SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

inline image

Melanoma has a wide spectrum of histologic features which mimic epithelial, hematologic, mesenchymal, and neural tumors. Immunohistochemistry has been the primary tool to distinguish melanomas from these other tumors; it has also been studied for use as an adjunct to distinguish benign and malignant melanocytic tumors and to elucidate prognosis. Furthermore, there has been extensive effort to find a suitable marker to differentiate spindle cell and desmoplastic melanoma from other tumors. We have reviewed the literature investigating melanocytic differentiation markers, proliferation markers, immunomodulatory markers, signaling molecules, and nerve growth factors and receptors. Despite the proliferation of immunohistochemical markers, S-100 remains the most sensitive marker for melanocytic lesions, while markers such as HMB-45, MART-1/Melan-A, tyrosinase, and MITF demonstrate relatively good specificity but not as good sensitivity as S-100. No marker has proven useful in distinguishing spindle cell and desmoplastic melanomas from other tumors. Ki67 remains the most useful adjunct in distinguishing benign from malignant melanocytic tumors. None of the markers reviewed has been shown conclusively to have prognostic value for melanocytic neoplasms.

Melanomas mimic the histologic features of a wide array of tumors including lymphomas, poorly differentiated carcinomas, neuroendocrine tumors, sarcomas and germ cell tumors.1 Melanoma cells range from epithelioid to spindled and include such diverse cytoplasmic morphologies as clear cell, signet ring shape, rhabdoid, ballooning and plasmacytoid appearance.1 Melanoma cells form diverse architectural patterns including nests, whorls, trabeculae, sheets, nodules, rosettes, glands and papillary formations.1 They undergo Schwannian, fibroblastic, myofibroblastic, rhabdoid, osteoid, cartilaginous, ganglionic and smooth muscle differentiation.1 Immunohistochemical staining is extensively used to differentiate melanomas from tumors that they mimic in conventionally stained sections. This paper discusses markers of melanocytic differentiation, the dilemma of the spindle cell melanoma and adjunct markers including cell cycle regulators, immune modulatory markers and signaling markers. Markers that are investigatory in nature will be discussed at the end of each section.

Established markers of melanocytic differentiation

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

S100 is a 21 kDa acidic calcium-binding protein that was first discovered in glial cells; its name is because of its solubility in 100% saturated ammonium sulfate solution.2–4 It is a commonly used sensitive marker for melanoma as well as nerve sheath and granular cell tumors and myoepitheliomas.2,4 In melanomas, S100 is present in the nucleus and cytoplasm and has a sensitivity of 97–100%.3–7 However, the specificity of S100 for melanocytic lesions is limited, it being expressed in nerve sheath cells, myoepithelial cells, adipocytes, chondrocytes and Langerhans cells and the tumors derived from these cells.4,8 Dennis Frisman’s Immunoquery website lists 62 separate non-melanocyte-derived tumors for which S100 is 100% sensitive and 135 separate non-melanocyte derived tumors for which S100 is 50% sensitive.9 Even spindle cells in dermal scars may express S100, a diagnostic pitfall when evaluating re-excisions of melanomas, especially desmoplastic melanomas.10 The specificity of S100 is reported to be 75–87%.7,11,12 More specific immunohistochemical markers including markers specific for non-melanocytic lesions should be used in parallel with S100 to reliably distinguish melanoma from other S100 positive malignancies. (Tables 1 and 2)

Table 1.  Antibodies useful in the diagnosis of epithelioid, pleomorphic and metastatic melanomas – positive markers
MarkerSensitivity/specificityAlso seen inComment
S100Sensitivity – 97–100%, specificity 75–87%3–7,11,12Numerous tumors including but not limited to those derived from nerve sheath cells, myoepithelial cells, adipocytes, chondrocytes, Langerhans cells4,8Nuclear and cytoplasmic stain.3–7 Most sensitive marker for spindle cell/desmoplastic melanomas.11,14–16,30,31,33,34,36–38,42,43
HMB45Sensitivity – 69–93% (77–100% in primary melanomas, 56–83% in metastatic melanomas)5,6,11–17PEComas (angiomyolipomas, lymphangiomyomatosis, pulmonary ‘sugar’ tumors), meningeal melanocytomas, clear cell sarcoma of the tendons and aponeurosis, some ovarian steroid cell tumors sweat gland tumors, some breast cancers, renal cell carcinoma with a t(6,11)(p21;q12) translocation8,14,18–25Cytoplasmic stain. Decreased sensitivity in metastatic melanoma.6–11,17 Can be used to help distinguish nevi from melanoma5,6,14
MART-1/Melan-ASensitivity – 75–92%, specificity – 95–100%7,14–17,30–34PEComas (angiomyolipomas, lymphangiomyomatosis, pulmonary ‘sugar’ tumors), some clear cell sarcomas.22,24,25 Clone A103 (Melan-A) also stains adrenal cortical tumors and gonadal steroid tumors31,32More intense and diffuse staining than HMB4514,15,17,31,32
TyrosinaseSensitivity – 84–94%, specificity – 97–100%7,14,33,35–37Rare angiolipomas, a minority of angiomyolipomas and clear cell sarcomas of the tendon sheath, and pigmented nerve sheath tumors33,37–40Sensitivity decreases with increased clinical stage and in metastatic lesions14,33,35–37
MITFSensitivity – 81–100%, specificity – 88–100%, lower in spindle cell lesions33,34,36,41–43Spindle cell tumors, lymphoid neoplasms, angiomyolipomas, rare breast carcinomas and renal cell carcinomas.33,34,39,43 Can also stain histiocytes, lymphocytes, fibroblasts, Schwann cells, smooth muscle cells and mast cells33,42,43Nuclear stain – increased ease of interpretation, but stains tumors of many other lineages3
NKI/C3Sensitivity – 86–100%, poor specificity14,17,44–48Neurothekeomas, medullary carcinomas of the thyroid, granular cell and neuroendocrine tumors, subset of breast, prostate, lung, clear cell, colorectal, bladder and ovarian carcinomas, lymphomas, neurofibromas and schwannomas.17,44–46,48 Also stains histiocytes, dendritic, mast, and endothelial cells, eosinophils, salivary, bronchial, sweat, pancreas and prostate glands14,17,44,48,49Poor specificity and high cost have severely limited use in clinical practice49
VimentinSensitivity – 96%123Too numerous to listIf negative, can be used to rule out melanoma
Table 2.  Antibodies useful in the diagnosis of epithelioid, pleomorphic and metastatic melanomas – negative markers
Tumor classMarkers and percent seen in melanomaReactivity for melanoma (%)
CarcinomaKeratin AE1:34123 (%)
Pan-Keratin1123 (%)
Keratin – LMW2123 (%)
EMA2123 (%)
CAM 5.21123 (%)
Smooth muscle tumorsSmooth muscle actin9123 (%)
Skeletal muscle tumorsDesmin0123 (%)
MyogeninNot well studied in melanoma
MyoD1Not well studied in melanoma
Vascular tumorsCD310123 (%)
CD340123 (%)
LymphomaCD450123 (%)
MyelomaCD13820123 (%)
MDM-225123 (%)
CD450123 (%)
SeminomaOCT40123 (%)
Neural tumorsGFAP2123 (%)
CD9916123 (%)
Neuroendocrine tumorsChromogranin0123 (%)
Synaptophysin12123 (%)
MesotheliomaCalretinin13123 (%)
Thrombomodulin0123 (%)
OthersCD99 (Ewings sarcoma/PNET, atypical fibroxanthoma, synovial sarcoma, solitary fibrous tumor, thymoma, meningioma, malignant peripheral nerve sheath tumor and gastrointestinal stromal tumor)16123 (%)
Inhibin (Adrenal cortical carcinoma, sex cord stromal tumors, leydig cell tumors, granulosa cell tumors, placental tumors including choriocarcinoma and hemangioblastomas)18123 (%)
CD30 (lymphoma, CD30 lymphoproliferative disease, Hodgkin disease and embryonal carcinoma)4123 (%)

HMB45, a marker of the cytoplasmic premelanosomal glycoprotein gp100, was one of the first melanoma ‘specific’ markers discovered.8 It is not as sensitive as S100 but has greater specificity. In positive melanocytic lesions, HMB45 is expressed in the cytoplasm.5,11 The reported sensitivity of HMB45 for melanoma ranges from 69% to 93%,5,6,11–17 and expression is maximal in primary melanoma specimens (77–100%) and less in metastases (58–83%).6,11–17 Staining may be patchy, and melanoma cells are less diffusely positive than with other markers.11,14,15,17 There may be strong staining with HMB45 in the epidermal component of a primary melanomas with gradually weaker staining in the deeper vertical growth phase.5,6,14 (Figure 1) There is some dispute as to whether HMB45 is less sensitive in amelanotic melanomas.5,11 HMB45 is very specific for melanomas and has been reported to be absent from non-melanocytic tumors by Wick et al. (133/133), Ordonez et al. (98/98), Kaufman et al. (150/150) and Trefzer et al. (84/84).7,11,12,13 However, HMB45 is expressed in PEComas (i.e. angiomyolipomas, lymphangiomyomatosis and pulmonary ‘sugar’ tumors), sweat gland tumors, meningeal melanocytomas, clear cell sarcoma of the tendons and aponeuroses, some ovarian steroid cell tumors and some breast cancers as well as renal cell carcinomas with a t(6,11)(p21;q12) translocation.8,14,18–25 Many of these tumors are fortunately histologically distinct from melanomas. There has been a report that HMB45 also stains pheochromocytomas, but this finding has not been corroborated.26–29 Decreased sensitivity in metastatic melanoma where the diagnosis is more likely in doubt, necessitates that other markers be used in conjunction with HMB45.

image

Figure 1. Hematoxylin and eosin (H&E), MART-1, Tyrosinase, HMB45, S100, p27, p53 and Ki67 stains, each performed on histologic sections of (A) intradermal nevus, (B) dysplastic (Clark’s) nevus, (C) nevoid melanoma, (D) primary melanoma, Not otherwise specified (NOS), (E) metastatic melanoma. MART-1, Tyrosinase, and S100 all strongly stain both benign and malignant melanocytic neoplasms. In benign and dysplastic nevi, HMB45 shows a gradient of strong staining in the superficial tumor cells and weak to negative staining in the deeper tumor cells; in melanoma, HMB45 shows strong staining even in the deep tumor cells. P27 can stain both benign nevi and melanomas and has not been shown to be of clinical utility. P53 is generally negative in common nevi but shows staining in both dysplastic nevi and melanomas. Ki67 stains junctional melanocytes in common nevi, may show increased staining in dysplastic nevi, and generally shows increased staining in melanomas, including staining toward the base of the lesion. (H&E stain photographed at 20× original magnification; HMB45 stain photographed at 40× original magnification; MART-1, Tyrosinase, S100, p27, p53 and Ki67 all photographed at 100× original magnification). MART-1, melanoma antigen recognized by T-cells-1.

Download figure to PowerPoint

Melanoma antigen recognized by T-cells-1 (MART-1) and Melan-A are synonyms for a cytoplasmic protein of melanosomal differentiation recognized by T-cells.3,7,8,30 Two clones of the antibody to this protein are available: M2-7C10, generally referred to as MART-1, and A103, generally referred to as Melan-A.16,31 Although there has been an almost 100% correlation established between the two clones with regard to melanoma staining, the A103 clone labels adrenal cortical tumors and gonadal steroid tumors, whereas the M2-7C10 clone does not stain these other tumors.31,32 Both clones also stain PEComas and can show staining in clear cell sarcomas.22,24,25 These antibodies show sensitivity (75–92%) and specificity (95–100%) for melanoma that is similar to HMB45.7,14–17,30–34 There is a decrease in staining in metastatic melanomas relative to primary melanomas.14,16 However, these antibodies generally show more diffuse and intense staining than HMB45 and do not show reduced staining in the dermal component of melanomas; this property makes them easier to interpret especially in metastatic melanomas.14,15,17,31,32

Tyrosinase is an enzyme that hydroxylates tyrosine as the first step in the synthesis of melanin.3 In melanomas, tyrosinase can be seen as fine granular cytoplasmic staining.35 Positive staining tends to be strong and diffusive.14,15 The sensitivity of tyrosinase for melanoma is somewhat better than HMB45 at 84–94%.7,14,33,35–37 Sensitivity decreases with increasing clinical stage and in metastatic lesions (79–93%).14,33,35–37 The specificity of tyrosinase for melanoma is 97–100%.6,33,37 Tyrosinase has been found in rare angiolipomas, a minority of angiomyolipomas (a type of PEComa) and clear cell sarcomas of the tendon sheath, and pigmented nerve sheath tumors.33,37–40

Microphthalmia transcription factor (MITF) is a transcription factor protein necessary for the development of melanocytes during embryogenesis.3 As a nuclear stain it is easier to interpret, especially as melanoma tends to be suspected when cytoplasmic pigment is present, pigment that may overlay reaction product making interpretation of cytoplasmic immunohistochemical stains difficult.3 Sensitivity for melanoma has been reported as 81–100%, and positivity has been reported in S100 negative melanomas.33,34,36,41,42 However, while initial reports claimed 100% specificity, more recent studies showed specificities as low as 88%, with even lower specificity in spindle cell neoplasms.33,34,41–43 False positives have been reported in diverse tumors, including spindle cell tumors, lymphoid neoplasms, occasional breast carcinomas and renal cell carcinomas.33,34,43 Like other melanocytic markers, MITF is positive in angiomyolipomas.39 MITF has been reported to stain histocytes and mast cells, potentially troubling pitfalls.33,42,43 Busam et al.43 reported that MITF staining was found in histiocytes, lymphocytes, fibroblasts, Schwann cells and smooth muscle cells. The advantages of nuclear staining must be weighed against the relative non-specificity of MITF as a marker.

NKI/C3 is an antibody to a 25–110 kD glycoprotein located on the inner membrane of cytoplasmic vesicles in melanocytes.5 This immunohistochemical stain, relatively widely used in the 1980s, is now less popular because of the availability of other more practical reagents.14 In melanomas, NKI/C3 should show peripheral membrane staining to be considered positive.5 NKI/C3 has a sensitivity of 86–100% for melanoma in paraffin embedded sections, but a very poor specificity, staining neurothekeomas, medullary carcinomas of the thyroid, granular cell and neuroendocrine tumors.14,17,44–48 It also stains a subset of breast, prostate, lung, clear cell, colorectal, bladder and ovarian carcinomas as well as lymphomas, neurofibromas and schwannomas.17,44–46,48 Vennegoor et al.44 found that NKI/C3 stained 16 of 18 neuroendocrine tumors and 33 of 196 other non-melanocytic, non-neuroendocrine tumors. In addition, NKI/C3 also stains histiocytes, dendritic, mast and endothelial cells, eosinophils and salivary, bronchial, sweat, pancreas and prostate glands.14,17,44,48,49 The non-specificity of this marker and its high cost have limited its use in clinical practice.49

Newer markers of melanocytic differentiation

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

Other melanocytic differentiation markers are currently being investigated and are not yet in widespread clinical use. (Table 5) Multiple myeloma-1 (MUM-1) is a protein with a role in regulating gene expression in response to cytokines.16 Although its primary use has been to differentiate between lymphoproliferative lesions, it is a sensitive and specific marker for melanoma when a lymphoid or plasma cell lesion has been ruled out.16 Sundram et al.16 reported positive nuclear staining in 33/36 melanomas. Of 944 malignancies tested, only melanomas and hematologic malignancies had positive staining.16

Table 5.  Markers under investigation: not in routine clinical use
MarkerStaining in melanomasComment
Melanocytic differentiation
 MUM-1Sundram et al.16 report positive staining in 33/36 melanomas. Out of 944 malignancies, positive staining was only seen in hematologic malignancies and melanomas16Primarily used in the workup of hematologic malignancies
 Melanocortin-1Salazar-Onray et al.50 reported positive staining in 26/26 melanomasAlso stains cerebellar neurons, hepatocytes, renal tubular cells, and the cells of the adrenal medulla, appendiceal mucosa, myocardium and myometrium50
 SM5-1Trezfer et al.12 reported positive staining in 98% of melanomas (401 cases) and negative staining in 84 non-melanoma malignanciesAlso stains dendritic cells, myofibroblasts, renal tubular cells, plasma cells and secretory epithelium of the thyroid, larynx and prostate12
 PNL2Busam et al.36 reported positive staining in 33 of 38 metastatic melanomasAlso stains angiomyolipomas and mature myeloid cells in CML patients36
 TRP-1 and TRP-2Newer antibodies to tyrosinase51Under investigation
Proliferation markers
 HDM2Polsky et al.99 reported >20% of lesional cells stained positive in 67 of 102 primary melanomas as opposed to 3 of 11 melanoma-insitu and 1 of 16 dysplastic neviPolsky et al.100 report increased positive staining paradoxically correlates with decreased recurrence rates and increased survival
 GADDKorabiowska et al.96 report that GADD 34, 45 and 153 stain an average of 82–92% of lesional cells in benign nevi and only 19–31% of lesional cells in melanomaNo significant data
Immune modulatory markers
 HLA Class I and IIIncreased positive staining in melanomas as compared with benign nevi101–103Several small studies have linked decreased expression to decreased survival, but Erricsson et al. (2001) linked decreased expression to increased survival!101–106
 CD40Increased expression in melanomas relative to benign nevi107,108B cell marker, not convincing as a prognostic marker in melanoma107,108
 CD26Slightly increased staining in melanomas109No prognostic significance109
 FAS and FAS-ligandConflicting reports110–112No prognostic significance110–112
 FLIPBullani et al. (2001) reported positive staining in 24 of 29 melanomas but only 1 of 32 benign nevi113Data too limited for any conclusions
Signaling molecules
 AktNo distinction between nevi and melanomas117,118Dai et al.117 reported Akt as an independent risk factor in thin melanomas
 PTENNo distinction between nevi and melanomas119–121 
 EzrinNo distinction between nevi and melanomas122Ilmonen et al.122 report statistically insignificant inverse correlation between positive staining and survival

Melanocortin-1 is a receptor for alpha-melanocyte stimulating hormone present on melanocytes but also on some keratinocytes and monocytes. Salazar-Onfray et al.50 showed that antibodies to melanocortin-1 were present in 26/26 melanoma cases studied. However, melanocortin-1 expression is also found, albeit less intensely, in cerebellar neurons, hepatocytes, renal tubular cells and the cells of the adrenal medulla, appendiceal mucosa, myocardium and myometrium.50

Trezfer et al.12 reported that SM5-1, a monoclonal antibody developed from mice immunized with the melanoma cell line SMMU, interacted with 98% of 401 melanomas tested and did not react with 84 non-melanoma malignancies tested. However, SM5-1 additionally stains dendritic cells, myofibroblasts, renal tubular cells, plasma cells and secretory epithelium of the thyroid, larynx and prostate.12

PNL2 is as yet uncharacterized antibody that Busam et al.36 found to react with 33 of 38 metastatic melanomas. PNL2 also stained angiomyolipomas and mature myeloid cells in patients with chronic myeloid leukemia.36

TRP-1 and TRP-2 are new antibodies to tyrosinase that are under investigation.51

This is not an exhaustive list of markers currently in use or under investigation; however, none of these newer markers has yet shown significant advantages over the immunostains currently in clinical use.

The dilemma of spindle cell and desmoplastic melanoma

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

Spindle cell and desmoplastic melanomas often pose a diagnostic dilemma because their morphology is atypical and their immunohistochemical profiles are different from and not as distinctive as those of other types of melanomas. S100 has excellent sensitivity in these lesions but also stains numerous non-melanocytic spindle cell lesions that may have similar histologic features. Unfortunately, all the markers that are more specific for ‘usual’ melanoma also show very poor sensitivity for spindle/desmoplastic lesions. We reviewed a series of papers that studied markers for their capacity to detect spindle cell and desmoplastic melanomas. (Table 3) While S100 was positive in 77/78 (98.7%) spindle cell and desmoplastic melanomas, the other immunohistochemical markers showed the following rates of positive staining: HMB45 – 16/91 (17.6%), Melan-A – 8/37 (21.6%), tyrosinase – 23/87 (26.4%), MITF – 9/58 (15.5%).11,14–16,30,31,33,34,36–38,42,43 Newer antibodies such as MUM-1 and PNL2, tested against spindle/desmoplastic and conventional melanomas have not yielded more promising results.16,36

Table 3.  Immunohistochemistry of spindle cell/desmoplastic melanoma
MarkerSensitivityComment
S100Sensitivity – 98.7%11,14–16,30,31,33,34,36–38,42,43Most sensitive marker for spindle cell/desmoplastic melanomas
HMB45Sensitivity – 17.6%11,14–16,30,31,33,34,36–38,42,43Very limited value
MART-1/Melan-ASensitivity – 21.6%11,14–16,30,31,33,34,36–38,42,43Very limited value
TyrosinaseSensitivity – 26.4%11,14–16,30,31,33,34,36–38,42,43Very limited value
MITFSensitivity – 15.5%11,14–16,30,31,33,34,36–38,42,43Very limited value
P75Stained 26/29 spindle cell melanomas by Kanik et al. and Satori et al.52,53Also stained peripheral nerve sheath tumors, dermatofibrosarcoma pertuberans, synovial sarcoma, rhabdomyosarcoma, ‘neurotized’ nevi54

There have been a few papers describing investigations of the role of nerve growth factors and their receptors in melanoma. The p75 nerve growth factor receptor, a 75 kDa protein which functions as a low affinity receptor for nerve growth factor, was found by Kanik et al.52 to be positive in 10/13 spindle cell melanomas and only 3/8 non-spindle cell melanomas. They also reported that p75 was positive in 5/5 ‘neurotized’ nevi, nevi that have undergone neural/Schwannian differentiation.52 Satori et al.53 found that p75 stained 16/16 spindle cell melanomas as opposed to only 6/10 non-spindle cell melanomas. However, Fanburg-Smith and Miettinen54 showed that p75 stains many other malignant spindle cell tumors including more than 90% of malignant peripheral nerve sheath tumors, dermatofibrosarcoma protuberans, synovial sarcomas and rhabdomyosarcomas. Therefore, p75 is not an effective marker for distinguishing spindle cell melanomas from other spindle cell tumors.

Markers of tumor cell proliferation

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

An increasing literature describes the role of proliferation markers in the evaluation of melanocytic tumors. (Table 4) The goal of many of these studies has been elucidation of the mechanisms of tumorigenesis of malignant melanoma. However, there are two clinical applications for which these markers have been studied: differentiation of benign nevi from malignant melanoma and assessment of clinical prognosis for patients with malignant melanoma.

Table 4.  Proliferation markers for distinguishing melanoma from benign nevi and prognosis for melanoma
MarkerStainingPrognosis
Ki67Usually <5% staining of cells in nevi, 13–30% in melanomas, although individual cases can be higher. Ki67 is also increased in Spitz neoplasms55–59,63–66Conflicting reports as to whether or not Ki67 is an independent risk factor for adverse outcome in melanoma57,60–63,67–74
PCNAIncreased staining in melanoma and Spitz neoplasms as compared to benign nevi56,76,77Niezabitowski et al. (1999) report PCNA as an independent prognostic factor, but this has not been confirmed in multiple other studies72,74,75,78
Cyclin ARarely expressed in benign nevi, positive in 42–99% of melanomas63,65,73Florenes et al.63 reported inverse correlation between cyclin A staining and decreased survival in superficial spreading melanomas only
Cyclin BRarely expressed in benign nevi, expressed in approximately 50% of melanomas65,73Not well studied
Cyclin D1Rarely expressed in benign nevi, commonly expressed in melanomas73,81No prognostic significance81
Cyclin D3Rarely expressed in benign nevi, commonly expressed in melanomas73,81Florenes et al.81 reported direct correlation between cyclin D3 staining and early relapse and decreased survival in superficial spreading melanomas only
P16Positive in benign nevi, loss of staining in 50–98% of melanomas73,83,85–88Loss of staining associated with decreased survival but not verified as an independent variable71,73,89
P21Rarely stains nevi, increased staining in melanomas73,88,90,91Conflicting studies as to whether positive staining is associated with a better, worse, or unaffected outcome73,78,90
P27Conflicting reports as to its ability to distinguish benign nevi from melanoma93,94Florenes et al.92 reported that <5% positivity is associated with risk of early relapse in nodular melanoma only. No correlation with overall survival
P53Does not stain most benign nevi, positive in 25–58% of melanomas. However, Chorney et al. (2003) reported that Spitz neoplasms and minimal deviation melanomas both show approximately 9% staining55,65,66,88,96,97Most reports state that positive staining has no impact on outcome. A minority of studies show a negative outcome in cases with positive staining not independent of tumor thickness57,72,74,89,90,95,97

The proliferation marker most widely used in pathology is Ki-67, a nuclear antigen present in all active phases of cell cycle proliferation (G1, S, G2 and M) but absent in the quiescent phase (G0).55–62 Ki-67 staining has been shown in multiple studies to be positive in <5% of tumor cells in most benign nevi, although there have been reports of up to 15% positivity in Spitz and dysplastic nevi.55,56,57,59,63–66 Conversely, Ki-67 staining is reported as positive in 13–30% of the cells in a malignant melanoma, although individual cases can show almost 100% nuclear positivity.55,56,58,59,63–66 There have been mixed results using Ki-67 as a prognostic marker in malignant melanoma. Some studies have shown an independent positive correlation between Ki-67 staining and metastatic potential and mortality in thick melanomas only; others have shown a similar correlation in thin melanomas only! 57,62,67–69 Many studies have shown an increase in recurrence rate and mortality that directly correlates with increasing positivity for Ki-67; however, others have shown that this correlation is not independent of Breslow thickness. 60,61,63,69–73 Reddy et al.74 did not find any correlation at all, although they only studied 14 cases.

Proliferating cell nuclear antigen (PCNA) is a 36-kDa protein that is a co-factor of DNA polymerase δ expressed in all phases of cell cycle proliferation (G1, S, G2,M).56,75–77 There is increased expression of PCNA in malignant melanoma compared to benign nevi, although Spitz nevi also have increased expression.56,76,77 Niezabitowski et al. found increased PCNA expression to be an independent prognostic factor for increased mortality and decreased disease-free survival, although others have been unable to confirm this finding.72,74,75,78

Cyclins

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

Cyclins are proteins, approximately 100 amino acids long, which bind and activate cyclin dependent kinases causing the cell to progress through the various stages of the cell cycle.63,65,79–81 Cyclins C, D1, D2, D3 and E help the cell progress from G1 to S phase; cyclin A from S to G2 phase; cyclin B from G2 to mitosis.63,65,80,81 Cyclin A is rarely expressed in benign nevi but expressed in 42–99% of melanomas.63,65,73 Florenes et al.63 reported an inverse correlation between cyclin A staining and disease-free survival in superficial spreading melanomas but not in nodular melanomas. Cyclin B is rarely expressed in benign nevi but expressed in about 50% of melanomas.65,73 Little is known about cyclin B’s prognostic utility. Cyclins D1 and D3 are rarely expressed in nevi but commonly expressed in melanomas.73,81 Cyclin D1 has not been shown to have any prognostic significance; however, increased cyclin D3 staining was reported by Florenes et al.81 to be associated with early relapse and decreased survival in superficial spreading melanomas but not nodular melanomas. Cyclin E has been reported by Alonso et al.82 to be inversely correlated with survival in melanoma.

Cyclin dependent kinase inhibitors inhibit progress through the cell cycle.79 P16 inactivates cyclin D/cdk4 complexes, inhibiting phosphorylation of the retinoblastoma protein that enables cell cycle progression from G1 to S phase.71,82–87 P16 staining is present in most benign nevi but lost in 50–98% of melanomas.73,83,85–88 The loss of P16 staining in melanomas is associated with decreased survival, although it has not been completely established as an independent variable.71,73,89 P21 inhibits cyclin/cdk complexes and also binds PCNA to directly inhibit DNA polymerase δ involved in DNA synthesis.78,88,90,91 P21 is anomalous in that it rarely stains nevi but shows increased staining in melanomas.73,88,90,91 There are conflicting reports as to whether increased P21 staining is correlated with a better or worse outcome or has no correlation with prognosis at all.73,78,90 P27 inhibits the cyclin D/cdk4 and cyclin E/cdk2 complexes, preventing cell cycle progression from G1 to S phase.92–94 There are conflicting reports as to its ability to distinguish benign nevi from melanomas.93,94 Florenes et al.92 report that nodular melanomas (but not superficial spreading melanomas) with <5% staining for P27 have an increased risk of early relapse; however, extent of P27 expression has no impact on overall survival.

P53 is a 53-kDa tumor suppressor protein that controls arrest of the cell cycle at G1 and G2 so that DNA damage can be repaired.55,66,95,96 P53 also induces the expression of P21 that helps inhibit DNA synthesis.88,91 The gene encoding P53 is the most commonly mutated gene in cancer.66,95 Mutations of the P53 gene cause an abnormal P53 protein to be formed which is unable to inhibit the cell cycle.95 The wild-type P53 protein has a very short half-life and is usually not detected by immunohistochemistry; however, the abnormal p53 has a much longer half-life and is readily detected by immunohistochemistry.55,66,95,97 P53 does not stain most common nevi but is present in 25–58% of melanomas; however, Chorney et al. reported that both Spitz nevi and minimal deviation melanoma show approximately 9% staining by P53.55,65,66,88,96,97 Most reports claim that there is no correlation between P53 staining and likelihood of metastasis, disease recurrence or overall melanoma-specific survival; the minority of reports that claim a linkage between P53 staining and outcome state that such staining is not independent of tumor thickness as a prognostic factor.57,72,74,89,90,95,97

Some of the more recently studied proliferation markers include HDM2 and the Growth arrest DNA damage (GADD) proteins. HDM2 is a 90-kDa zinc finger protein that binds to the transcription activation domain of P53 to inhibit its function and ubiquinates p53 so that it is degraded by proteosomes.98–100 Polsky et al.99 reported that >20% of lesional cells stained for HDM2 in 1 of 16 dysplastic nevi, 3 of 11 melanomas in situ, and 62 of 107 invasive primary melanomas. Polsky et al.100 claim that increased HDM2 staining is an independent prognostic factor paradoxically associated with decreased recurrence rates and increased survival in the 134 melanoma patients that they studied. GADD proteins control transcription factors associated with cell cycle arrest, apoptosis and some forms of cell differentiation.96 Korabiowska et al.96 report that GADD 34, 45 and 153 stained an average of 82–92% of lesional cells in benign nevi and 19–31% of tumor cells in melanoma. There is little data on the prognostic implications of the GADD proteins.

In summary, Ki-67 has the most data to support its use as an adjunct that assists in the distinction of benign nevi from melanomas. Ultimately, none of the proliferation markers discussed has been definitely shown to have independent prognostic value, although with further study HDM2, and GADD may have real promise as prognostic markers.

Immune modulatory markers

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

There is increasing interest in immunohistochemical staining of immunomodulatory markers. Studies of HLA Class I and Class II markers have shown increased staining in melanomas compared with nevi and several small studies have linked decreased expression of these markers in melanomas with decreased survival.101–103 Paradoxically, patients with choroidal melanomas that show decreased HLA Class I and Class II staining have been reported by Erricsson et al. to have increased survival.101,102,104–106 CD40, a B cell marker that is also involved in tumor suppression, has increased expression in melanomas relative to nevi but has not been convincing as a prognostic marker.107,108 van den Ord109 studied CD26, an adenosine deaminase receptor, and found slightly increased staining in melanomas relative to benign nevi but no prognostic significance. There are conflicting reports of the expression of FAS and FAS-ligand (tumor suppressor proteins of the tumor necrosis family) in melanomas and nevi and neither has been shown to be an independent prognostic factor.110–112 Flice inhibitory protein was studied by Bullani et al.113 and identified in 24 of 29 melanomas but only 1 of 32 benign nevi. Cancer/testis antigens are proteins normally expressed only in the testis but aberrantly expressed in many types of malignancies.114–116 Luftl et al.116 studied six of these antigens in 19 nevi and 38 primary melanomas and showed that when used as a group, they can be of value in distinguishing benign nevi from melanomas. They do not show prognostic significance.

Signaling molecules

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

Immunohistochemical staining of signaling molecules has also been evaluated in melanomas. The product of the oncogene Akt is a serine-threonine kinase that inhibits apoptosis through the phosphatidylinositol-3 kinase pathway.117,118 It does not distinguish dysplastic nevi from melanomas but was reported by Dai et al. in a study of 170 primary melanomas to be an independent risk factor for thin melanomas.117,118 PTEN is another tumor suppressor gene that is also involved in the phosphatidylinositol-3 kinase pathway that does not distinguish benign nevi from melanoma.119–121 Ilmonen et al.122 describe ezrin, a 70 kDa protein involved in the phosphatidylinositol-3 kinase pathway, and report its expression to be inversely correlated with survival in 95 melanomas studied, although the correlation did not reach statistical significance.

Conclusion

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References

In current practice, the most clinically useful stains are melanoma differentiation markers. Most suspected melanomas are diagnosed with the most sensitive marker S100 and one or more of the more specific markers such as Melan-A, tyrosinase or HMB45. Most spindle cell or desmoplastic lesions stain only with S100, and the other markers have limited value. While p75 has been shown in small studies to be more sensitive than other confirmatory markers currently in clinical use, it is also very non-specific. Thus, the search for a specific marker for these lesions continues. As for the other markers, Ki-67 is commonly used as an adjunct in distinguishing benign nevi from melanoma and as an aid in prognosis. None of the other proliferation, immunoregulatory or signaling markers has proven to significantly improve diagnostic or prognostic accuracy; therefore, these markers have not yet found a niche in clinical practice.

References

  1. Top of page
  2. Abstract
  3. Established markers of melanocytic differentiation
  4. Newer markers of melanocytic differentiation
  5. The dilemma of spindle cell and desmoplastic melanoma
  6. Markers of tumor cell proliferation
  7. Cyclins
  8. Immune modulatory markers
  9. Signaling molecules
  10. Conclusion
  11. References