Lessons from a consultation practice

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1 Pitfalls in the Application of Immunohistochemistry in Diagnostic Pathology Lessons from a consultation practice Kevin O. Leslie, MD Professor and Consultant Mayo Clinic Arizona Scottsdale, Arizona

2 Presenter Disclosures Kevin O. Leslie, MD Personal financial relationships with commercial interests relevant to this presentation during the past 12 months: Personal financial relationships with non-commercial interests (e.g., government or other nonprofit funding) relevant to this presentation, within past 12 months: Relevant institutional financial interests Personal financial relationships with tobacco industry entities within the past 3 years: No Disclosures

3 Mayo Clinic

4

5

6 The Territory Ahead Introduction The critical role of sample size and quality Tumors can be triaged by pattern Not all antibodies are created equal Top 10 Pitfalls you can avoid

7 Why is this presentation useful? Today, immunohistochemistry (IHC) is fully integrated into contemporary diagnostic pathology Technical challenges of the past have been largely eliminated by commercialization and automation The expertise of application is highly variable in practice

8 Why is this presentation useful? Problems in selection of antibodies and interpretation of staining results play a major role in the consultation practice A limited set of recurrent pitfalls emerge Examining these in detail may help us hone our individual mastery of this broad and complex ancillary technique in our daily practice

9 A word about cost containment The reality of contemporary IHC is that multiple antibody determinations are the rule. The trick is to avoid wasteful avenues of investigation. Today, it is becoming progressively difficult to identify proliferative histopathological lesions that rely on routine morphology alone!

10 AAH Metaplasia PB AdenoCa+BAC Metaplasia PB Metaplasia PB AdenoCa BAC AAH AdenoCa+BAC AdenoCa+BAC AdenoCa BAC

11 A word about cost containment The reality of contemporary IHC is that multiple antibody determinations are the rule. The trick is to avoid wasteful avenues of investigation. Today, it is becoming progressively difficult to identify proliferative histopathological lesions that rely on routine morphology alone! So, for all of those cases where we must rely on IHC, we should try to use a consistent strategy

12 IHC is most helpful when it Clarifies the phenotype of a neoplasm or pathogen Clarifies the origin of a metastatic tumor Clarifies tumor behavior, and possibly therapy Clarifies patient prognosis Clarifies cellular relationships

13 Case example Case courtesy of Dr. Fuad Al Dayal, Saudi Arabia A 10 year old boy presented with a hemorrhagic left pleural effusion. The child is hearing and speech impaired. The past medical history is significant for recurrent lung infections, recurrent hemorrhagic pleural effusions and a cerebrovascular accident. KOL03

14 KOL03

15 A surgical wedge lung biopsy was performed KOL03

16 KOL03

17 KOL03

18 KOL03

19 EMA KOL03

20 Factor VIII KOL03

21 TTF-1 KOL03

22 ASMA KOL03

23 MIB1 KOL03

24 Diagnosis Diffuse Pulmonary Lymphangiomatosis KOL03

25 The Territory Ahead Introduction The critical role of sample size and quality

26 The critical role of sample size There is a natural tendency for clinicians to expect more and more information from smaller and smaller biopsy samples. Case example: A 66 year old woman is found to have a 3 cm lung mass. A transbronchial biopsy is performed

27 You decide the included cell group is malignant, and nonsmall cell. and if it is please send for Her2 You attempt IHC to confirm lung origin. Results: TTF-1 neg, CK 7 pos neu. Oh, and if it is a lung cancer, we CK20, synapto, chromogranin, and P63 insufficient tumor in the recuts need to know if it is squamous. If not please send for EGFR After signing the case out as nonsmall cell carcinoma, the clinician calls to ask if it could be from her breast cancer analysis.

28 The Territory Ahead Introduction The critical role of sample size and quality Neoplasms can be triaged by pattern

29 A practical approach Neoplasms are the largest source of targets for diagnostic IHC 4 general morphological categories emerge Neoplasms of lymphoid cells (hematolymphoid) Neoplasms with organoid features Neoplasms with spindled features Neoplasms with undifferentiated epithelioid cells

30 Lymphoid Purpose: Confirm hematolymphoid, confirm neoplastic; subclassify The panel (s) (first consult a local hematopathologist!) CD45 CD20 CD3 CD43 Kappa and lambda (if cytoplasmic) BCL2 (if nodular) Unstained slides (many) CD5 CD10 Cyclin D1 CD79a CD138 CD30 CD15 Myeloperoxidase CD68 Lysozyme S100 protein CD21/CD35 Histiocytic and dendritic cell

31 A 47 year old man presents with weightloss, malaise and abdominal pain. Imaging shows multiple enlarged lymph nodes. A needle core biopsy is performed Another DDX: H&E Immunopanel: Lymphoma Carcinoma Diagnosis CD30 Melanoma CD3 CD45 CD20 CD3 CD43 Kappa and lambda Anaplastic large cell lymphoma, ALK-1 Other? negative (see discussion) CD43, kappa, lambda ALK-1CD20 LCA CD30 ALK-1 LCA

32 The panel CK7/20 Synaptophysin TTF-1 CDX2 PSA Organoid Purpose: Determine primary origin of metastasis, guide therapy Pattern assists general localization Neuroendocrine carcinoma, paraganglioma Lung and thyroid, other small cell Intestinal adenos, other mucinous, endometrioid Prostate, breast, salivary, sweat gland ca, ER/PR --other adeno, melanoma Breast, ovary, endometrium. ER in some lung, stoma and thyroid adenos Calret-CK5/6-WT1 Combined specificity for mesothelioma

33 A 62 year old patient with back pain is found to have a lytic lesion involving T11. A core biopsy is performed DDX: Diagnosis Metastatic adenocarcinoma Metastatic adenocarcinoma consistent with lung (TTF1) or breast (ER) origin of unknown primary origin panck and CK7 CK20 panck CK20 CDX2 TTF1 or and ER! CK 7 TTF1 or ER!

34 The panel Spindled cells Purpose: Determine primary origin/ phenotype, guide therapy PanCK S100 protein Defines epithelial phenotype, spurious in sarcomas, melanoma, dendritic cells. plasma cells Melanocytic, neural, myoepithelial, histiocytic/dendritic, Langerhans cells, liposarcoma, chondrosarcoma Melan-A Desmin CD99 CD31/34 Melanocytic, pre-melanosomes, adrenocortical, sex-cord ovarian Myogenous tumors, PNETs, epithelioid sarcoma Don t forget ER/PR for metastatic Lymphoblastic lymphoma, synovial sarc, EWS/PNET spindled cell tumors in women! Vascular tumors (CD34 better for KS); LFT/SFT (CD34) CD117 GIST

35 A 49 year old woman presents with chest pain and cough.. A 10 cm tumor is identified and removed from the RLL. DDX: Sarcomatoid Ca Primary sarcoma Diagnosis: S100, Desmin, CD117, ER Metastatic sarcoma Localized fibrous tumor Sarcomatoid mesothelioma CD34 + BCL2 Nerve sheath tumor Localized fibrous tumor CD34 + BCL2 S100, Desmin, CD117, ER

36 The panel Undifferentiated epithelioid Purpose: Determine primary origin of metastasis, guide therapy Pan CK S100 protein Synaptophysin CD45 ER/PR (female) Defines epithelial phenotype Melanocytic, neural, myoepithelial, histiocytic/dendritic, Langerhans cells Neuroendocrine cells Hematolymphoid cells Breast, ovary, endometrium. ER in some lung, stomach, and thyroid adenos

37 A 61 year old man presents with a large left axillary mass. His past medical history is remarkable for a previously resected skin appendage tumor from the left hand (said to have been a malignant poroma outside hospital). DDX The Panel panck Inflammatory Diagnosis: panck pseudotumor S100 protein K Lymphoma Metastatic LCA carcinoma Plasma cell Synaptophysin myeloma Melanoma SYN, LCA. Kappa S100 + lambda L panck

38 The Territory Ahead Introduction The critical role of sample size and quality Neoplasms can be triaged by pattern Not all antibodies are created equal

39 Who to trust. Sensitivity and specificity issues Example: synaptophysin and chromogranin Certain antigens in tissue are more resistant to fixation, processing, and tissue degredation Example: panck versus S100 protein The utility and specificity of some antibodies requires context Example: CD30 in ALCL versus carcinoma, or melanoma!

40 A 71 year old man, smoker, is found to have a large central lung mass. A transbronchial biopsy is performed TTF-1 and MIB-1 TTF-1 and MIB1 Small Cell Carcinoma

41 The Territory Ahead Introduction The critical role of sample size and quality Neoplasms can be triaged by pattern Not all antibodies are created equal Top 10 Pitfalls you can avoid

42 TOP TEN PITFALLS IN IHC 10. Incorrect panel of antibodies

43 A 57 year old woman presents with an enlarged groin lymph node. She has a history of node + breast cancer. A needle core biopsy is performed DDX: MelanA Diagnosis: Carcinoma Lymphoma Metastatic melanoma, amelanotic Melanoma Paraganglioma Sarcoma panck, HMB45, EMA, CK7, CK20 LCA S100 Protein LCA panck, HMB45, EMA, CK7, CK20 S100 Protein

44 A 26 year old African woman presents to the emergency room with cough and chest pain, 1 month after delivering a healthy baby. Bronchoscopy yields this biopsy PanCK Synapto, CD31 CD34, TTF1, Melan-A DDX Carcinoid Melanoma Sarcoma Sarcomatoid Ca S100 Vascular PanCK Smooth muscle IMFT CD31

45 TOP TEN PITFALLS IN IHC 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

46 Some diagnoses require a combination of IHC results for validity A 72 year old man, long time smoker, presents with right sided chest pain The and surgeon breathlessness. finds diffuse thickening of the pleura without a definite mass in underlying Imaging lung. reveals He a feels right the pleural changes are quite effusion. typical The for mesothelioma underlying lung in is his not experience well visualized. He gives a history of asbestos exposure. A VATS procedure obtains tissue

47 Some diagnoses require a combination of IHC results for validity A limited battery of IHC stains is performed, including calretinin CALRET A diagnosis of malignant mesothelioma, epithelial type. The family swears that the patient had no asbestos exposure CK 5/6 and requests that the biopsy be sent out for review BerEP4 B72.3 CALRET WT-1 BerEP4 CALRET B72.3

48 TOP TEN PITFALLS IN IHC 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

49 Thyroidectomy from a young patient with Hashimoto thyroiditis

50 TOP TEN PITFALLS IN IHC 7. Incorrect histopathological DDX 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

51 A 37 year old man presents with chest discomfort and is found to have several nodular lung lesions. A VATS biopsy is performed DDX: The case was sent out for consultation. The lesion was recognized as epithelioid hemangioendothelioma and confirmatory IHC was performed Carcinoma Mesothelioma Melanoma PanCK Chordoma TTF1 TTF1 CD34/CD31 PanCK Hamartoma? CD34/CD31

52 TOP TEN PITFALLS IN IHC 6. Undue pressure on speed of diagnosis 7. Incorrect histopathological DDX 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

53 A 22 year old college student notices a persistent swelling above her right knee. Imaging reveals involvement of the distal femur and additional lytic bone lesions are present. She is brought into the hospital for a percutaneous needle core biopsy She is the only daughter of the Chief of Surgery.

54

55 DDX: Ewing sarcoma/pnet Large cell lymphoma Melanoma of soft parts Epithelioid sarcoma Other? Recommended panel panck LCA S100 protein Synaptophysin 8 unstained Their panel Vimentin panck TdT CD99

56 DDX: Ewing sarcoma/pnet Large cell lymphoma Melanoma of soft parts Epithelioid sarcoma Other? Recommended panel panck LCA S100 protein Synaptophysin 8 unstained

57 DDX: Ewing sarcoma/pnet Large cell lymphoma Melanoma of soft parts Epithelioid sarcoma Other? Secondary panel CD20 CD3 MIB1 Final Diagnosis: Malignant lymphoma, diffuse large B-cell type, CD20 positive.

58 TOP TEN PITFALLS IN IHC 5. Overconfidence in the value of IHC 6. Undue pressure on speed of diagnosis 7. Incorrect histopathological DDX 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

59 A 52 year old man has a serum PSA drawn during a routine physical exam. This results in sextant needle core biopsies

60 TOP TEN PITFALLS IN IHC 3. Relying on tissue from another lab 4. Relying on the IHC of another lab 5. Overconfidence in the value of IHC 6. Undue pressure on speed of diagnosis 7. Incorrect histopathological DDX 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

61 Lymphoid Case in point A 65 year old man presents with a soft tissue mass adjacent to his clavicle and eroding bone. A biopsy is performed. VIM After a first round of IHC the diagnosis remained uncertain and the case was sent for consultation. LCA panck, S100 VIM LCA panck, S100

62 Lymphoid Case in point We restained the tissue block in our laboratory with the following results panck, S100, melana Repeat LCA Our diagnosis: Diffuse large B-cell lymphoma, CD20 positive. CD20 panck, S100, melana Repeat LCA CD20

63 From tissue acquisition to coverslip on your IHC slide, there are so many potential areas for mishap that it is remarkable how frequently IHC is successful! Crush injury Antigen retrival problems Delay in fixation IHC technical failures Improper fixative -reagent sequence Processing damage -incomplete slide flooding Overheating in paraffin -poor humidity control Infiltration contaminants -pipetting inaccuracy Rehydration damage -outdated reagents Poor section adherence -poor antibody quality Incomplete deparaffinization of sections

64 A 48 year old woman present with leg pain and is found to have a cystic lesion in the proximal tibia. Curettings of the lesion are performed at another hospital and a frozen section is requested by the surgeon. SYN

65

66 TOP TEN PITFALLS IN IHC 2. Not knowing the expected staining pattern 3. Relying on tissue from another lab 4. Relying on the IHC of another lab 5. Overconfidence in the value of IHC 6. Undue pressure on speed of diagnosis 7. Incorrect histopathological DDX 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

67 Before ordering, check the expected positive staining reaction! All of these are NUCLEAR stains! ER MyoD1 P63 Ki67 PR WT-1 P53 FLI-1 TTF-1 TDT PCNA Myogenin CDX-2 Cyclin D1

68 TOP TEN PITFALLS IN IHC 1. Not recognizing the histopathology 2. Not knowing the expected staining pattern 3. Relying on tissue from another lab 4. Relying on the IHC of another lab 5. Overconfidence in the value of IHC 6. Undue pressure on speed of diagnosis 7. Incorrect histopathological DDX 8. Excessive panel of antibodies 9. Incomplete panel of antibodies 10. Incorrect panel of antibodies

69 A 66 yr old man presents to the emergency room with left sided chest pain and is found to have a large left pleural effusion. His past medical history is remarkable for left sided pneumonia 3 months earlier for which he was hospitalized and treated empirically with broad spectrum antibiotics. Calret, CK5/6, TTF-1. CK,7, CK20, CEA all negative PanCK Our diagnosis: PanCK WT-1 Malignant mesothelioma, desmoplastic type Calret, CK5/6, TTF-1. CK7. CK20, CEA all neg WT-1

70 The Trail Behind Introduction The critical role of sample size and quality Tumors can be triaged by pattern Not all antibodies are created equal Top 10 Pitfalls you can avoid

71 Questions?

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