1 Immunohistochemical Diagnosis Of Human Alveolar Echinococcosis (HAE) With the Monoclonal Antibody Em2G11* Beate Grüner, MD Comprehensive Infectious Diseases Center (CIDC), University Hospital and Medical Center, Ulm, Germany Good morning, i am glad to be here and Id like to thank the organizers for the invitation, Im really glad to be here, and like to talk about New trends in the managment and treatment of Infection with E. multilocularis, the cause oh Human Alveolar Echinococcosis. What enables me, to talk to you about this field? 1Gruener, Beate, 2*Barth, Thomas FE, 2*Herrmann Tobias S, 3Tappe Dennis, 2Stark, Lorenz, 4Buttenschön, Klaus, 5Hillenbrand, Andreas; 6 Juchems, Markus, 5Henne-Bruns, Doris, 7 Kern, Petra , 8Seitz Hanns M, 2Moeller, Peter, 9†Rausch, Robert L, 1Kern, Peter; 10Deplazes, Peter *equally contributed. *published in: PLoS Negl Trop Dis. 2012;6(10):e1877

2 The puzzle of AE-DiagnosisClinical findings Medical History Imaging - with typical liver lesion PCR/ Histo- pathology Serology * Brunetti et al. / Acta Tropica 114 (2010) 1–16 Peter Kern: Clinical features and treatment of alveolar echinococcosis. Current Opinion in Infectious diseases 2010, 23:

3 Echinococcosis in Germany : Reports to the Robert-Koch-Institut CE Cystic Echinococcosis Echinococcosis is a rare diagnosis! The differential diagnosis is diffult for the pathologist due to the low frequency. AE Alveolar Echinococcosis First Id like to give you a quick overview of the diseases caused by echinococcocus spp. Inzidenz ~0,05 / (Süddeutschland) 0,02-2,4 / (Mitteleuropa) Bis 80/ in Zentralchina Lange Inkubationszeit (5-15 Jahre) Primärer Leberbefall Maligner Charakter Unbehandelt hohe Letalität Therapieindikation Chance der kurativen Resektion in ca. 30% Meist Benzimidazole langjährig On he right side AE caused by E. multi is a rare disease that only exists in the northern hemispere AE behaves in human like a malignant process with characteristic tissue destruction and invasion and the potency to form distant metastases According to the TNM-system for cancer a classification system was developed with the objective to help clinicians with therapy-decisions, prognostic-indication and assess therapy- results and simplify the exchange of information between different centers PNM - system for the Classification of Human Cases of Alveolar Echinococcosis was developed Caused by echi mult One the most life threatening helminthic disease Occurs by ingestion of contaminated food witg eggs of the of em from the feces of infected foxes, cats or dogs…ore from close contacts with these animals.the distribution of thi cestode is geografically restricted to the northern hemisphere, predominantly endemic areas: Alaska/Canada/ Zentral Europe, Siberia and Japan Literatur McManus Lancet 2003 Echinococcosis Non-differentiated Source Infektionsepidemiologisches Jahrbuch für 2012: 27. Juni 2013 – Infektionsepidemiologisches Jahrbuch meldepflichtiger Krankheiten für

4 The Study Aim: Improving diagnosis of AE on paraffin sectionsof infected human tissue by immunohistochemical testing of a specific antibody Methods: Paraffin blocks of 96 patients were investigated Standard staining procedures: Haematoxylin&eosin (H&E) and Periodic acid-Schiff (PAS) Immunohistochemistry / Immunocytology: using the mAb Em2G11 96 patients were included: according to medical records 47 with AE 49 with CE 12/96 with uncertain diagnosis according to standard histology The samples were first analysed by two pathologists (TFEB&TSH)consensus diagnosis. Barth TFE, Herrmann TS, Tappe D, Stark L, et al. (2012) Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11. PLoS Negl Trop Dis 6(10): e1877. doi: /journal.pntd

6 Em2G11 – a species specific monoclonal antibody to the Em2 antigen of Echinococcus multilocularis Controlls 2 tubes with antibody from Zurich; Prof. Dr. P. Deplazes Immunohistochemistry with mAbEm2G binding activity to the laminated layer E. multilocularis from gerbils (Meriones unguiculatus (PD Dr. D. Tappe, Universität Würzburg)

7 Immunohistochemical staining modalities of the monoclonal antibody (mAb) Em2G11 for metacestodes of Echinococcus multilocularis. 2mm! spems: Small mAb Em2G11- positive particles of E. multilocularis Immmunohistochemistry controls zB tissue achieved from paraffin samples caseous necrosis as: Tb, rheumatoid noduel, sarkoidodis, sarcoma, colon carcinoma… Positice controlls ware sections from AE tissue from gerbils, mongolian jird Em2: Major antigen of the laminated layer of E. multilocularis Barth TFE et al. (2012) PLoS Negl Trop Dis 6(10): e1877.

8 Immunohistochemical staining modalities of the monoclonal antibody (mAb) Em2G11 for metacestodes of Echinococcus multilocularis. A: E. multilocularis lesion in human liver The antigen is detected in the laminated layer and in the necrotic area around the lesion. Small particels of E. multilocularis , spems are detected 1,5 mm away from the main lesion and in the lymphatic tissue of a draining lymph node B: No staining in TB necrosis and bronchial epithel tissue C : Aspirate from the liver, PAS-staining C`: Aspirate from the liver, mAb Em2G11 D: Brain tissue, PAS staining D`: Brain tissue, mAb Em2G11 SPEMs Brain tissue of an Inuit, St. Lawrence Island, Alaska, operated in 1950, provided by Prof. R. Rausch, University of Washington Barth TF, et al. (2012) PLoS Negl Trop Dis 6(10): e1877.

9 The Study: Results Aim: Improving diagnosis of AE on paraffin sectionsof infected human tissue by immunohistochemical testing of a specific antibody Methods: Paraffin blocks of 96 patients were investigated Standard staining procedures: Haematoxylin&eosin (H&E) and Periodic acid-Schiff (PAS) Immunohistochemistry / Immunocytology: using the mAb Em2G11 96 patients were included: (according to medical records) 47 with AE 49 with CE 12/96 with uncertain diagnosis according to standard histology The samples were first analysed by two pathologists (TFEB&TSH)consensus diagnosis. In 6/12 diagnosis was conclusively adjusted by using immunohistology with mAb Em2G11 Barth TFE, Herrmann TS, Tappe D, Stark L, et al. (2012) Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11. PLoS Negl Trop Dis 6(10): e1877. doi: /journal.pntd

10 Results and ConclusionsImmunohistochemistry using the mAb Em2G11: allows a 100%- differentiation between E. multilocularis und E. granulosus. The mAb Em2G11-positive laminated layer is the crucial immunohistologic hallmark for the diagnosis of AE. Positive signals can also be found in the necrotic zone surrounding the main lesion and in draining lymph nodes. Spems (small mAb Em2G11-positive acellular particels of E. multilocularis, < 1µm); suggesting that the interface of infection is much larger than assumed. Diagnosis also on aspirates from liver lesions: „immunocytology“ The Em2 antigen is highly stable for long periods in formalin  retrospective analysis of archived tissue is possible . Barth et al. (2012) PLoS Negl Trop Dis 6(10): e1877.

11 T. Barth, Pathologie, Universität Ulm TT. Barth, Pathologie, Universität Ulm T. Herrmann, Pathologie, Universität Ulm L. Stark, Pathologie, Universität Ulm P. Möller, Pathologie, Universität, Ulm P. Deplazes, Institut für Parasitologie, Universität Zürich P. Kern, Innere Medizin III, Infektiologie, Universität Ulm P. Kern, Institut für Epidemiologie und Medizinische Biometrie, Universität Ulm D. Tappe, Bernhard-Nocht-Institut für Tropenmedizin, Hamburg HM Seitz, Institut für medizinische Parasitologie, Universität Bonn R. Rausch†, University of Washington W. Kratzer, Innere Medizin I, Zentraler Ultraschall, Universitätsklinikum Ulm T. Gräter, Diagnostische und Interventionelle Radiologie, Universitätsklinikum Ulm Hillenbrand, Klinik für Allgemein-und Viszeralchirurgie, Universitätsklinikum Ulm D. Henne-Bruns, Klinik für Allgemein-und Viszeralchirurgie, Universitätsklinikum Ulm Beer, Klinik für Nuklearmedizin, Universitätsklinikum Ulm J. Bampoe-Addo; Klinik für Nuklearmedizin, Universitätsklinikum Ulm

12 The puzzle of AE-DiagnosisPossible versus probable versus confirmed case* Possible case. Any patient with clinical and epidemiological history and imaging findings or serology positive for AE. Probable case. Any patient with clinical and epidemiological history, and imaging findings and serology positive for AE with two tests Confirmed case. The above, plus (1) histopathology compatible with AE and/or (2) detection of E. multilocularis nucleic acid sequence(s) in a clinical specimen. Clinical findings Medical History Imaging - with typical liver lesion PCR/ Histo- pathology Serology * Brunetti et al. / Acta Tropica 114 (2010) 1–16 Peter Kern: Clinical features and treatment of alveolar echinococcosis. Current Opinion in Infectious diseases 2010, 23:

13 Immunohistochemistry as a part of the diagnostic AE-puzzle?Discussion: And now- Immunohistochemistry as a part of the diagnostic AE-puzzle? First presentation of AE-patients in Ulm 63 new AE patients 26 surgery 12/26 surgery in Ulm Em2G11 AE-confirmation 10/63 biopsy/ partial resectionEm2G11 confirmation In > 22 patients Immunihstochemistry Em2G11 used 10 new AE patients 5 with typical lesions (AE diagnosis „probable“) 5 conventional histology/ 4 Em2G11 immunohistochemistry 01/ /2014 >>27 samples investigated by immunohistochemistry mAb Em2g11 from patients seen in Ulm spezialized outpatient center

14 The Study Aim: Improving diagnosis of AE on paraffin sectionsof infected human tissue by immunohistochemical testing of a specific antibody Table: Patients characteristics and localisation of probe First Id like to give you a quick overview of the diseases caused by echinococcocus spp. Inzidenz ~0,05 / (Süddeutschland) 0,02-2,4 / (Mitteleuropa) Bis 80/ in Zentralchina Lange Inkubationszeit (5-15 Jahre) Primärer Leberbefall Maligner Charakter Unbehandelt hohe Letalität Therapieindikation Chance der kurativen Resektion in ca. 30% Meist Benzimidazole langjährig On he right side AE caused by E. multi is a rare disease that only exists in the northern hemispere AE behaves in human like a malignant process with characteristic tissue destruction and invasion and the potency to form distant metastases According to the TNM-system for cancer a classification system was developed with the objective to help clinicians with therapy-decisions, prognostic-indication and assess therapy- results and simplify the exchange of information between different centers PNM - system for the Classification of Human Cases of Alveolar Echinococcosis was developed Caused by echi mult One the most life threatening helminthic disease Occurs by ingestion of contaminated food witg eggs of the of em from the feces of infected foxes, cats or dogs…ore from close contacts with these animals.the distribution of thi cestode is geografically restricted to the northern hemisphere, predominantly endemic areas: Alaska/Canada/ Zentral Europe, Siberia and Japan Literatur McManus Lancet 2003 Barth TFE, Herrmann TS, Tappe D, Stark L, et al. (2012) Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11. PLoS Negl Trop Dis 6(10): e1877. doi: /journal.pntd

15 Table 3. Cases with difficult histological/cytological diagnoses.Barth TFE, Herrmann TS, Tappe D, Stark L, et al. (2012) Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11. PLoS Negl Trop Dis 6(10): e1877. doi: /journal.pntd Table 3. Cases with difficult histological/cytological diagnoses.

16 Fallbericht: Inuit, St. Lawrence Island, AlaskaProf. R. Rausch University of Saskatchewan University of Washington School of Public Health's Department of Pathobiology and the School of Medicine's Department of Comparative Medicine Fallbericht: Inuit, St. Lawrence Island, Alaska

18 AE: Atypical cases 2014 RF *1972: CT MRTSeite 18 MRT MR* FDG-PET CT: 2 lesions in the right liver lobe RF *1972: CT LT: PET-CT Images: courtesy Prof. Dr. A. Beer, Nuklearmedizin