Chapter 14 Immunohistochemical Diagnostic and Prognostic Markers for Melanoma Mehdi Nosrati and Mohammed Kashani-Sabet Abstract Recent studies in our laboratory have identified novel molecular diagnostic and prognostic markers based on analyses in large cohorts of melanoma patients. These markers were initially derived from gene expression profiling analyses of distinct stages of melanoma progression. Immunohistochemical analyses confirmed the differential expression of these markers, and immunohistochemistry-based multimarker assays were developed to assess melanoma diagnosis and prognosis at the molecular level. In this chapter we review the development of these assays and the methodologies used to assess marker expression in both nevi and primary melanomas. Key words Immunohistochemistry, Molecular diagnosis, Prognostic markers, ARPC2, FN1, RGS1, SPP1, WNT2, NCOA3

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Introduction

1.1 Rationale for Development of Molecular Diagnostic Markers for Melanoma

The diagnosis of primary cutaneous melanoma at the pathological level can represent a daunting task [1]. Several features are used to diagnose melanoma, such as cytologic atypia, maturation with descent, poor circumscription, presence of mitoses, and asymmetry. However, the pathologic diagnosis of melanoma remains challenging, resulting in a high degree of interobserver variability [2–6]. As a result, the misdiagnosis of melanoma is a major cause of inappropriate therapy as well as malpractice claims involving pathologists and dermatologists in the United States [7]. These issues have created an unmet need in the molecular diagnosis of melanoma. While certain molecular aberrations have been described to differentiate benign from malignant melanocytic neoplasms [8, 9], no molecular assays are routinely performed to assist in the differential diagnosis of nevus versus primary melanoma. Moreover, several of these studies may require sophisticated assays (such as comparative genomic hybridization), which may not be

Magdalena Thurin and Francesco M. Marincola (eds.), Molecular Diagnostics for Melanoma: Methods and Protocols, Methods in Molecular Biology, vol. 1102, DOI 10.1007/978-1-62703-727-3_14, © Springer Science+Business Media New York 2014

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available in routine diagnostic pathology laboratories. In addition, the standard immunohistochemical markers used to determine melanocytic lineage, such as S100, HMB-45, MART-1, and MITF, are unable to distinguish primary melanomas from nevi [10–14]. Thus, the development of a multimarker assay using immunohistochemical methods and reagents would represent an important advance in the pathologist’s armamentarium in assisting with this differential diagnosis. 1.2 Development of a Multimarker Diagnostic Assay for Melanoma

cDNA microarray analyses of distinct stages of melanoma progression demonstrated a specific gene expression signature for nevi when compared with that of primary melanomas, and identified several markers that could potentially aid in the diagnosis of melanoma [15]. Five markers [actin-related protein 2/3 complex, subunit 2 (ARPC2), fibronectin (FN1), regulator of G-protein signaling 1(RGS1), osteopontin (SPP1), and wingless-type MMTV integration site family member 2 (WNT2)] derived from this original profiling analysis were incorporated into a multimarker immunohistochemical diagnostic assay using commercially available antibodies and analyzed in a cohort of 693 melanocytic neoplasms, composed of a training set and four validation sets [16]. Each marker was assessed for its staining intensity on a four-point (0–3) scale. In addition, marker expression was assessed at the junctional zone of the lesion (termed the “top”) and the base of the lesion (termed the “bottom”), and differences in “top-to-bottom” expression were evaluated for each marker. In this analysis, nevi were observed to lose expression (with the “top” score greater than the “bottom” score), whereas in melanomas, this “top-to-bottom” loss of marker expression was not readily observed. A diagnostic algorithm that combined (a) marker intensity scores with (b) top-tobottom differences in expression was shown to achieve a specificity of 95 % and a sensitivity of 91 % in melanoma diagnosis in the training set. Applying the same diagnostic algorithm to the validation sets, the multimarker assay achieved 95 % specificity and 97 % sensitivity in melanomas arising in a nevus and correctly identified 95 % (37/39) of dysplastic nevi and 95 % (20/21) of Spitz nevi. Finally, the assay correctly diagnosed 18/24 (75 %) of initially misdiagnosed lesions. These studies established the diagnostic accuracy of this multimarker immunohistochemical assay for melanoma. The assay is undergoing additional validation prior to its commercial application and availability for use in the molecular diagnosis of melanocytic neoplasms.

1.3 Rationale for Development of Molecular Prognostic Markers for Melanoma

The clinical behavior of malignant melanoma can be capricious. Even though the thickness of the primary tumor is an important prognostic factor to determine survival, it is by itself inadequate to accurately predict the outcome of individual melanoma patients. This highlights the need for additional markers to improve predictive algorithms for melanoma patients. The development of molecular prognostic

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markers for melanoma, based on the identification of the molecular determinants of melanoma tumor progression, would allow further improvements in the prognostic assessment of melanoma patients. Moreover, the development of a molecular prognostic assay for primary melanomas should identify patient subsets at higher risk of relapse and death, as well as those who may benefit from systemic adjuvant therapies. Despite the identification of several prognostic factors that may correlate with melanoma outcome (reviewed in [17]), no molecular factors are routinely used in the prognostic evaluation of melanoma patients. 1.4 Development of a Multimarker Prognostic Assay for Melanoma

We also developed a multimarker immunohistochemical prognostic assay for primary cutaneous melanoma [18] combining the expression levels of three markers (nuclear receptor coactivator 3 (NCOA3), SPP1, and RGS1) in an initial cohort of 395 patients with primary cutaneous melanoma. Each marker was shown to significantly predict sentinel lymph node (SLN) status and disease-specific survival (DSS) [19–21]. The cumulative impact of marker overexpression was assessed using DSS as the primary endpoint. A multimarker index was developed, and reflected the total number of markers overexpressed or under-expressed. Increasing multimarker scores were significantly predictive of SLN metastasis and reduced DSS. Multimarker overexpression was associated with significantly reduced DSS by Kaplan-Meier analysis. Multivariate logistic regression analysis indicated that the multimarker assay was independently predictive of SLN status, and was more significant than tumor thickness. Multivariate Cox regression analyses of DSS identified the multimarker index as the most significant predictor of DSS. Even with the inclusion of SLN status in the model, the multimarker expression level remained the most significant factor predicting DSS. The prognostic role of this immunohistochemical assay was then confirmed using a digital imaging analysis of the stained specimens. The prognostic impact of this multimarker assay was also demonstrated in tissues from 141 patients collected from Germany. Multivariate Cox regression analysis indicated that the multimarker expression index was independently predictive of DSS in this cohort, confirming the prognostic impact of this assay. These studies described the first independently predictive molecular assay for primary melanoma. Importantly, the significant prognostic impact of this multimarker assay was demonstrated in a tissue set drawn from a completely different patient population (Germany), across different tissue platforms, and using both pathologist scoring and digital imaging analysis. Based on these results, the prognostic impact of this multimarker assay is undergoing additional evaluation on tissues collected from the Eastern Cooperative Oncology Group (ECOG) E1690 randomized trial of observation versus high-dose and low-dose interferon in the setting of high-risk melanoma.

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Materials

2.1 Solutions for Immunohistochemical (IHC) Staining

1. 3 % H2O2 in PBS: Add 25 mL 30 % H2O2 to 225 mL 1× PBS (10 mM, pH 7.4). 2. 10 mM Citrate Buffer for steaming or microwave antigen retrieval. Add 1.05 g of Citrate Monohydrate to dH2O and adjust the final volume to 500 mL; then adjust to pH 6.0 using 1 N NaOH. 3. Diluent for antibody solutions-1 % Bovine Serum Albumin (BSA) in PBS: Add 1 g of BSA to 100 mL of 1× PBS. 4. Avidin/biotinylated enzyme staining complex-Horseradish peroxidase(ABC-HRP) (1:100 Dilution): Add 30 μL Avidin to 30 μL Biotin-HRP and 2,940 μL Diluent. Prepare the ABCHRP reagent 30 min before use. 5. 3,3′-Diaminobenzidine(DAB)/H2O2 HRP substrate: Add 10 mg DAB tablet to 20 mL PBS and 20 μL 30 % H2O2. Add 30 % H2O2 just before use. 6. PBS Tween-20 (PBST): Add 500 μL Tween-20 to 800 mL of 1× PBS, adjust pH to 7.4, and adjust the final volume to 1 L with PBS for a final concentration of 0.05 % Tween-20. Always prepare freshly before use. PBST is a more stringent wash buffer. Alternatively, it can be used in place of PBS in the protocols in this chapter.

2.2 Antibodies and Dilutions

1. ARPC2 primary Rabbit Polyclonal Antibody (Upstate Cell Signaling). For 1:25 dilution, add 80 μL antibody to 1.92 mL Diluent. 2. Fibronectin primary Rabbit Polyclonal Antibody (Dako): For 1:400 dilution, add 5 μL Fibronectin Antibody to 1.995 mL Diluent. 3. NCOA3 primary Mouse Monoclonal Antibody (Abcam): For 1:10 dilution, add 300 μL NCOA3 to 2.7 mL Diluent. 4. RGS1 primary Chicken Polyclonal Antibody (GeneTex): For 1:50 dilution add 60 μL RGS1 Antibody to 2.94 mL Diluent, and for 1:100 dilution add 30 μL RGS1 Antibody to 2.97 mL Diluent. 5. SPP1/Osteopontin primary Rabbit Polyclonal Antibody (Abcam): For 1:200 dilution add 15 μL SPP1 Antibody to 2,985 mL Diluent. 6. Wnt-2 primary Rabbit Polyclonal Antibody (Biovision): For 1:5 dilution, add 400 μL Wnt-2 Antibody to 1.6 mL Diluent. 7. Normal Goat Serum (Vector Labs; 1:10 Dilution): Add 300 μL Normal Goat Serum to 2.7 mL Diluent. 8. Normal Horse Serum (Vector Labs): For 1:10 dilution, add 300 μL Normal Horse Serum to 2.7 mL Diluent.

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9. Biotinylated goat anti-Rabbit secondary antibody (Vector Labs). For 1:200 dilution, add 15 μL Biotinylated Goat Anti-Rabbit to 2,985 mL Diluent. 10. Biotinylated Horse Anti-Mouse secondary antibody (Vector Labs): For 1:200 dilution, add 15 μL Biotinylated Horse AntiMouse to 2,985 mL Diluent. 11. Biotinylated Goat anti-Chicken secondary antibody (Vector Labs): For 1:200 dilution, add 15 μL Biotinylated Goat AntiChicken to 2,985 mL Diluent. 2.3

Kits

1. ABC-HRP Kit (Vectastain, Vector Labs). 2. Avidin Biotin Blocking Kit (Vector Labs).

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Methods

3.1 Actin-Related Protein 2/3 Complex, Subunit 2 (ARPC2) (Fig. 1) (See Note 1)

1. Bake slides in oven at 60 °C for 30 min prior to staining. 2. Deparaffinize and rehydrate (Subheading 3.7). 3. Steaming in 10 mM Citrate Buffer: Heat up 1 L Citrate buffer in a 2 L beaker in the microwave at high power for 6 min. Transfer the heated Citrate buffer to the plastic bowl in a steamer and steam the buffer to 97 °C. Place the slide rack in the Citrate buffer and steam at 95–100 °C for 20 min. Check the temperature of the buffer with a thermometer and regulate the temperature by positioning the lid of the steamer. After

Fig. 1 Photomicrograph of ARPC2 immunostaining in primary melanoma

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steaming, allow the slides to cool at room temperature for 20 min. Pressure cooker appears to results in superior antigen retrieval compared to microwave or steamer. The user is encouraged to use pressure cooker in the protocols that were previously optimized with microwave or steamer. 4. Wash in dH2O for 5 min. 5. Wash in PBS for 5 min. 6. Block in 3 % H2O2 in PBS for 15 min. 7. Wash in PBS three times, each for 3.5 min. 8. Incubate with Normal Goat Serum (1:10) at room temperature for 30 min. 9. Incubate with ARPC2 primary antibody (1:25, item 1 of Subheading 2.2), and coverslip overnight at 4 °C (see Note 1). 10. Wash off coverslip with PBS for 8 min. 11. Wash in PBS three times, each for 3.5 min. 12. Incubate with Biotinylated Goat anti-Rabbit (1:200) at room temperature for 30 min. 13. Wash in PBS three times, each for 3.5 min. 14. Incubate with ABC reagent at room temperature for 30 min. 15. Wash in PBS three times, each for 3.5 min. 16. Incubate with DAB/H2O2 at room temperature for5 min. 17. Wash in running H2O for 5 min. 18. Counterstain with Hematoxylin Gill #2 for 10 s. 19. Wash in running H2O for 3 min. 20. Blue in Scott’s H2O for 1 min. 21. Wash in running H2O for 3 min. 22. Dehydrate through graded alcohols and xylene (Subheading 3.8). 23. Coverslip with permount medium. 3.2 Fibronectin 1 (FN1) (Fig. 2) (See Note 2)

1. Bake slides in oven at 60 °C prior to staining for 30 min. 2. Deparaffinize and Rehydrate (Subheading 3.7). 3. Wash in PBST for 5 min. 4. Block in 3 % H2O2 in PBS for 15 min. 5. Wash in PBST for 5 min. 6. Wash in dH2O for 5 min. 7. Steaming in 10 mM Citrate Buffer as in step 3 of Subheading 3.1. 8. Wash in dH2O for 5 min. 9. Wash in PBST for 5 min. 10. Block in Avidin blocking reagent for 15 min. 11. Wash in PBST three times, each for 3.5 min.

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Fig. 2 Photomicrograph of FN1 immunostaining in primary melanoma

12. Block in Biotin blocking reagent for 15 min. 13. Wash in PBST three times, each for 3.5 min. 14. Incubate with Normal Goat Serum (1:10) at room temperature for 30 min. 15. Incubate with Fibronectin primary antibody (1:400, item 2 of Subheading 2.2), and coverslip overnight at 4 °C (see Note 2). 16. Wash off coverslip with PBS for 8 min. 17. Wash in PBST three times, each for 3.5 min. 18. Incubate with Biotinylated Goat anti-Rabbit (1:200) at room temperature for 30 min. 19. Wash in PBST three times, each for 3.5 min. 20. Incubate with ABC reagent at room temperature for 30 min. 21. Wash in PBST three times, each for 3.5 min. 22. Incubate with DAB/H2O2 at room temperature for 5 min. 23. Wash in running H2O for 5 min. 24. Counterstain with Hematoxylin Gill #2 for 10 s. 25. Wash in running H2O for 3 min. 26. Blue in Scott’s H2O for 1 min. 27. Wash in running H2O for 3 min. 28. Dehydrate through (Subheading 3.8).

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29. Coverslip with permount medium.

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3.3 Nuclear Receptor Coactivator 3 (NCOA3) (Fig. 3) (See Note 3)

1. Bake slides in oven at 60 °C for 30 min prior to staining. 2. Deparaffinize and rehydrate (Subheading 3.7). 3. Microwave in 10 mM Citrate Buffer: Place slides in three plastic coupling jars filled with 10 mM Citrate Buffer; four slides in each jar, filling empty spaces with blank slides. Microwave (900 W, high power) slides in coupling jars for 10 min, stop and check to see if dH2O needs to be replenished. After microwaving, allow the slides to cool for 30 min at room temperature. Pressure cooker appears to results in superior antigen retrieval compared to microwave or steamer. The user is encouraged to use pressure cooker in the protocols that were previously optimized with microwave or steamer. 4. Wash in dH2O for 5 min. 5. Wash in PBST for 5 min. 6. Block in 3 % H2O2 in PBS for 30 min. 7. Wash in PBST three times, each for 3.5 min. 8. Block in Avidin blocking reagent for 15 min. 9. Wash in PBST three times, each for 3.5 min. 10. Block in Biotin blocking reagent for 15 min. 11. Wash in PBST three times, each for 3.5 min. 12. Incubate with Normal Horse Serum at room temperature for 30 min. 13. Incubate with NCOA3 primary antibody (1:10, item 3 of Subheading 2.2) at room temperature for 60 min (see Note 3).

Fig. 3 Photomicrograph of NCOA3 immunostaining in primary melanoma

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14. Wash in PBST three times, each for 3.5 min. 15. Incubate with Biotinylated Horse Anti-Mouse (1:200) at room temperature for 30 min. 16. Wash in PBST three times, each for 3.5 min. 17. Incubate with ABC reagent at room temperature for 30 min. 18. Wash in PBST three times, each for 3.5 min. 19. Incubate with DAB/H2O2 at room temperature for 5 min. 20. Wash in running H2O for 5 min. 21. Counterstain with Hematoxylin Gill #2 for 10 s. 22. Wash in running H2O for 3 min. 23. Blue in Scott’s H2O for 1 min. 24. Wash in running H2O for 3 min. 25. Dehydrate through graded alcohols and xylene (Subheading 3.8). 26. Coverslip with permount medium. 3.4 Regulator of G-Protein Signaling 1 (RGS1) (Fig. 4) (See Note 4)

1. Bake slides in oven at 60 °C for 30 min prior to staining. 2. Deparaffinize and Rehydrate (Subheading 3.7). 3. Pressure Cook with Citrate (Subheading 3.9). 4. Wash in dH2O for 5 min. 5. Wash in PBS for 5 min. 6. Block in 3 % H2O2 in PBST for 15 min. 7. Wash in PBST three times, each for 3.5 min.

Fig. 4 Photomicrograph of RGS1 immunostaining in primary melanoma

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8. Incubate with Normal Goat Serum (1:10) at room temperature for 30 min. 9. Incubate with RGS1 primary antibody (1:50 or 1:100, item 4 of Subheading 2.2), and coverslip overnight at 4 °C (see Note 4). 10. Wash off coverslip with PBST for 8 min. 11. Wash in PBST three times, each for 3.5 min. 12. Incubate with Biotinylated Goat anti-Chicken (1:200) at room temperature for 30 min. 13. Wash in PBST three times, each for 3.5 min. 14. Incubate with ABC reagent at room temperature for 30 min. 15. Wash in PBST three times, each for 3.5 min. 16. Incubate with DAB/H2O2 at room temperature for 5 min. 17. Wash in running H2O for 5 min. 18. Counterstain with Hematoxylin Gill #2 10 s. 19. Wash in running H2O for 3 min. 20. Blue in Scott’s H2O for 1 min. 21. Wash in running H2O for 3 min. 22. Dehydrate through graded alcohols and xylene (Subheading 3.8). 23. Coverslip with permount medium. 3.5 Secreted Phosphoprotein 1 (SPP1, or Osteopontin) (Fig. 5) (See Note 5)

1. Bake slides in oven at 60 °C for 30 min prior to staining. 2. Deparaffinize and Rehydrate (Subheading 3.7). 3. Microwave in 10 mM Citrate Buffer as in step 3 of Subheading 3.3).

Fig. 5 Photomicrograph of SPP1 immunostaining in primary melanoma

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4. Wash in dH2O for 5 min. 5. Wash in PBST for 5 min. 6. Block in 3 % H2O2 in PBS for 30 min. 7. Wash in PBST three times, each for 3.5 min. 8. Incubate with Normal Goat Serum (1:10) at room temperature for 30 min. 9. Incubate with SPP1 primary antibody (1:200, item 5 of Subheading 2.2), and coverslip overnight at 4 °C (see Note 5). 10. Wash off coverslip with PBST for 8 min. 11. Wash in PBST three times, each for 3.5 min. 12. Incubate with Biotinylated Goat anti-Rabbit (1:200) at room temperature for 30 min. 13. Wash in PBST three times, each for 3.5 min. 14. Incubate with ABC reagent at room temperature for 30 min. 15. Wash in PBST three times, each for 3.5 min. 16. Incubate with DAB/H2O2 at room temperature for 5 min. 17. Wash in running H2O for 5 min. 18. Counterstain with Hematoxylin Gill #2 10 s. 19. Wash in running H2O for 3 min. 20. Blue in Scott’s H2O for 1 min. 21. Wash in running H2O for 3 min. 22. Dehydrate through graded alcohols and xylene (Subheading 3.8). 23. Coverslip with permount medium. 3.6 Wingless-Type MMTV Integration Site Family Member 2 (WNT2) (Fig. 6) (See Note 6)

1. Bake slides in incubator at 37 °C for 60 min. Then bake slides in oven at 60 °C for 30 min prior to staining. 2. Deparaffinize and Rehydrate (Subheading 3.7). 3. Pressure Cook with Citrate (Subheading 3.9). 4. Wash in dH2O for 5 min. 5. Wash in PBS for 5 min. 6. Block in 3 % H2O2 in PBST for 15 min. 7. Wash in PBST three times, each for 3.5 min. 8. Incubate with Normal Goat Serum (1:10) at room temperature for 30 min. 9. Incubate with Wnt-2 primary antibody (1:5, item 6 of Subheading 2.2), and coverslip overnight at 4 °C (see Note 6). 10. Wash off coverslip with PBS for 8 min. 11. Wash in PBST three times, each for 3.5 min.

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Fig. 6 Photomicrograph of WNT2 immunostaining in primary melanoma. All sections were scanned by using a whole slide high-resolution scanning system, and the digital snapshots represented were captured at the final magnification of 400× (20× objective × 20× software) at a resolution of 0.32 μm per pixel

12. Incubate with Biotinylated Goat anti-Rabbit (1:200) at room temperature for 30 min. 13. Wash in PBST three times, each for 3.5 min. 14. Incubate with ABC reagent at room temperature for 30 min. 15. Wash in PBST three times, each for 3.5 min. 16. Incubate with DAB/H2O2 at room temperature for5 min. 17. Wash in running H2O for 5 min. 18. Counterstain with Hematoxylin Gill #2 10 s. 19. Wash in running H2O for 3 min. 20. Blue in Scott’s H2O for 1 min. 21. Wash in running H2O for 3 min. 22. Dehydrate through (Subheading 3.8).

graded

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23. Coverslip with permount medium. 3.7 Deparaffinize and Rehydrate

1. Place the tissue section sequentially in the following solutions for the specified time: xylene twice, each for 5 min; 100 % ethanol twice, each for f5 min; 95 % ethanol twice, each for 5 min 70 % ethanol twice, each for 5 min; deionized water (dH2O) for 5 min. 2. After deparaffination the blocking step (3 % H2O2 in PBS) can be performed before or after the antigen retrieval.

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3.8 Dehydrate Through Graded Alcohols

3.9 Pressure Cooking for Antigen Retrieval (See Note 7)

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Place the tissue section sequentially in the following solutions for the specified time: deionized water (dH2O) for 5 min; 70 % ethanol twice, each for 5 min; 95 % ethanol twice, each for 5 min; 100 % ethanol twice, each for 5 min; xylene twice, each for 5 min. 1. In a Pascal Pressure Cooker (Dako), place 500 mL of dH2O in the chamber, and place the heat shield at the center of the chamber. 2. Place slides in a Coupling Jar or staining container filled with the appropriate buffer (e.g. Trilogy or Citrate Buffer). No need to put on the lid. 3. Place all the jars or containers in the pressure cooker chamber. 4. Place Pascal Quality Strip (Dako) on top rim of the container. 5. The chamber can accommodate a maximum of four staining containers, i.e., 4 × 24 slides at one time. There is no need to put in blank slides to make up the total number of slides. 6. Place the lid on the chamber and close by aligning “OPEN” on the lid to the “white dot” on the brown handle and close the lid clockwise. 7. Check that there is no gap in the lid and the handles are all lined up. 8. Turn POWER on. 9. Press “start”, SP1 Light will illuminate. The temperature will automatically rise to the preset 125 °C. It will take approximately 15 min. 10. The chamber will hold the temperature (125 °C) for 30 s. 11. The chamber will beep. Record the temperature and pressure for quality control. 12. Press “Stop”, SP2 light will illuminate, the chamber will start cooling off. It takes about 25 min to cool to 90 °C. 13. When the temperature falls to 90 °C, it will hold the temperature for 10 s, and the alarm will sound. 14. Press “Stop” to end the alarm and the program. 15. Confirm the pressure reach 0 psi. 16. Release the pressure of the chamber by pressing on one edge of the brown knob on the lid. 17. Turn the lid anti-clockwise and lift the lid with the concave side facing away from self. 18. Turn POWER off. 19. Check the Temperature/Pressure Quality strip for proper color (dark gray to charcoal black). 20. Cool the slides in the buffer solution by adding room temperature dH2O slowly and in small amounts until the slides reach the room temperature.

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21. The slides are done with antigen retrieval and are ready for further processing. 22. The chamber has to be cooled before use for another run. 3.10 Immunohistochemical Scoring System

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Prior to scoring, the significance of cellular localization of each specific antibody—cytoplasmic for the above protocols—was assessed based on the pattern and positivity of staining, and biological relevance of the antigen. During the process of scoring all other areas (such as background, nuclear, and membrane staining) were ignored. Each marker was graded on a 4-point scale for cellular intensity using the following scale: no staining (0), weak staining (1), moderate staining (2), and intense staining (3).

Notes 1. ARPC2 dilution of 1:25 should be used for both regular section and the tissue microarray (TMA) slides. The positive controls for immunohistochemical staining of ARPC2 in cell lines are LOX, FEM, Jurkat, and HeLa, and in tissues spleen, thymus, and primary melanoma. The histological evaluation of this antibody was assessed based on cytoplasmic localization. 2. FN1 dilution of 1:400 should be used for both regular section and the tissue microarray (TMA) slides. The positive controls for IHC staining of FN1 in tissues are primary melanoma, normal kidney, and tonsil. The histological evaluation of this antibody was assessed based on cytoplasmic localization. 3. NCOA3 dilution of 1:10 should be used for both regular section and the tissue microarray (TMA) slides. The positive controls for IHC staining of NCOA3 in cell lines are melanoma (LOX, FEM), breast (BT474, SKBR3), and in tissues are breast tumor, metastatic melanoma, or ovarian tumor. The histological evaluation of this antibody was assessed based on cytoplasmic localization. 4. RGS1 dilution of 1:50 should be used for regular section and 1:100 for the tissue microarray (TMA) slides. The positive controls for IHC staining of NCOA3 in cell lines is Jurkat, and in tissues are primary or metastatic melanoma, and non-Hodgkin lymphoma. The histological evaluation of this antibody was assessed based on cytoplasmic localization. 5. SPP1 dilution of 1:200 should be used for both regular section and the tissue microarray (TMA) slides. The positive controls for IHC staining of SPP1 in tissues are breast carcinoma or metastatic melanoma. The histological evaluation of this antibody was assessed based on cytoplasmic localization. 6. WNT2 dilution of 1:5 should be used for both regular section and the tissue microarray (TMA) slides. The positive controls for IHC staining of WNT2 in tissues are primary or metastatic

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melanoma, and breast tumor. The histological evaluation of this antibody was assessed based on cytoplasmic localization. 7. In the optimization process of RGS1 and WNT2, better antigen retrieval was achieved by using EDTA instead of citrate, but the harsher pH treatment caused tissue wash off in the subsequent steps. This problem was circumvented by fixing the tissue on to the glass slide in a pressure cooker (Subheading 3.9) rather than by microwave. However, after baking the tissue with pressure cooker, switching back to citrate did not result in any difference in antigen retrieval. It is conceivable that stronger treatment in pressure cooker would cause the same level of antigen retrieval for both citrate and EDTA. Hence, these two protocols differ from the rest in this regard. References 1. Ming ME (2000) The histopathologic misdiagnosis of melanoma: sources and consequences of “false positives” and “false negatives”. J Am Acad Dermatol 43:704–706 2. Piepkorn MW et al (1994) A multiobserver, population-based analysis of histologic dysplasia in melanocytic nevi. J Am Acad Dermatol 30:707–714 3. Farmer ER, Gonin R, Hanna MP (1996) Discordance in the histopathologic diagnosis of melanoma and melanocytic nevi between expert pathologists. Hum Pathol 27:528–531 4. Corona R et al (1996) Interobserver variability on the histopathologic diagnosis of cutaneous melanoma and other pigmented skin lesions. J Clin Oncol 14:1218–1223 5. Barnhill RL et al (1999) Atypical Spitz nevi/ tumors: lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome. Hum Pathol 30:1523–1526 6. Brochez L et al (2002) Inter-observer variation in the histopathological diagnosis of clinically suspicious pigmented skin lesions. J Pathol 196: 459–466 7. Troxel DB (2003) Pitfalls in the diagnosis of malignant melanoma: findings of a risk management panel study. Am J Surg Pathol 27: 1278–1283 8. Wettengel GV, Draeger J, Kiesewetter F, Schell H, Neubauer S, Gebhart E (1999) Differentiation between Spitz nevi and malignant melanomas by interphase fluorescence in situ hybridization. Int J Oncol 14:1177–1183 9. Bastian BC, Wesselmann U, Pinkel D, Leboit PE (1999) Molecular cytogenetic analysis of Spitz nevi shows clear differences to melanoma. J Invest Dermatol 11:1065–1069 10. Busam KJ et al (1998) Expression of melan-A (MART1) in benign melanocytic nevi and primary cutaneous malignant melanoma. Am J Surg Pathol 22:976–982

11. King R, Weilbaecher KN, McGill G, Cooley Mihm M, Fisher DE (1999) Microphthalmia transcription factor. A sensitive and specific melanocyte marker for melanoma diagnosis. Am J Pathol 155:731–738 12. Fullen DR, Reed JA, Finnerty B, McNutt NS (2001) S100A6 preferentially labels type C nevus cells and nevic corpuscles: additional support for Schwannian differentiation of intradermal nevi. J Cutan Pathol 28: 393–399 13. King R, Googe PB, Weilbaecher KN, Mihm MC Jr, Fisher DE (2001) Microphthalmia transcription factor expression in cutaneous benign, malignant melanocytic, and nonmelanocytic tumors. Am J Surg Pathol 25:51–57 14. Kucher C et al (2004) Expression of Melan-A and Ki-67 in desmoplastic melanoma and desmoplastic nevi. Am J Dermatopathol 26: 452–457 15. Haqq C et al (2005) The gene expression signatures of melanoma progression. Proc Natl Acad Sci U S A 102:6092–6097 16. Kashani-Sabet M et al (2009) A multi-marker assay to distinguish benign nevi from malignant melanomas. Proc Natl Acad Sci U S A 106: 6268–6272 17. Gogas H et al (2009) Biomarkers in melanoma. Ann Oncol 20(Suppl 6):vi 8–13 18. Kashani-Sabet M et al (2009) A multi-marker prognostic assay for melanoma. Clin Cancer Res 15:6987–6992 19. Rangel J et al (2006) Prognostic significance of NCOA3 overexpression in primary cutaneous melanoma. J Clin Oncol 24:4565–4569 20. Rangel J et al (2008) Osteopontin as a molecular prognostic marker for melanoma. Cancer 112:144–150 21. Rangel J et al (2008) Novel role for RGS1 in melanoma progression. Am J Surg Pathol 32: 1207–1212

Immunohistochemical diagnostic and prognostic markers for melanoma.

Recent studies in our laboratory have identified novel molecular diagnostic and prognostic markers based on analyses in large cohorts of melanoma pati...
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