore, intermittent shedding of melanoma cells into the circulation, or low number of circulating cells lying below the threshold of detection, lead to a high false-negative rate [208,210,211]. Nonetheless, some markers exhibit promising results as prognostic factors in the early detection of disease progression, such as the melanomaassociated antigen S-100-β, immune complex tumor associated antigen 90 (TA90-IC) and RT-PCR detection of MRPs [208], nested PCR [212], or cell enrichment methods [213,214]. If confirmed by further studies, these markers could direct future therapeutic strategies and could help to select patients who would benefit most from more aggressive adjuvant therapies. S-100-β S-100 protein is a 21 kda acidic calcium-binding protein composed of a heterodimer of two isomeric subunits, α and/ or β, that is soluble at neutral ph in 100% saturated ammonium sulfate [215]. Its IHC detection is widely used for diagnosis of melanocytic tumors [9] and quantification of its β-subunit can be determined in serum by an immunoradiometric (IRMA) or luminoimmunometric assay (LIA) [69,202]. LIA is preferred over IRMA due to its lack of radioactivity, good reproducibility and higher sensitivity compared with IRMA [69,202]. Levels of S-100-β have been found to rise in a stage dependent manner (Stage I/II %, Stage II %, Stage IV %) [ ], correlate with metastatic tumor burden [220,221], have been proposed to be an independent prognostic factor [ ] and decrease in response to therapy [220,222,227,228]. In the detection of early metastatic disease, S-100-β levels have a sensitivity and specificity of approximately 74 94% and 83 91%, respectively [218,226,229,230], which was found to be more predictive than levels of melanoma inhibitory activity (MIA), LDH and albumin [226]. In the case of MIA (a small 11 kda soluble autocrine growth inhibitor [231]), other investigators have found its sensitivity roughly equal to that of S-100-β [228,232]. TA90-IC Patients with early-stage disease and a low tumor burden rarely have detectable tumor antigen (TA90) but often have high levels of TA90-IC [233] that can be detected by ELISA, a test that does not require pretreatment of test samples [234]. This assay can stratify melanoma patients with Stage II III disease into high-risk groups in the early postsurgical period. Moreover, patients with recurrent melanoma are identified early, well before routine clinical and radiological studies are able to detect metastases, approximately 19 ± 7 months before recurrence. In addition, PET scanning of TA90-IC positive patients may aid in localization of recurrent melanoma [153]. This long lead time could allow for earlier treatment interventions for patients with metastatic melanoma. Prospective validation of the TA90-IC results is currently underway [234,235]. 318 Expert Rev. Mol. Diagn. 3(3), (2003)

17 Biomarkers in melanoma Figure 4. The status of the sentinel lymph node (SLN) is the single most predictive factor in outcome in primary melanoma patients [31,263]. However, the majority of patients with positive SLN biopsies do not have additional regional lymph node metastases, suggesting that subsequent therapeutic complete regional lymph node dissection may not be necessary for all SLN positive patients. The pattern of distribution of melanoma within the SLN appears to predict involvement of other regional nodes [66,200]. For example, subcapsular melanoma metastasis is not associated with additional regional lymph node metastasis, however, parenchymal or extensive nodal disease may be associated with further regional metastases [66]. Intermediates of melanogenesis Melanogenesis is a metabolic pathway that is unique to cells of melanocytic lineage and is often deregulated in disseminated melanoma, leading to elevated extracellular and serum levels of melanin precursors, such as DOPA, 5-S-CD, DHI, its carboxylic form (DHICA) and O-methyl derivatives of DHI and DHICA [24,85,194]. Indeed, serum and/or urine levels of tyrosinase and melanin precursors, such as 5-S-CD or of 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) or the ratio of L-dopa/L-tyrosine, are elevated before conventional methods detect metastases, correlate positively with melanoma progression and demonstrate potential in the monitoring or therapy of melanoma patients, or detection of an occult disease [230, ]. Of all precursors to melanin, 5-S-CD appears to hold the most promise in the detection of melanoma metastases/progression of melanoma [239,245] with a positive predictive value of 94% [247]. However, MIA was found to be superior to 5-S-CD during follow-up of primary melanoma patients in the detection of melanoma recurrence [246]. Since intermediates of melanogenesis can act as potent immunosuppressors [24], the finding of increased serum levels of intermediates of melanogenesis and tyrosinase activity may serve as an indicator of impaired host immune response to melanoma [24,25]. MRPs Tyrosinase, TRP-1, TRP-2, MART-1/Melan-A and gp100 are MRPs that represent recent diagnostic and therapeutic additions into the pathology arsenal [194]. They are involved in the regulation of melanogenesis, the main differentiated function of the melanocyte, and, under certain conditions, can be released into the extracellular environment [24]. Thus, serum levels of tyrosinase and melanin precursors correlate positively with melanoma progression. Serum tyrosinase can be detected with assays for measuring either enzyme activity or enzyme concentration (RIA) [85,194]. Since extracellular tyrosinase can oxidate tyrosine to form lymphocytotoxic and mutagenic precursors of melanin, its serum concentration may help to predict natural history of melanoma progression [25]. RIA assays can also be used to measure serum concentrations of other MRPs, such as TRP-1, TRP-2, MART-1 and gp100. However, their role as serum markers of melanoma progression, recurrence, responsiveness to therapy or marker of occult disease would still require clinical testing. Nonetheless, the expression of these MRP or melanoma differentiation associated antigens on metastatic melanoma cells from Stage IV patients is significantly predictive of improved OS [249]. RT-PCR for CMCs Detection of CMCs, so-called molecular staging, has the greatest potential to asses an individual patient s risk for disseminated melanoma and ultimately guide decisions for adjuvant treatment. Genes coding proteins involved with melanin synthesis provide an invaluable means to detect CMC by RT-PCR as melanocytes do not normally circulate in the peripheral blood and melanoma specific mrnas are rarely detected in negative (healthy) controls (tyrosinase positivity in 2/521 negative controls) [207]. The most widely employed CMC biomarker is tyrosinase. Other melanoma specific biomarkers include the melanosomal antigen recognized by T-cells (MART-1/Melan- A), gp100/pmel17, the family of TRPs and tumor antigens of the melanoma antigen-encoding gene (MAGE) family [85,128,194,208,250]. Some of these potential melanoma biomarkers, such as MUC-18 and p97 have been proven to be nonspecific 319