A major study of the use of immunohistochemistry to distinguish adenocarcinoma from mesothelioma was published in the April 2006 issue of Modern Pathology by PhenoPath Laboratories pathologists Todd S. Barry, Harry Hwang, Steven Kussick, and Allen M. Gown, in conjunction with pathologists Hector Battifora, Carlos Bacchi, and Hadi Yaziji, and the statistician Martin W. McIntosh. While there have been numerous previous attempts to define the immunophenotype of mesothelioma, in this study the most up-to-date panel of mesothelial and adenocarcinoma markers was employed, combined with a unique statistical method called logic

regression analysis. The latter was used to determine which combinations of the positive and negative markers of mesothelioma were most powerful in separating these tumors. The most powerful discriminatory markers were found to be calretinin, a positive marker of mesothelioma, and the antibodies Bg8 and MOC-31, which identify glycoproteins that are positive markers of adenocarcinoma. The conclusions of this paper have important diagnostic implications, as many laboratories continue to use antibodies in this clinical setting (e.g., B72.3 and antibodies to CEA) that do not have the high discriminatory properties of antibodies to calretinin, and the antibodies Bg8 and MOC-31.

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T H E N E W S L E T T E r O F P H E N O PAT H L A b O r AT O r i E S

Evaluation of 12 antibodies for distinguishingepithelioid mesothelioma from adenocarcinoma:identification of a three-antibodyimmunohistochemical panel with maximalsensitivity and specificity

Hadi Yaziji1, Hector Battifora2, Todd S Barry3, Harry C Hwang3, Carlos E Bacchi4,Martin W McIntosh5, Steven J Kussick3 and Allen M Gown3

1Ancillary Pathways, Miami, FL, USA; 2Pathology Consultation Services, Arcadia, CA, USA; 3PhenoPathLaboratories and Immunohistochemistry and Molecular Pathology Research Institute of Seattle, Seattle,WA, USA; 4Consultoria em Patologia, Botucatu, Brazil and 5Fred Hutchinson Cancer Research Center,Seattle, WA, USA

We evaluated the sensitivity and specificity of 10 monoclonal and two polyclonal antibodies for distinguishingepithelioid mesothelioma from adenocarcinoma (AdCA) using immunohistochemistry (IHC). The antibodieswere directed against the mesothelial-associated antigens mesothelin, calretinin, cytokeratin 5, thrombomo-dulin, Wilms’ tumor-1 (WT-1) gene product and HBME-1, and the nonmesothelial antigens Lewis-Y blood group(antibody BG8), MOC-31, BerEp4, CD15, and carcinoembryonic antigen (CEA) family. The 133 tumors evaluatedincluded 65 malignant epithelioid mesotheliomas, 22 lung AdCAs, 27 ovarian serous carcinomas, 24 breastcarcinomas, and five gastric carcinomas. Diagnoses were based on clinical, histologic, ultrastructural, and/orIHC findings. Calretinin had the best sensitivity for mesothelioma (95%), followed by HBME-1 (84%), WT-1 (78%),cytokeratin 5 (76%), mesothelin (75%), and vimentin and thrombomodulin (68%). Thrombomodulin had the bestspecificity for mesothelioma (92%), followed by cytokeratin 5 (89%), calretinin (87%) vimentin (84%), and HBME-1 (45%). When ovarian carcinomas were excluded from the analysis, the specificity of mesothelin and WT-1 forthe diagnosis of mesothelioma increased to 90 and 81%, respectively. The sensitivity of the nonmesothelialantigens for AdCA was organ dependent, with BG8 performing best in the breast cancer group (96%), andBerEp4, BG8, MOC-31 performing best in the lung cancer group (100%). The specificity of the nonmesothelialantigens for AdCA was 98% for BG8 and CEA, 97% for CD15, 95% for BerEp4, and 87% for MOC-31. A novelstatistical analysis technique employing logic regression analysis identified a three-antibody immunohisto-chemical panel including calretinin, BG8, and MOC-31, which provided over 96% sensitivity and specificity fordistinguishing epithelioid mesothelioma from AdCA.Modern Pathology (2006) 19, 514–523. doi:10.1038/modpathol.3800534

Keywords: mesothelioma; immunohistochemistry; logic regression; pleura; asbestos; calretinin

The histologic distinction between malignantepithelioid mesothelioma (MEM) and adenocarci-noma (AdCA) is often difficult and requires ancil-lary studies. For many years, electron microscopywas the preferred ancillary method in this differ-ential diagnosis. Typical ultrastructural features ofMEM include long slender microvilli devoid of

gylocalyx over much of the cell membrane, andaberrant microvilli projecting through deficienciesin the basal lamina.1,2 However, these features areoften absent in the less-differentiated MEM.

The development of antibodies to molecularmarkers of tissue differentiation has enabledimmunohistochemistry (IHC) to supplant electronmicroscopy in distinguishing MEM from AdCA.Initially, antibodies to antigens commonly ex-pressed by AdCA, such as carcinoembryonic antigen(CEA), LeuM1 (CD15), BerEp4, and B72.3 were usedto distinguish MEM from AdCA. Thus, the diagnosisof MEM was based on the absence of expression of

Received 28 June 2005; revised 28 September 2005; accepted 29September 2005

Correspondence: Dr H Yaziji, MD, Ancillary Pathways, PO Box43-0777, Miami, FL 33243-0777, USA.E-mail: ancillarypath@mac.com

Modern Pathology (2006) 19, 514–523& 2006 USCAP, Inc All rights reserved 0893-3952/06 $30.00

www.modernpathology.org

H&EH&E of a pleural-based tumor shows a malignant epithelial tumor with mesothelioma in the differen-tial diagnosis.

calretinin WT-1 podoplaninThe tumor is positive for expression of calretinin (nuclear and cytoplasmic) and WT-1 (nuclear), two positive mesothelial markers which featured prominently in our published study. PhenoPath Laboratories is also now offering antibodies to podoplanin (previously known as the D2-40 antigen), a highly sensitive mesothelial marker identified after submittal of our study.

PhenoPath Pathologists Publish Major Mesothelioma Study

Modern Pathology 19:514-523, 2006

PhenoPath Laboratories offers many services of particular interest to dermatopathologists, assisting them with ancillary services essential to arriving at an accurate diagnosis.

CD34Strong, uniform immunostaining with antibodies to CD34, characteristic of a dermatofibrosarcoma protuberans.

Desmin Prominent immunoreactivity with antibodies to desmin in a cutaneous leiomyosarcoma.

The differential diagnosis of Spindle Cell Lesions of the Skin generally includes spindle cell carcinoma, melanoma, dermatofibroma, dermatofibrosarcoma protuberans, leiomyoma, etc., and immunohistochemical studies can be quite useful in ruling in or out most of these diagnoses. As usual, the approach at PhenoPath Laboratories is not “one size fits all” and we recognize that the histology and clinical context will often restrict the differential diagnosis. We take a very selective approach to the application of antibody panels in this (and all other) settings.

Hematolymphoid Processes in the Skin can pose particularly difficult diagnostic problems given their protean histologic appearances, the considerable morphologic overlap between benign and malignant processes in the skin, and the rapidly evolving nomenclature and classification schemes for lymphomas presenting in the skin. PhenoPath Laboratories has on staff two board-certified hematopathologists, Dr. Todd Barry and Dr. Steven Kussick, with extensive expertise in the evaluation of these processes. This evaluation frequently requires the application of immunohistochemical studies employing antibodies to lymphoid subsets (e.g., B and T cell-associated markers) and/or myelomonocytic cells, mast cells, and Langerhans cells. Flow cytometric and/or gene rearrangement studies can also be of assistance in this clinicopathologic setting. All of these specialized testing services are provided at PhenoPath.

Direct Immunofluorescence Studies looking for the presence of deposits of immunoglobulin and/or complement in specific locations within a skin or mucosal biopsy can play an important role in the identification of selected skin disorders. For example, granular deposition of IgA at the dermal-epidermal junction is the sine qua non of dermatitis herpetiformis, whereas the presence of linear IgG deposits in a ‘chicken wire’ pattern within the epidermis is characteristic of the pemphigus family of disorders. PhenoPath Laboratories’ pathologists have more than two decades of experience reading skin immunofluorescence studies.(Note: these studies require fresh tissue that should be sent in ammonium sulfate-based transport media; please contact PhenoPath Laboratories and we will gladly ship media to you.)

Specialized Dermatopathology ServicesPhen

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These images demonstrate a very unusual case of ALK-positive anaplastic large cell lymphoma (ALCL) presenting in the skin. The ALK positivity raises a concern that this represents a cutaneous manifesta-tion of a systemic ALCL.

ALK CD30

Sher yl Ferrero Billing Lead par excellence, Sheryl joined PhenoPath Laboratories in 1998, one year after its inception. Many of us regard overseeing all aspects of coding, reimbursement and collection as one of the most challenging jobs at the company; dealing with patients who are not only ill but also facing potentially large financial bur-dens requires skill, patience, good judgment and compassion. With over 10 years of medical billing experience, Sheryl works tirelessly to help clients and patients understand the intricacies of medical billing and how to navigate today’s complex insurance systems for the maximum benefit of the patient. In the past year, Sheryl has also been instrumental in the implementation of PhenoPath’s new medical billing system. Should you have any questions or concerns regarding PhenoPath’s medical billing policies or procedures, we encourage you to contact Sheryl directly at 206-374-1480 or toll free, at866-927-4366.

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Breast Pathology: Current Concepts and Controversies June 5-6, 2006 The Fairmont Copley Plaza Hotel, Boston, MA Beth Israel Deaconess Medical Center, Department of Pathology

Dr. Allen M. Gown is a featured speaker at the Breast Pathology: Current Concepts and Controversies course to be held June 5-6, 2006 at the Fairmont Copley Plaza Hotel in Boston, MA. Dr. Gown will give the following presentations:

Monday, June 5 11:30 AM – 12 PM Special Lecture: Current Status of ER/PR Testing 1:15 PM – 1:45 PM Special Lecture: Current Status of HER2 Testing

For additional information about this meeting, please visit http://cme.hms.harvard.edu/.

See our pathologists at the following upcoming meetings:

2006 ASCO Annual Meeting, June 2-6, 2006Georgia World Congress Center, Atlanta, GeorgiaAdvocating Survivorship, Clinical Science, & Oncology Quality Care

Dr. Gown will present a poster entitled “Multivariate analysis of epres-sion of the microtubule-associated protein, tau, predicts improved progression free and overall survival in patients with metastatic HER-2-negative breast cancers treated with docetaxel and vinorelbine plus filgrastim” on Saturday, June 3, 2006 from 8:00 AM to 12:00 PM in Building B, Level 4, Room B401. Check our website www.phenopath.com for further updates. For additional information about this meeting, please visit http://www.asco.org/.

PhenoPath’s requisition form has been expanded PhenoPath’s requisition form has been expanded. The back of the form now includes a listing of antibodies for flow cytometry and immunohistochemistry, sorted by diagnostic setting. In addition, in response to your feedback, we have removed the requirement for the ordering physician’s signature. If you have any questions regarding the new form or would like to obtain forms with your institution’s name, address, phone number, and fax number preprinted, contact a member of our Client Services Staff at 888-92-PHENO (888-927-4366) or e-mail lab@phenopath.com.

Name (Last, First,MI)

Social Security # DOB Gender Male Female

Address

( )

PhoneInpatient Outpatient Non-Hospital Patient

Medical Record # Patient #

PATIENT INFORMATION

SPECIMEN INFORMATION

IMMUNOFLUORESCENCE (IF)

CLINICAL HISTORY/CLINICAL QUESTION

Collection Date Collection Time

Tissue Source(s) A) B) C) D)

Specimen ID #(s) A) B) C) D)

Multiple specimen submitted: Test all Select best block

Paraffin: Tissue block Cell blockFormalin Bouins B-5 Prefer Other

Slides: Unstained # Stained #

Smears: Air-dried Fixed Stained: # H&E Diff Quik Wright PAP

Bone marrow: green top(s) purple top(s) otherBlood: green top(s) purple top(s) other

Name of person completing form:

Date: Phone:

Skin IF (Direct) Serum IF (Indirect) Other (specify) Differential Diagnosis

PCR (GENE REARRANGEMENT STUDIES)

B cell (IgH) T cell (TCR-γ)

BREAST IHC STUDIES

ER/PR ER only p53 MIB-1HER-2/neu by IHC (and in addition): If HER-2/neu by IHC is indeterminate (2+), run FISH

Myoepithelial markers to r/o invasive carcinomaIf invasive CA, run the above selected markers:Regardless, run the above selected markers:

E-cadherin to differentiate ductal from lobular CAR/O basal-like breast CA

LEVEL OF SERVICE (REQUIRED)

Perform & interpret tests determined medically necessary by PhenoPath pathologist(s) Perform & interpret only test(s) as requested below

INSTITUTIONAL INFORMATION

Institution Address

Ordering Physician UPIN #

( ) ( )Phone FAX

Treating Physician UPIN #

( ) ( )Phone FAX Send copy of report

Expe r t i s e , I nnova t i on and Exce l l enceL A B O R A T O R I E SPhenoPath lab@phenopath.com

www.phenopath.com551 N. 34th Street, Suite 100Seattle, WA 98103

Toll-free: (888) 92-PHENOP (206) 374-9000F (206) 374-9009

PhenoPath pathologists, based on their medical judgement, will select antibodies that arenecessary to answer your clinical question.

Met CA of unknown primary Pancreatic endocrine neoplasm Undifferentiated malignant neoplasm Pituitary neoplasm Hematolymphoid marker studies R/O prostate CA Spindle cell tumor/sarcoma subtyping Small round cell tumor Adeno CA vs. mesothelioma Spindle cell dermal tumor R/O and/or subtype germ cell tumor Sentinel lymph nodes Neuroendocrine neoplasm R/O occult met CA Amyloid type analysis R/O occult met melanoma R/O hydatidiform mole Microsatellite instability

Provisional/Differential Diagnosis to be addressed by IHC

IMMUNOHISTOCHEMISTRY (IHC) - NON-BREAST

BONE MARROW MORPHOLOGY (CBC/WBC differential results required)

Comprehensive morphologic evaluation CBC/WBC DIFFERENTIAL RESULTS (Please Complete) Hgb MCV HCT WBC RBC PLT

%Neutrophils %Lymphocytes %Monocytes

BILLING INFORMATION (Must be completed or Institution will be billed) Attach copy of Insurance Card

BILL: Patient/Insurance Medicare Medicaid (WA DSHS only) Institution

Referral/Authorization # ICD-9 (required for other than institution billing)

Medicare #Advance Beneficiary Notice Yes (provide copy) No

Healthplan

Address

Policy/Cert # Group/Plan #

Employer’s Name

Name of Insured

Relationship to insured

Secondary Insurance Yes (Please attach separate sheet) No

Send me more: Req Forms Airbills PhenoBoxes Flow Media (RPMI) IF Media (Michel’s) Other (specify)

Diagnosis under consideration: CML/MPDLymphoma/Chronic lymphoproliferative disorder MDS

B cell T cell ALLPlasma cell dyscrasia B cell T cellAML Other

FLOW CY TOMETRY

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

HER-2/neu/Breast CA Inv(16), t(16;16) AML (FAB M4)SYT Syn Sarcoma 11q23/MLL AML, ALLEWSR PNET/Ewing Sarcoma t(9;22) BCR/ABL CML, ALL1p/19q Oligodendroglioma t(14;18) Follicular lymphomaX,Y (BMT) c-MYC Burkitt lymphomat(8;21) AML (FAB M2) t(11;14) Mantle cell lymphomat(15;17) AML (FAB M3) t(11;18) MZBCL/MALT lymphoma

Last updated: 16MAR06

For the most up-to-date listing of tests, see our website: www.phenopath.com

All of the above listed tests can be performed on paraffin-embedded tissue sections, with the following exceptions:1. CD52 and skin IF tests require fresh tissue specimens2. Hematolymphoid tests – can be run by either IHC or flow, except as follows:

* = Performed by Flow cytometry only and require fresh specimens+ = Performed by IHC only and can be performed on paraffin-embedded tissue sections

CarcinomaCD10 (CALLA)CDX2CEA (CD66E)CEA FAMILY (CD66)Chorionic gonadotropinChromogranin ACytokeratin 1/10 (34βB4)

Cytokeratin 5Cytokeratin 7Cytokeratin 8Cytokeratin 17Cytkeratin 19Cytokeratin 20Cytokeratin, high MW(34βE12)

Cytokeratins (pan)EMAEstrogen receptor (ER)GCDFP-15 (Brst2)HBME-1HepPar1Inhibin-alphaMammaglobinp63p504s (AMACR)PAX-2Progesterone receptor (PR)Prostate specific antigenProstatic acid phosphataseSurfactant ApoA1SynaptophysinThyroglobulinTTF-1UroplakinVillinVimentinWT-1

HormonesACTHCalcitoninFSHGastrinGlucagonGrowth hormoneInsulinLeutinizing hormonePancreatic polypeptideParathormoneProlactinSerotoninSomatostatinThyroid stimulatng hormoneVIP

Spindle cell and SBRCTlesions/Undifferentiatedneoplasms

Actin, muscle specific (HHF-35)Actin, smooth muscle alphaBeta-cateninCaldesmonc-kit (CD117)CD31CD34CD35CD99CD117 (c-kit)Collagen, type IVD2-40DesminEMA

Spindle cell and SBRCTlesions/Undifferentiatedneoplasms (continued)

Factor XIIIaFLI-1Ki-67 antigenMyoD1MyogeninMyoglobinNB84 antigenp75NTR

Prognostic markersAndrogen receptorCOX-2 (cyclo-oxygenase-2)DCCEGFR (31G7)EGFR (PharmDx)Estrogen receptor (ER)HER-2/neu by FISHHER-2/neu gene productHER-2/neu HercepTestKi-67 antigenp53Progesterone receptor (PR)Thymidylate synthaseTopoisomerase IIVEGF

Hematolymphoid+ALK protein (p80)

Bcl-2+Bcl-6+Bob-1

c-kit (CD117)CD1aCD2CD3CD4CD5CD7CD8

* CD9CD10 (CALLA)

* CD11c* CD13* CD14

CD15* CD16* CD19

CD20+CD21* CD22

CD23CD25 (IL-2 R β)

CD30 (Ki-1 antigen)+CD31* CD33

CD34+CD35* CD41+CD43

CD45 (LCA)CD52 (CAMPATH 1H)CD56

* CD59+CD57* CD61* CD64* CD66b+CD68* CD71+CD79a* CD90

Hematolymphoid (continued)* CD103* CD123

* CD133+CD138* CD158a* CD158b* CD158e+Cyclin D1+DBA.44+Fascin* FMC7+Hemoglobin A* HLA-DR+IgA+IgD+IgG+IgM+Ki-67 antigen

Kappa light chainsLambda light chains

+Lysozyme+MUM1

Myeloperoxidase+Oct-2+PAX-5+Pan-TCR-β

* TCR-α/β

* TCR-γ/δ

* TCR-β isoforms (24

antibodies)TdT

+TIA-1+TRAcP+Tryptase+vWF

ZAP-70

BreastAndrogen receptorCalponinE-cadherinEstrogen receptor (ER)HER-2/neu by FISHHER-2/neu by IHC

HER-2/neu HercepTestKi-67Maspinp63Progesterone receptor (PR)SMMHC

Adenocarcinoma versusmesothelioma

Ber-Ep4Bg8CalretininCytokeratin 5D2-40HBME-1MesothelinMOC-31ThrombomodulinWT-1

OrganismsAdenovirusBK virusChlamydiaCytomegalovirusEBV (EBER1 ISH)EBV-LMP1Helicobacter pyloriHepatitis B core Ag

Organisms (continued)Hepatitis B surface AgHerpes virusHHV8 (human Herpes virus 8 -

KSHV)

HPVJC virusLegionellaParvovirusPneumocystisPolyomavirusRespiratory syncytial virusSV-40 virusToxoplasmaVaricella zoster

Microsatellite InstabilityMLH1MSH2MSH6PMS2

Melanomagp100 (HMB-45)MART-1 antigenMicrophthalmia transcriptionfactor (MTF)S100Tyrosinase

Amyloid SubtypingAmyloid A (AA)Amyloid BetaAmyloid P (P component)Beta-2 microglobulinTransthyretin (prealbumin)KappaLamda

Germ Cell MarkersPLAPAFPCD30βHCG

Skin Immunofluorescence(IF)

Complement (C3)IgAIgGIgM

Floater/tissue ContaminantBlood group ABlood group BBlood group H(O)

MiscellaneousAlpha-1 antitrypsinAndrogen receptorCaspase 3 fragmentGFAP (glial-fibrillary acidicprotein)MitochondriaNeurofilamentsp16p57TFE3Vimentin

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Spring Quarterly Conference

Dr. Torsten O. Nielsen of the University of British Columbia in Vancouver, Canada, will present “Translating Expression Profiles into Clinical Care” at the Quarterly Pathology/Immunohistochemistry Conference on Thursday, May 11, 2006 at PhenoPath Laboratories. The format of the conference is a social hour commencing at 6:30 p.m., followed by the lecture at 7:30 p.m. A catered light dinner will be provided during the social hour.

Dr. Nielsen’s research interests involve understanding how molecular changes in cancer cells impact diagnosis, prognosis, and treatment, and in developing new and clinically practical molecular diagnostics for targeted therapies for human cancer, particularly sarcomas and breast cancer. His active research areas are gene expression profiling, tissue microarrays and experimental therapeutics. As one of the directors of the Genetic Pathology Evaluation Centre in Vancouver, he leads several projects focusing on the validation and clinical correlation of results from gene expression profiling of sarcomas and breast cancer (including the basal-like phenotype). Dr. Nielsen will speak on how expression profile results can be applied in practice using techniques (immunohistochemistry, FISH, PCR) that are applicable on standard paraffin material and accessible/affordable for hospital laboratories.

Torsten Nielsen completed his MD/PhD at McGill University and is currently an Assistant Professor and clinician-scientist in the Departments of Pathology and Orthopaedics at the University of British Columbia. He is based at the Vancouver General Hospital and British Columbia Cancer Agency. The spring Quarterly Conference will be co-sponsored by Dako Corporation.

Featuring Dr. Torsten O. Nielsen