Antibody selection and optimization. Part III

Transcription

1 Antibody selection and optimization Part III Lymphoma markers Take Home Messages Søren Nielsen Project coordinator & Scheme Manager NordiQC Aalborg University Hospital, Denmark

2 Use NordiQC homepage as additional checklist Identify the Abs with highest proportion of optimal score Identify the recommended controls tissue and ICAPCs Use efficient HIER in an alkaline buffer (very few exceptions) No marker assessed in NordiQC provided optimal result by proteolysis Only Abs for IgM, IgK, IgL and CD30 had superior result by HIER ph 6 Use sensitive 3-step polymer / multimer based detection system General aspects potential to affect the IHC result Platform depending Abs Epitopes deterioated by endogenous peroxidase blocking Lot-to-lot variations.

3 Most common pitfalls and reasons for insufficient result Too low concentration of the primary Ab (LDT) BCL6, CD4, CD10, CD23, CD30, IgM, MUM1, PAX5 Use of low sensitive detection systems BCL6, CD10, CD15, CD23, CD30, MUM1, PAX5 Insufficient HIER (too short time and/or use of non-alkaline buffer) BCL6, CD10, CD15, CD23, CD30, MUM1, PAX5 Inappropriate epitope retrieval (use of proteolysis, omission) CD10, IgK, IgL, IgM Less successful primary Ab CD5, CD15, CD19, CD138, TdT

4 Use NordiQC homepage as additional checklist Identify the Abs with highest proportion of optimal score Identify the recommended controls tissue and ICAPCs

5 Antibody selection and optimization Part III Unknown primary tumor Epithelial markers Søren Nielsen Project coordinator & Scheme Manager NordiQC Aalborg University Hospital, Denmark

6 CD10 Uroplakin II Bladder IHC Protocols and controls - Part III Ep-CAM Generel CGA Neuroendocrine diff. Claudin 4 SYP CDX2 GI GATA3 Breast CEA Mammaglobin Cadherin 17 GCDFP 5 SMAD4 CD117 PAX8 Fem. Gen. PSA Prostate CA125 Prostein WT1 AMACR Arginase Liver NKX3.1 Glypican 3 ERG Glutamin synthease Melan A Melanoma Hepatocellular antigen SOX10

7 UPT EPCAM Recommendable clones (conc.)* mab Ber-EP4 mab BS14 mab MOC31 mab SPM491*** mab VU-1D9 Less successful clones (conc.) rmab E144 pab 7196*** RTU plug and play ** giving optimal result Dako: mab Ber-EP4 Clau. 4 mab 3E2C1*** * Potential to provide optimal result by a laboratory developed test (LDT) ** Using the protocol settings as recommended by the vendor incubation, retrieval, detection kit. *** In-house data - Aalborg University Hospital

8 UPT EPCAM Clau. 4 Positive tissue control HE* Appendix: Columnar epithelial cells Appendix: Columnar epithelial cells Positive tissue control LE** Kidney: Epithelial cells of proximale tubules + Bowman capsule Placenta: Throphoblasts Negative tissue control NE*** Liver: Hepatocytes Liver: Hepatocytes * HE = High expression ** LE = Low expression *** NE = No expression

9 EP-CAM reaction pattern ICAPCs Kidney Appendix Liver A moderate to strong membranous staining of virtually all epithelial cells of collecting ducts. An at least weak staining of epithelial cells of proximal tubules and Bowman capsule. A moderate to strong membranous staining of virtually all columnar epithelial cells both luminal and crypt base. A moderate to strong membranous staining of virtually all epithelial cells of the bile ducts. No staining reaction in hepatocytes.

10 All: 3-step pol/mul. Ber-EP4 & MOC31: HIER in TRS ph 6.1 (Dako) or DIVA ph 6.2 (Biocare) HIER in CC1, ER1, ER2 or use of protease less robust! BS14: HIER in CC1 (+/- proteolysis) or HIER in standard high ph buffer

11 ICAPCs: epithelial cells of the prox. tubules & Bowm.

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13 Claudin-4 reaction pattern ICAPCs Kidney Appendix Placenta A weak to strong predominantly membranous but also cytoplasmic staining reaction of epithelial cells of collecting ducts and proximal tubules. A moderate to strong membranous staining reaction of virtually all columnar epithelial cells both luminal and crypt base. A weak to moderate predominantly membranous staining reaction of virtually all throphoblasts. No reaction in stromal cells.

14 Placenta x Claudin-4 Placenta x 200 EP-CAM Pancreas x Claudin-4 Pancreas x 200 EP-CAM

15 Colon ad. carc. x Claudin-4 Colon ad. carc. x 200 EP-CAM Thyroid fol. carc. x Claudin-4 Thyroid fol. carc. x 200 EP-CAM

16 Ovarian ser. carc. x Claudin-4 Ovarian ser. carc. x 200 EP-CAM Seminoma x Claudin-4 Seminoma x 200 EP-CAM

17 Renal clear cell carc. x Claudin-4 Renal clear cell carc. x 200 EP-CAM Renal clear cell carc. x Claudin-4 Renal clear cell carc. x 200 EP-CAM

18 UPT II: EPCAM Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab Ber-EP4 mab MOC31 mab BS14 HIER TRS 6.1 & Diva HIER TRS 6.1, Diva (High ph??) HIER high ph 1: step Dako 3-step 1: step - - 1: step - - UPT II: Claudin-4 In-house pre-liminary data for best technical result Retrieval Titre Detection RTU Detection mab 3E2C1 HIER TRS 6.1 1:100 3-step - -

19 UPT CEA CDX2 Recommendable clones (conc.)* mab II-7 mab CEA31 mab COL1 mab DAK-CDX2 rmab EPR2764Y Less successful clones (conc.) mab mab PARLAM4 mab TF3H8-1 mab AMT28 mab CDX2-88 RTU plug and play ** giving optimal result Leica: mab II-7 VMS: mab CEA31 Dako: mab DAK-CDX2 VMS: rmab EPR2764Y CAD17 SMAD4 rmab SP183*** mab BC8 (non-vms) rmab EP618Y * Potential to provide optimal result by a laboratory developed test (LDT) ** Using the protocol settings as recommended by the vendor incubation, retrieval, detection kit. *** Aalborg University Hospital data

20 UPT CEA CDX2 CAD17 SMAD4 Positive tissue control HE* Appendix: Brushborder of columnar epith. cells Appendix: Columnar epithelial cells Appendix: Columnar epithelial cells Tonsil: Dispersed squam. epith. cells + plasma cells Positive tissue control LE** Appendix: Cytoplasm. compartm. of col. epith. cells. Pancreas: Epith. cells of intercalating ducts Pancreas: Scatered col. epith. cells of large ducts Tonsil: Lymphocytes Negative tissue control NE*** Liver: Hepatocytes Liver: Hepatocytes Liver: Hepatocytes Tumour: Neoplasia with loss of SMAD4 * HE = High expression ** LE = Low expression *** NE = No expression

21 CEA reaction pattern ICAPCs Appendix / Colon A strong accentuation of the brushborder / glycocalyx of the luminal epithelial cells. Appendix / Colon An at least weak to moderate and distinct cytoplasmic staining reaction of the vast majority of epithelial cells. Liver No staining reaction. No staining of bile canaliculi, leucocytes and Kupffer cells (NCA, BGP)

22 COL-1 & CEA31 Optimal on all platforms II-7 less successful on VMS; 0/35 opt. Dako; 5/25 opt. Leica; 3/6 opt. Clone! HIER Titre

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24 CDX2 reaction pattern ICAPCs Appendix / Colon A strong nuclear staining reaction of virtually all epithelial cells. A weak to moderate cytoplasmic staining reaction can be expected. Pancreas An at least weak to moderate and distinct nuclear staining reaction of the vast majority of epithelial cells of intercalating ducts. Tonsil No staining reaction. Few lymphocytes may show a faint nuclear staining reaction.

25 AMT28 and CDX2-88 lower pass rate used by a significantly lower proportion 18% in this run vs 70% in run DAK-CDX2 & EPR2764Y Conc & RTU Control tissue!

26 Dako conc. and RTU format of mab clone DAK-CDX2 will show an inferior pass-rate on a VMS platform Frequently Dako RTU format was applied on VMS..! < use of mab clones CDX2-88 and AMT28

27 Inappropriate RTU settings use of modified protocol settings of otherwise successful RTU product Dilution of RTU format, use of RTU format out of system!!!! 27

28 Cadherin 17 reaction pattern ICAPCs Appendix Pancreas Liver A moderate to strong membranous staining of virtually all columnar epithelial cells both luminal and crypt base.. ICAPCs A weak to moderate membranous staining of dispersed columnar epithelial cells of large ducts and exocrine acini. No staining reaction should be seen.

29 Colon adenocarcinomas Cadherin 17, rmab SP183 CDX2, rmab EPR2764Y

30 SMAD4 reaction pattern ICAPCs Pancreas A moderate to strong nuclear staining reaction of the majority of all cells. A weak to moderate cytoplasmic staining reaction can be expected. Tonsil A moderate to strong nuclear staining reaction of the majority of all cells. A weak to moderate cytoplasmic staining reaction can be expected. Tumour with loss No nuclear staining reaction in neoplastic cells. Stromal cells serving as internal positive control.

31 SMAD4 reaction pattern Pancreas; reaction affected by autolysis / delayed fixation

32 SMAD4 staining: mab BC8, HIER TE ph 9, FLEX+

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34 UPT II: CDX2 Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab DAK-CDX2* HIER high ph 1: step Dako 2- & 3-step rmab EPR2764Y HIER high ph 1: step Ventana 2- & 3-step UPT II: CEA Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab II-7* HIER high ph 1: step Dako, Leica 3-step mab CEA31 HIER high ph 1: & 3-step Ventana 2- & 3-step mab COL-1 HIER high ph 1: & 3-step * Inferior performance on VMS stainer platform

35 UPT II: SMAD4 Basic protocol settings for an optimal staining result (Internal data) Retrieval Titre Detection RTU Detection mab BC8* HIER high 1: step - - rmab EP618Y HIER high 1: step - - UPT II: Cadherin 17 Retrieval Titre Detection RTU Detection rmab SP183 HIER high ph 1: step - - * Inferior performance on VMS stainer platform

36 UPT PAX8 CA125 WT1 Recommendable clones (conc.)* mab BC12 mab MRQ-50 rmab ZR-1 pab AP mab M11 rmab OV185:2 mab 6F-H2 mab WT49 Less successful clones (conc.) pab 363A pabs in general RTU plug and play ** giving optimal result Dako: mab M11 Dako: mab 6F-H2 Leica: mab WT49 * Potential to provide optimal result by a laboratory developed test (LDT) ** Using the protocol settings as recommended by the vendor incubation, retrieval, detection kit. *** Aalborg University Hospital data

37 UPT PAX8 CA125 WT1 Positive tissue control HE* Fallopian tube: Secretory epithelial cells Fallopian tube: Brushborder of columnar epith. cells. Fallopian tube: Columnar epithelial cells Positive tissue control LE** Fallopian tube: Ciliated epithelial cells Fallopian tube: Brushborder of columnar epith. cells. Kidney: Parietal epithelial cells and podocytes Negative tissue control NE*** Appendix: Columnar epithelial cells Appendix: Columnar epithelial cells Tonsil: Lymphocytes, endothelial cells (nuclei) * HE = High expression ** LE = Low expression *** NE = No expression

38 PAX8 reaction pattern ICAPCs ICAPCs Fal. tube A strong nuclear staining reaction of virtually all secretory epithelial cells. A weak to moderate nusclear staining reaction of the majority of ciliated cells. Kidney An at least weak but distinct nuclear staining of the majority of epithelial cells of proximal tubules. Moderate to strong nuclear staining of epithelial cells of distale tubules and Bowman. Appendix No staining reaction of epithelial cells.

39 Choose clone depending on platform Use 3-step system

40 Dako AS48: ZR-1, BC12, MRQ-50 or Prot. Tech VMS, Dako OMNIS: ZR1 or Prot. Tech

41 Tonsil stained for PAX8 = Same pattern as for PAX5

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43 PAX8 N-term PAX8 C-term mab clone BC12 (C-term.) mab clone MRQ-50 (N-term.)

44 PAX8 antibodies towards N-terminal (most likely..): mab clone MRQ-50 (Roche/Cell Marque) pab AP (Protein Tech group) pab A363 (Cell Marque) pab CP 379 (Biocare) PAX8 antibodies towards C-terminal (most likely..): mab clone BC12 (Biocare) rmab clone ZR-1 (Abcam)

45 PAX8 antibodies towards N-terminal (most likely..): Fallopian tube secretory & ciliated cells Kidney epithelial cells lining the collecting tubules Thyroid epithelial cells lining the follicles Tonsil B-lymphocytes Pancreas neuroendocrine cells PAX8 antibodies towards C-terminal (most likely..): Fallopian tube secretory & ciliated cells Kidney epithelial cells lining the collecting tubules Thyroid epithelial cells lining the follicles

46 UPT II: PAX8 Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab MRQ-50* HIER high ph 1: step Ventana 3-step (OP) mab BC12* mab ZR-1 pab AP HIER high ph 1: step - - HIER high ph ** 1: step - - HIER high ph 1: step * Inferior performance on VMS stainer platform ** VMS: P3 4 min + HIER CC1 32M

47 WT1 reaction pattern (mab 6F-H2, HIER) ICAPCs ICAPCs Fal. tube A moderate to strong nuclear staining reaction of virtually all epithelial (and stromal ) cells. A weak to moderate cytoplasmic staining in many cells will be seen. Kidney A moderate to strong nuclear staining reaction in parietal epithelial cells and podocytes of the Bowman capsule... Tonsil No nuclear staining reaction of lymphocytes, endothelial cells etc.

48 WT1 reaction pattern (mab WT49 and 6F-H2, HIER+proteolysis) ICAPCs ICAPCs Fal. tube A moderate to strong nuclear staining reaction of virtually all epithelial (and stromal ) cells. Kidney A moderate to strong nuclear staining reaction in parietal epithelial cells and podocytes of the Bowman capsule. Tonsil No nuclear staining reaction of lymphocytes, endothelial cells etc.

49 Optimal but challening to read Optimal and easy to read Insufficient: - Too low titre - Short incubation time - (WT49 on VMS)

50 UPT III: WT1 Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab 6F-H2 HIER high ph 1: & 3-step Dako 2- & 3-step mab 6F-H2 mab WT1 HIER high ph + proteolysis* 1: step Ventana 3-step (OP) HIER high ph 1: step Leica 3-step * e.g. VMS: HIER in CC1 32 min + P3 for 4-8 min.

51 UPT Recommendable clones (conc.)* Less successful clones (conc.) RTU plug and play ** giving optimal result GATA3 mab L mab HG3-31 VMS: mab L Mamgl. GCDFP mab 304-1A5 rmab 31A5 mab 23A6 mab D6 rmab EP1582Y Dako: mab 304-1A5 VMS: rmab 31A5 Dako: mab 23A6 VMS: rmab EP1582Y * Potential to provide optimal result by a laboratory developed test (LDT) ** Using the protocol settings as recommended by the vendor incubation, retrieval, detection kit. *** Aalborg University Hospital data

52 UPT GATA3 Mamgl. GCDFP Positive tissue control HE* Kidney: Podocytes and epithelial cells of collecting tubules Breast: Columnar epith. cells of ducts Breast: Columnar epith. cells of ducts Positive tissue control LE** Tonsil: T-cells Skin: Columnar epith. cells of sweat glands Skin: Columnar epith. cells of sweat glands Negative tissue control NE*** Appendix: Columnar epithelial cells Appendix / tonsil: Columnar / squamous epithelial cells, etc Appendix / tonsil: Columnar / squamous epithelial cells, etc * HE = High expression ** LE = Low expression *** NE = No expression

53 GCDFP15 / Mammaglobin reaction pattern A moderate to strong, distinct cytoplasmic staining reaction in scattered ductal epithelial cells and in apocrine metaplastic cells. Breast Skin Tonsil A moderate to strong, distinct cytoplasmic staining reaction of the majority of the epithelial cells of the eccrine sweat glands No staining reaction should be seen.

54 GCDFP15 / Mammaglobin reaction pattern Antigen diffusion Carc. 4 Carc. 4 54

55 Local diffusion

56

57 Ins.: Omission of HIER and/or too low conc. RTU > Conc. (difficult to calibrate what is best control )

58 GATA3 reaction pattern ICAPCs Tonsil Kidney App. An at least weak nuclear staining reaction of the majority of T-cells in the T-zones in the tonsil. An at least moderate, distinct nuclear staining reaction of virtually all epithelial cells in collecting ducts and podocytes in glomeruli in the kidney. No staining reaction in epithelial cells should be seen.

59 Optimal result basis: mab L Careful calibration HIER high ph Insufficient result: RTU superior to LDT Titre of L Clone HG3-31

60 Breast panel: GCDFP15 & Mammaglobin Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab 23A3 HIER TE 1: step Dako 2- & 3-step mab D6 HIER TE 1: step - - rmab EP1582Y HIER TE 1: step Ventana 2- & 3-step mab HIER TE 1: & 3-step Dako 2-step mab 31A5 HIER TE - - Ventana 2-step rmab clone EP1582Y can show positive staining reaction in smooth muscle cells

61 Breast panel: GATA3 Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab L HIER TE 1: & 3-step Ventana 2- & 3-step GATA3: Highly sensitive for Breast carcinomas (& Urothelial carcinoma). But also seen in other neoplasias Lobular carc. Urothelial carc. Leiomyosarcoma

62 Questions???...

63 Markers for the UPT : Marker Protocol Control SOX10 BC34, SP267, EP268 Uroplakin II BC21 NKX3.1 EP353 HIER high ph HIER high ph HIER high ph Skin, appendix Bladder / Utrethra Prostate

64 SOX10 reaction pattern ICAPCs ICAPCs Skin A moderate to strong nuclear staining reaction of virtually all melanocytes (and myoepithelial cells of sweat glands) Appendix A moderate to strong nuclear staining of Schwann cells. Tonsil No staining reaction (apart from Schwann cells).

65 Melanomas x 200 SOX 10

66 Cerv. uteri, ad. carc. x 200 SOX 10 Ovarian ser. carc. x 200 SOX 10

67 Optimal result basis: mab BC34 rmab EP268 (rmab SP267) + 3-step system Insufficient result basis: pabs. 2-step systems

68 Uroplakin II reaction pattern Bladder A moderate to strong predominantly cytoplasmic staining reaction of the vast majority of umbrella cells. Bladder A moderate to strong predominantly cytoplasmic staining reaction of the vast majority of umbrella cells. Tonsil No staining reaction.

69 Uroth. carc. x 200 Uroplakin II Uroth. carc. x 200 GATA3

70 Lung. sq. carc. x 200 Uroplakin II Lung. sq. carc. x 200 GATA3

71 NKX3.1 reaction pattern Prostate A moderate to strong nuclear staining reaction of the vast majority of luminal epithelial cells. Prostate A moderate to strong nuclear staining reaction of the vast majority of luminal epithelial cells. Prostate A moderate to strong nuclear staining reaction of the vast majority of luminal epithelial cells..

72 NKX3.1 reaction pattern Testis A weak to moderate nuclear staining reaction of dispersed germ cells. Tonsil No staining reaction of the vast majority of cells. Dispersed squamous epithelial cells can be demonstrated. Appendix / Colon No staining reaction.

73 NKX3.1 reaction pattern Prostate ad. carc. Internal studies: Prostate ad. carc. Colon ad. carc of 18 prostate adenocarcinomas - in 39 of 39 other neoplasias 10% cut-off

74 UPT II: SOX10 Retrieval Titre Detection RTU Detection mab BC34 HIER high ph 1: step - - rmab EP268 HIER high ph 1: & 3-step - - rmab SP267 HIER high ph 1:25-50* - VMS 3-step UPT II: Uroplakin II* Retrieval Titre Detection RTU Detection mab BC21* HIER TE 1: step - - UPT II: NKX3.1* Retrieval Titre Detection RTU Detection rmab EP353* HIER TE 1: step - - *In-house data for best technical result

75 Markers for the UPT (Liver): Marker Protocol Control HEPA OCH1E5 Arginase SP156 Glypican 3 1G12 HIER high ph HIER high ph HIER high ph Liver Liver Placenta

76 HEPA reaction pattern ICAPCs Liver A moderate to strong granular cytoplasmic staining reaction in virtually all hepatocytes (mitochodria) No-go for biotin based detection systems. Appendix No staining reaction of lymhocytes, stromal cells, muscle cells and vast majority of epithelial cells.

77 Arginase reaction pattern ICAPCs Liver A moderate to strong nuclear and cytoplasmic staining reaction in virtually all hepatocytes). Appendix No staining reaction.

78 Arginase HEPA Hepatocelluar carcinoma & undifferentiated carcinoma

79 Glypican 3 reaction pattern ICAPCs ICAPCs Placenta An at least moderate predominantly cytoplasmic staining reaction of the vast majority of throphoblastic cells. Weak reaction in stromal and endothelial cells. Appendix / Colon An at least weak but distinct cytoplasmic staining reaction of peripheral nerves. Liver No staining reaction.

80 UPT II: HEPA Basic protocol settings for an optimal staining result (NQC) Retrieval Titre Detection RTU Detection mab OCH1E5* HIER 1: & 3-step * Less successful performance on Bond stainer Dako Ventana 2- & 3-step 2- & 3- step UPT II: Glypican 3 & Arginase* NordiQC data and In-house pre-liminary data* Retrieval Titre Detection RTU Detection mab 1G12 HIER 1: & 3-step Ventana 2- & 3-step rmab SP156 HIER TE 1: step - -