Recent Advances in Pathologic Evaluation of Melanoma Sentinel Lymph Nodes.

2. Potential areas of challenges 1. Role of regional LN dissection in melanoma. 1. Role of regional LN dissection in melanoma. 2. Mapping, identification, and biopsy of true SLN. 1. Role of regional LN dissection in melanoma. 2. Mapping, identification, and biopsy of true SLN. 3. Relationship of SLN status with regional LNs. 1. Role of regional LN dissection in melanoma. 2. Mapping, identification, and biopsy of true SLN. 3. Relationship of SLN status with regional LNs. 4. Evaluation of SLN for melanoma metastases. 1. Role of regional LN dissection in melanoma. 2. Mapping, identification, and biopsy of true SLN. 3. Relationship of SLN status with regional LNs. 4. Evaluation of SLN for melanoma metastases.

3. Background Primary lesion Sentinel Lymph node Regional lymph nodes

4. Primary lesion Sentinel Lymph node Regional lymph nodes Status of Sentinel lymph node accurately predicts the status of regional lymph nodes

5. Primary lesion Sentinel Lymph node Regional lymph nodes False negativity is low (0 to 8%)

6. Potential areas of challenges 1. Role of regional LN dissection in melanoma. 2. Mapping, identification, and biopsy of true SLN. 3. Relationship of SLN status with regional LNs. 4. Evaluation of SLN for melanoma metastases.

7. Evaluation of SLN for melanoma metastases a. How to process the SLNs-? Grossing SLN- How to cut? b. Which immunomarkers to choose? c. How many levels to study? d. How to evaluate intraopearively?

8. Evaluation of SLN for melanoma metastases a. How to process the SLNs-? Grossing SLN- How to cut? b. Which immunomarkers to choose? c. How many levels to study? d. How to evaluate intraopearively?

9. SLN- 2.6X1.0X0.5 cm 1.0 X 0.5 cm (11 slices) 2.6X0.5 cm (4 slices) 2.6X1 cm (2 slices)

10. Area: K =Pi ab Ciecumference: C =Pi(a+b) {1+3x 2 /[10+Sqrt(4-3x 2 )]} approx http://mathforum.org/dr.math/faq/formulas/faq.ellipse.html (as on 3-5-05) Pi = (22/7) = 3.14 K: 5.5 sq cms C: 17.4 cms K: 5.2 sq cms C: 8 cms K: 5.2 sq cms C: 3.1 cms C a b K 2.6X1 cm (2 slices) 1.0 X 0.5 cm (11 slices) 2.6X0.5 cm (4 slices)

11. SLN- 2.6X1.0X0.5 cm 17.4 cms 8 cms 3.1 cms 1.0 X 0.5 cm (11 slices) 2.6X0.5 cm (4 slices) 2.6X1 cm (2 slices)

12. Evaluation of SLN for melanoma metastases a. How to process the SLNs-? Grossing SLN- How to cut? b. Which immunomarkers to choose? c. How to evaluate intraopearively? d. How many levels to study?

13. Immunohistochemistry for melanoma SLN S-100 protein and HMB45 - Conventionally used Non-discriminatory pattern * *

14. S-100 HMB-45 S-100 HMB-45 a b d c

15. Shidham et al. Am J Surg Pathol 2001;25:1039-46.

16. Negative SLN- immunostained with MCW melanoma cocktail

17. Shidham et al. BMC Cancer 2003 May 7;3(1):15 (p1-9) http://www.biomedcentral.com/qc/1471-2407/3/15

18. The monoclonal antibodies in ‘ MCW Melanoma Cocktail ’ * Final Dilution in the cocktail. Applied as primary antibody for 30 minutes . Shidham et al. BMC Cancer 2003 May 7;3(1):15 (p1-9) http://www.biomedcentral.com/qc/1471-2407/3/15 Antigen retrieval- ‘Dako pH 6.0 target retrieval solution’® (Dako Corporation, Carpinteria, CA) at 95-100 o C for 35 minutes and cooled at room temperature for 20 minutes. Marker Clone Source Dilution * MART-1 M2-7C10 Signet Laboratories, Inc. Dedham, MA 1:500 Melan-A A103 Dako Corporation, Carpinteria, CA 1:100 Tyrosinase T311 Novocastra, Newcastle upon Tyne, UK 1:50

19. H & E Cocktail SLN Positive for melanoma micrometastases- immunostained with MCW melanoma cocktail

20. SLN Positive for melanoma micrometastases- immunostained with MCW melanoma cocktail

21. Other Melanoma Cocktails reported- 1. Eudy GE et al. Rapid immunohistochemistry of sentinel lymph nodes for metastatic melanoma. Hum Pathol 2003;34:797-802. 2. Parker DC et al . Improved detection of melanoma metastases in sentinel lymph nodes. USCAP Annual Meeting, March 2001, Abstract 406, Page 71A. Mod Pathol 2001;14:1A-239A. Marker Components Source Eudy GE et al (2003) 1 Monoclonal HMB45 (Dako) Monoclonal melan A (Dako), Monoclonal tyrosinase (Novacastra, Newcastle-upon-Tyne, U.K.). Home brewed by the author’s lab. Parker DC et al (2001) 2 HMB-45 + DT101 + BC199 + T311 Biocare Medical, Walnut Creek, CA, USA

22. Melanophages Hematoxylin counterstain 40X 100X zoomed 100X zoomed Hematoxylin & Azue B counterstain 40X 100X zoomed 100X zoomed

23. 100X DAB Melanin Hematoxylin with Azue B counterstain

24. A B D C X10 Zoomed MCW melanoma cocktail H&E

25. Evaluation of SLN for melanoma metastases a. How to process the SLNs-? Grossing SLN- How to cut? b. Which immunomarkers to choose? c. How many levels to study? d. How to evaluate intraopearively?

26. From: Cserni G. J Clin Pathol 52:922-924,1999. Distribution of metastases in SLN **5 step sections 50-100 μ m

27. Effect of increasing the number of step sections evaluated on identification of metastases Modified from: Cserni G. J Clin Pathol 52:922-924,1999. Number of 3-5 μ m thick step sections examined (Interval between sections 50 to 100 μ m) 5 10 15 25 20 30 35 40 45 Deeper 100 50 75 25 5 10 15 20 30 35 40 45 55 60 65 70 80 85 90 95 Metastases identified (%) 5 10 15

28. Projected SLN positivity by different sectioning strategies Modified from: Spankenbel K et al. Am J Surg Pathol 2005;29:305-317. Variable numbers of levels in 1 mm of specimen 20 levels at 50 μm 21 (100%) 10 levels at 100 μm 15 (71%) 5 levels at 200 μm 8 (38%) 4 levels at 250 μm 5 (24%) 3 levels at 250 μm 15 (71%) 2 levels at 250 μm 6 (29%) 1 level * 4 (19%) 3 levels at variable sectioning interval 3 levels at 50 μm 9 (43%) 3 levels at 100 μm 12 (57%) 3 levels at 150 μm 12 (57%) 3 levels at 200 μm 13 (62%) 3 levels at 250 μm 15 (71%) 3 levels at 300 μm 13 (62%) 3 levels at 350 μm 13 (62%) 3 levels at 400 μm 10 (48%) 3 levels at 450 μm 11 (52%) * for comparison the first level from the block would be expected to yield 4 positive SLNs or 19% of patients studied (n = 14). No. of Levels x Sectioning Interval No. of Nodes with Metastatic Melanoma (n=21)

29. Cases (n=42) Mean 10.6 Mean 13 16 Step sectioning increases diagnostic yield of melanoma micrometastases in sentinel lymph nodes with MCW melanoma cocktail. Modern Pathology 2005; 18(Supplement 1):1a-358a . Abstract no. 1551 ( 94th Annual Meeting of United States and Canadian Academy of Pathology, February 26- March 4, 2005, San Antonio, TX .) Levels Positive for Melanoma metastases Slices (n=405) Blocks (n=116) SLN (n=83) 1 level 1 alone 50 Mean 42 17 Mean 17 11 Mean 12 10 4 alone 36 15 11 10 7 alone 40 19 14 12 2 levels 1+4 51 Mean 48.3 18 Mean 19.6 12 Mean 14.3 11 1+7 52 21 16 15 4+7 42 20 15 13 3 levels 1+4+7 80 30 22

30. Modified from: Spankenbel K et al. Am J Surg Pathol 2005;29:305-317. Disease-specific survival with reference to SLN status Disease-specific survival in months 24 48 72 20 96 120 100 50 75 25 5 10 15 20 30 35 40 45 55 60 65 70 80 85 90 95 Survival Probability (%) 144 SLN- Neg by immuno * w serials SLN- Pos by immuno * w serials SLN- Pos by FS & HE * Used S-100 & HMB45

31. Evaluation of SLN for melanoma metastases a. How to process the SLNs-? Grossing SLN- How to cut? b. Which immunomarkers to choose? c. How many levels to study? d. How to evaluate intraopearively?

32. Intraoperative evaluation of SLN Authors Intraoperative evaluation Sensitivity (%) Specificity (%) Gibbs et al . Ann Surg Oncol 1999; 6: 699-704. FS 43 100 Clary et al . Eur J Nucl Med 1999; 26: S68. FS 56 100 Tanis et al . Ann Surg Oncol 2001; 8: 222-26. FS 47 100 Stojadinovic A et al. Ann Surg 2002;235:92-8. FS 59 100 Creager et al. Cancer. 2002;94:3016-22. IS 38 100 Eudy et al. Hum Pathol 2003;34:797-802. FS with IHC 86 97 Our study ICIS 90 100

33. The discriminatory immunostaining pattern with ‘ MCW melanoma cocktail ’ facilitated the possibility of immunocytochemical evaluation of imprint smears of Sentinel Lymph Nodes Immunocytochemistry of imprint smears (continued)

34. Imprint smear Intraoperative Imprint / Scrape Cytology (continued) 1 2 OR 3

35. a1 b2 b3 c4 Imprint smears for intra-operative immunocytochemistry MCW Melanoma Cocktail Immunocytochemistry of imprint smears (continued) a c b a b c 1 4 2 3 2-3 mm

36. Shidham et al. Diag Cytopathol 2003 Oct;29(4):217-21. Immunocytochemistry of imprint smears (continued)

37. Air dried smears post-fixed in alcohol formalin after saline re-hydration Immunocytochemistry of imprint smears (continued) Shidham et al. Diag Cytopathol 2003 Oct;29(4):217-21.

38. ADS WFS WFS WFS

39. Rapid intra-operative immunostaining with MCW melanoma cocktail with 17 minute protocol. Immunocytochemistry of imprint smears (continued)

40. Shidham et al. CytoJournal 2004 Aug 06;1(1):2. 44

41. 17 minute protocol for rapid immunostaining Hydrate the smear in 0.2% Tween 20 in DW- 30 sec 3% H 2 O 2 in DW- 1 mt Protein blocking solution- 1 mt ‘ MCW melanoma cocktail’- 5 mt Rinse in 0.2% Tween 20 in DW HRP-linker Antibody- 5 mt Rinse in 0.2% Tween 20 in DW Chromogen (DAB)- 3 mt Rinse in 0.2% Tween 20 in DW Azure B- Blue solution of Diff-Quik- 1 mt Rinse in tap water Haris Hematoxylin- 30 sec Rinse in tap water Dehydrate in ascending alcohol Clear in xylene Apply mounting medium and coverslip Immunocytochemistry of imprint smears (continued)

42. HE section 40X ICC 10X ICC 40X ICC 100X SLN Positive for melanoma micrometastases- IS immunostained with MCW melanoma cocktail

43. SLN Positive for melanoma micrometastases- IS immunostained with MCW melanoma cocktail IHC section 40X ICC 40X ICC 100X HE section 40X

44. Melanoma cells Mast cells

45. Melanoma cells

46. Mast cells

47. Melanoma cell Mast cell Immunocytochemistry of imprint smears (continued)

48. Immunocytochemistry with MCW melanoma cocktail ( a combination of MART-1, Melan-A, & Tyrosinase ) can be used for rapid intra-operative evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma Air-dried imprint smears rehydrated and postfixed in alcohol formalin showed best results. Immunocytochemistry of imprint smears (Summary)

49. Regional lymph node dissection NOT indicated * To decrease the cost and avoid longer turn around time, the section number 2,5,&6 may be mounted on one slide and immuno-stained all together. However, if the sections are large, they may not be accommodated on one slide. Summary Modified from: Shidham et al. Expert Review of Molecular Diagnostics (In press) 1 4 2 3 2 mm Imprint smears for intra-operative immunocytochemistry 1 2 3 4 MCW Melanoma Cocktail Negative for metastases * FPTS 200 μ m 200 μ m A B C B C A H&E H&E H&E 200 μ m Discard 200 μ m Discard Positive for metastases Indeterminate or Negative for metastases FPTS 200 μ m 200 μ m A B C B C A 4. H&E 3. Immuno- Neg Cntrl 5. Immuno- MCW Mel Cocktail 1. H&E 2. Unstained for Immuno * 7. H&E 6. Unstained for Immuno * 200 μ m Discard 200 μ m Discard Proceed with immunostaining of levels 2 & 6. Negative for metastases Positive for metastases Positive for metastases Regional lymph node dissection may be completed

50. Possibility of studying more than one step sections on one slide 1 4 7 MCW Melanoma Cocktail

51. Future prospects One-step immunocytochemical staining with a shorter immunostaining time. Rapid selective blocking of endogenous peroxidase activity (such as in mast cells). Further characterization of metastatic cells, with recent techniques such as microdissection of cells detected by IC for molecular studies- RT-PCR, proteomics. Immunocytochemistry of imprint smears (continued)

52. Future prospects (contd) Routine availability of infrastructure for rapid immunocytochemistry comparable to cryostat in frozen section lab would facilitate wider application of rapid intraoperative immunocytochemical evaluation . The rapid molecular cytopathology protocols may be developed for intraoperative applications in future. Immunocytochemistry of imprint smears (continued)

53. Future prospects (contd) Objective marker for benign capsular melanocytic nevi . Long term follow-up studies evaluating relationship of various patterns and sizes of melanoma metastases with prognosis. Immunocytochemistry of imprint smears (continued)

54. Faculty, fellows, and residents Dept of Pathology, Medical College of Wisconsin and my previous institutions. Acknowledgements Supported by MCW Cancer Center Interdisciplinary grant

55. Thank you Milwaukee Art Museum [email_address]