External Quality Assessment of melanocytic marker analyses NordiQC experience Jan Klos MD, Department of Pathology Stavanger University Hospital Norway 1
Content 18 Runs = 2112 submissions between 2001-2014 MSA/HMB-45 MLA/Melan A S-100 protein Ki-67 Vimentin 2
MSA/HMB-45 Glycoprotein 10kDa (Pmel 17) localized in the matrix of premelanosomes (early forms do not containing melanin yet). It is a marker of melanogenesis not the cell lineage! Present in normal foetal and noenatal melanocytes. In adults the epitope is detected in reactive melanocytes and neoplasias, but not in resting melanocytes. 3
MSA/HMB-45 Data based on 3 runs: Run 7 in 2003 Run 20 in 2007 Run 40 in 2014 with 323 submissions from participants. 4
MSA/HMB-45 Last Run 2014 1. Skin, 2. Kidney, 3. Angiomyolipoma, 4. Malignant melanoma 5. Malignant melanoma 1 2 3 4 5 Only one clone used HMB45 Proportion of sufficient results for MSA in three NordiQC runs performed Run 7 2003 Run 20 2007 Run 40 2014 Participants, n= 66 99 158 Sufficient results 74% 97% 91% 5
MSA/HMB-45 High quality antibody Causes of insufficient staining: omission or insufficient HIER biotin based detection systems Recommended control tissue: PECOMA (i.e. angiomyolipona) or blue nevus 6
Optimal MSA staining of the melanoma blocks 4 and 5. Insufficient MSA staining of the melanoma too low concentration of primary Ab. 7
Optimal MSA (HMB-45) staining of the angiomyolipoma Insufficient staining of the angiomyolipoma - same protocol as on the left, but HIER omitted. Same time still sufficient staining 8 result in the melanoma (insert).
Aberrant MSA staining of the melanoma (right). A moderate, predominantly nuclear staining reaction of the neoplastic cells is seen. This staining pattern was observed by using efficient HIER in an alkaline buffer. The overall result assessed as good, as a coexisting cytoplasmic staining reaction was also present. 9
MLA Protein Melan-A/MART-1 The 18 kb long gene MLANA is regulated by the Microphtalmia-associated transcription factor (MITF). The gene sequenced in 1994, by two independent research groups, codes for the protein named as melanocyte lineage-specific protein Melan-A (Coulie P.G.) and Melanoma Antigen Recognized by T cells 1 (MART-1) (Kawakami Y.). The protein is localized in endoplasmic reticulum and melanosomes, but it is also present on cell membranes, where it is recognized by autologous cytotoxic T lymphocytes. The protein is considered a melanocyte differentiation antigen, but exact function is less known. 10
MLA Protein Melan-A/MART-1 Data based on 6 runs: Run 7 in 2003 Run 16 in 2006 Run 20 in 2007 Run 24 in 2008 Run 31 in 2011 Run 42 in 2014 with 682 submissions from participants. 11
MLA Protein Melan-A/MART-1 Last Run 2014 1. Adrenal gland 2. Kidney 3. Malignant melanoma I 4. Malignant melanoma II 5. Ovarian granulosa cell tumor 1 2 3 4 5 Clones with different spectrum of reactivity are in use: A103 constant crossreactivity with (?)epitope in cells producing steroid hormones. M2-7C10 and M2-9E3 which do not show this crossreactivity. Proportion of sufficient results for MLA in the six NordiQC runs 2003 2006 2007 2008 2011 2014 Participats, n= 35 79 90 115 165 198 % Sufficient results 69% 32% 48% 50% 66% 68% 12
MLA Protein Melan-A/MART-1 Clone A103 used by 95% (188/198) participants in the last run. Optimal results: clone A103 and M2-7C10 alone or in cocktails. Main causes of insufficient results: use of detection system with low sensitivity insufficient HIER too low concentration of primary Ab Recommended control: adrenal gland for protocols using clone A103 and non-biotin based detection systems angiomyolipoma for other protocols. 13
Results with concentrated clone A103 were related to the IHC platform used. Several parameters could contribute to this difference e.g. sensitivity of the detection systems impact of other reagents used too stringent washing 14
Optimal staining of the adrenal gland (upper) and melanoma I (lower) with A103 Reduced intensity of staining on the same tissue with A103 on BenchMark Ultra 15
Optimal staining of melanoma I and II (upper) and granulosa cell tumor (lower) with clone A103 Reduced staining intensity in melanoma I and II and false negative staining of granulosa cell tumor with clone A103 on BenchMark Ultra. 16
S-100β Is a family of acidic, small molecule (9-13 kda) proteins present in both cell nucleus and the cytoplasm. The name is derived from the fact that these proteins are soluble in 100% saturated ammonium sulfate at neutral ph. Antibodies are directed mostly against β-chains in the protein molecule. S100 proteins have been implicated in a variety of intracellular and extracellular functions like regulation of protein phosphorylation, transcription factors, Ca 2+ homeostasis, dynamics of cytoskeleton constituents, enzyme activities, cell growth and differentiation and the inflammatory response. S100 proteins are normally present in the cells derived from the neural crest (Schwann cells, melanocytes), chondrocytes, adipocytes, myoepithelial cells, macrophages, Langerhans cells, dendritic cells and some epithelial cells. 17
S-100 Data based on 3 runs: Run 7 in 2003 Run 20 in 2007 Run 34 in 2012 with 369 submissions from participants. 18
S-100 Last Run 2012 1. Skin 2. Appendix 3. Breast hyperplasia 4. Malignant melanoma 5. Schwannoma 1 2 3 4 5 Proportion of sufficient results for S100 in the three NordiQC runs Run 7 2003 Run 20 2007 Run 34 2012 Participants, n= 63 106 200 Sufficient results 71 % 75 % 64 % 19
S-100 Optimal results achieved only with concentrated pab Z0311, and only by 18% (36/200) of participants. Causes of insufficient staining: Inadequate/insufficient antigen retrieval - use of proteolysis or omission of HIER generally gave significantly inferior performance. Too low concentration of primary Ab Recommended control: appendix or tonsil. 20
Optimal S00 staining of the GC macrophages in appendix using the pab Z0311 with HIER in an alkaline buffer. Insufficient S100 staining of the appendix using the pab Z0311 by protocol settings providing a too low sensitivity. Optimal S100 staining of the malignant melanoma using same protocol. Insufficient S100 staining of malignant melanoma using same protocol. Insufficient S100 staining of malignant melanoma 21 - proteolytic pre-treatment.
Ki-67 Protein (encoded by the MKI67 gene) associated with and probably necessary for cell proliferation, involved also in transcription of ribosomal RNA. Nuclear staining often with nucleolar enhancement in G1, S and G2 phases, diffuse cytoplasmic in M phase, but absent in G0 phase. 22
Ki-67 Data based on 4 runs Run 5 in 2001 Run 19 in 2007 Run B7 in 2009 Run B13 in 2012 with 495 submissions from participants. 23
Last Run 2012 1. Tonsil, fixed 24 hours 2. Tonsil, fixed 48 hours 3. Breast carcinoma 1 9 % 4. Breast carcinoma 10 24 % 5. Breast carcinoma 50 % Ki-67 Proportion of sufficient results for Ki-67 in the four NordiQC runs Run 5 2001 Run 19 2007 Run B7 2009 Run B13 2012 Participants, n= 42 100 124 229 Sufficient results 71% 73% 77% 89% 24
Ki-67 Optimal results (72%) with clones: MIB1, MM1, K2 and SP6. The RTU systems showed superior pass rates compared to the in-house validated assays The most frequent causes of an insufficient staining: Too low concentration of the primary antibody Insufficient HIER (too short heating time) Excessive HIER Inadequate deparaffination Control tissue: normal tonsil or appendix (germinal centres) 25
Optimal Ki67 staining of the tonsil fixed for 24 hours in NBF using the mab clone MIB1 properly calibrated and with HIER in an alkaline buffer. Insufficient Ki67 staining of the tonsil fixed for 24 hours in NBF using the mab clone MIB1 with a protocol providing a too low sensitivity. Optimal Ki67 staining of the breast carcinoma using same protocol. Insufficient Ki67 staining of the breast carcinoma using 26 the same protocol.
Vimentin Intermediate filament (57 kda), encoded by the VIM gene. Has structural function in the cytoplasm supporting and anchoring the position of the organelles in the cytosol, maintaining cell shape, integrity of the cytoplasm and stabilizing cytoskeletal interactions. Present in the cytoplasm nearly all cells at early phases of development, but later replaced by other microfilaments. In adult life present mainly in mesenchymal cells, but also in a number of other cell types. 27
Vimentin Data based on 2 Runs: Run 12 2004 Run 30 2010 with 243 submissions from participants. 28
Vimentin Last Run 2010 1. Tonsil, 2. Kidney, 3. Seminoma, 4. Melanoma, 5. Renal cell carcinoma 1 2 3 4 5 Proportion of sufficient results for VIM in the two NordiQC runs Run 12 2004 Run 30 2010 Participants, n= 79 164 Sufficient results 94 % 83 % 29
Vimentin Optimal staining : mabs clones V9 and Vim 3B4, rmab clone SP20. 122/164 participants used clone V9 which gave 86% sufficient results The mab clone V9, both as concentrate and RTU format gave the highest proportion of optimal results. The most frequent causes of insufficient staining were: Too low concentration of the primary antibody Omission of HIER Inappropriate epitope retrieval (i.e. proteolysis) Irrespective of the clone applied, HIER is mandatory to obtain an optimal staining. Proteolysis should not be used! Recommended control: tonsil 30
Optimal Vimentin staining of the tonsil using the mab clone V9. Insufficient Vimentin staining of the tonsil, using the mab clone V9 in too low concentration. Optimal Vimentin staining of the melanoma using same protocol. Insufficient Vimentin staining in melanoma using the 31 same protocol as above.
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