The Newsletter of PhenoPath

henomenaP Laboratories 1-888-92-PHENO www.phenopath.com Volume12 No.2May 2009

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The PAX proteins are a family of nine proteins, critical during organogenesis, with expression of individual PAX genes helping guide development of specific tissue types. In recent years, however, several members of this nuclear transcription factor family have also emerged as useful lineage-restricted immunohistochemical markers for the analysis of tumors.

H&E stained section of clear cell tumor metastatic to liver in 56-year-old female with remote history of ovarian tumor.

Uniform expression of PAX8 confirms diagnosis of metastatic clear cell ovarian carcinoma.

PAX2 and PAX5 have been utilized for several years as markers of renal cell and B cell malignancies, respectively. PAX8 is expressed in thyroid and non-ciliated mucosal cells of the fallopian tubes and simple ovarian inclusion cysts, but not the ovarian surface epithelial cells. PAX8 is also emerging as an extremely sensitive, and in the appropriate context, specific, marker of ovarian carcinomas. Unlike WT-1, which is only positive in ovarian serous carcinomas, PAX8 is expressed by ovarian serous, endometrioid, and clear cell carcinomas, but only rarely in primary ovarian mucinous adenocarcinomas. A subset of endometrial adenocarcinomas are also PAX8-positive, but studies have demonstrated absence of expression of PAX8 in breast and other non-GYN carcinomas other than those primary to the thyroid. PAX8 is an important new marker of ovarian cancer.Ref. Nonaka D et al. Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol 32:1566-71, 2008; Bowen NJ et al. Emerging roles for PAX8 in ovarian cancer and endosalpingeal development. Gynecol Oncol 104:331-7, 2007

NEW TESTSNow Available at PhenoPathPAX8, MDM2, BRAF

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Examples of Embryonic Expression

Examples of Tumors Identified

PAX2 Kidney Renal cell carcinoma

PAX5 B lymphocytes B cell lymphoma

PAX8 Mullerian duct Ovarian carcinomas (non-mucinous)

New Molecular Testing Requisition FormWe have introduced a new requisition form specifically for ordering molecular tests (e.g., FISH, PCR). You can download this requisition form directly from our website, www.phenopath.com, or call us at 206-374-9000, and we will send you pre-printed forms with your information.

Well-differentiated liposarcoma/atypical lipomatous tumors are unique in harboring ring and giant marker chromosomes consisting of amplicons of the q13-15 region of chromosome 12, which includes the MDM2 gene. MDM2 is a nuclear phosphoprotein that binds and inhibits transactivation by the tumor suppressor gene product, p53. Recently, Weaver and colleagues have demonstrated that the technique of fluorescence in situ hybridization (FISH) can be employed to detect the presence of MDM2 gene amplification and hence assist in the diagnosis of these tumors. FISH testing for MDM2 has been validated and is now available at PhenoPath Laboratories. The assay employs a dual-color MDM2 FISH probe, which is composed of labeled probes to MDM2 as well as the centromere of chromosome 12 (SE12). The accompanying image demonstrates the finding in well-differentiated liposarcoma, with multiple orange MDM2 signals and eusomic green SE12 signals. As demonstrated by Weaver and colleagues, this FISH assay can be helpful in distinguishing well-differented liposarcoma from other soft tissue neoplasms. Most importantly, MDM2 amplification is not present in benign lipoma and angiolipoma. The histologic spectrum of lipomatous neoplasms that show MDM2 amplification includes not only well-differentiated liposarcoma/atypical lipomatous tumor, but also dedifferentiated liposarcoma and a minority of myxoid liposarcomas. Spindle cell/pleomorphic lipoma shows polysomy at this locus. This MDM2 FISH assay can prove especially helpful, given the dearth of immunostains which can be employed to identify liposarcoma and distinguish it from histologic mimics. Ref. Weaver J et al. Flourescence in situ hybridization for MDM2 gene amplification as a diagnostic tool in lipomatus neoplasms. Mod Pathol 21:943-9, 2008

MDM2 FISH studies require a paraffin block of formalin-fixed tissue or unstained 4 micron sections. Please contact Dr. Harry Hwang, Director of Molecular Pathology at PhenoPath Laboratories, if you have any specific questions about this, or any other molecular assay.

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Clusters of orange signals in tumor cell nuclei confirming presence of MDM2 gene amplification in this case of liposarcoma.

The serine/threonine kinase BRAF, in addition to KRAS, is also a key downstream component of the EGFR signaling cascade. As with KRAS, mutations in BRAF have been identified in colorectal and other cancers, with up to 15% of colorectal cancers having a BRAF mutation. The main mutation that occurs in BRAF is at codon 600 (V600D) and results in activated BRAF. In controlled clinical trials, patients with a colorectal tumor having wild-type KRAS, but mutated BRAF, were resistant to anti-EGFR antibody therapy, and had shorter progression-free survival (PFS) and overall survival (OS) than wild-type BRAF patients. Therefore, wild-type BRAF is required for patient responsiveness to anti-EGFR antibody therapy and evaluation of BRAF mutation status, in addition to KRAS mutation status, is of significant predictive value in assessing a patient for potential anti-EGFR therapy. At PhenoPath, we therefore recommend that in addition to assessing tumor specimens for one of the seven known activating KRAS mutations, that tumors are also assessed for the presence of the V600D BRAF activating mutation. Ref. Di Nicolantonia F et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 26(35):5668-70, 2008

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PCR MUTATION ANALYSES KRAS & BRAF KRAS__________Test: KRASPCR BRAF__________Test: BRAFPCR JAK2 __________Test: JAK2 PCR

GENE REARRANGEMENT BY PCR B cell (IgH)____Test: IgH PCR T cell (TCR- ) __Test: TCR PCR

Send: REQS: MOL PATH HEME DERM Airbills PhenoBoxes Ice packs Flow Media (RPMI) IF Media (Michel’s) Other______________

FLUORESCENCE IN SITU HYBRIDIZATION (FISH) & ISH 1p36/19q13 - Oligodendroglioma ____________Test: 1P19QP EGFR/CEP7 ________________________________Test: EGFRF

EWSR - PNET/Ewing, DSRCT, CCS, ExMyxChondro ___ Test: T11-22 HER2/CEP17 ________________________________Test: PATHVY

MDM-2/CEP12 ______________________________Test: XYZ p53/CEP17 ____________________________________Test: XYZ SMS/RARA ____________________________________Test: XYZ SS18 - SYT - synovial sarcoma _________________Test: TX-18 TOP2A/CEP17 _______________________________Test: TOP2A bcl-6 translocations_____________________Test: BCL6 FISHP IgH translocations___________________________Test: IGHF MALT1 translocations____________________Test: MALT1BRK MYC translocations ______________________Test: MYC FISH t(4;14) FGFR3/IGH __________________________Test: T4-14 t(11;14) CCND1/IGH________________________Test: T11-14 t(14;18) MALT1/IGH _________________________Test: T14-18M t(14;16) cMAF/IGH_________________________Test: T14-16 t(11;18) - MALT1/AP12______________________Test: T11-18 t(14;18) BCL2/IGH _________________________Test: T14-18 t(9;22) BCR/ABL ____________________________Test: T9-22 t(11q23) MLL _______________________________Test: MLL t(15;17) PML/RARA ________________________Test : T15-17

RARA translocations_______________________Test: RARABP Plasma Cell Myeloma FISH Panel (or order individually above): IgH (Test: IGHF) -AND- t(11;14) CCND1/IGH (Test: T11-14)

If IGH is positive, run: t(4;14) FGFR3/IGH (Test: T4-14) -&- t(14;16) cMAF/IGH ( Test: T14-16)

CEP-17 - Hydatidiform Mole _____________________Test: CEP17 CEP-X/Y_____________________________________Test: CEPXY EBV ____________________________________Test: EBV-ISH Other (list): ________________________________________

CLINICAL SPECIMEN INFORMATION

Hosp/Inst where specimen collected: _________________________Collection Date ___________________ Collection Time __________Specimen ID Block # / Sublabel Tissue Source(s)_______________ _____________ ____________________

_______________ _____________ ____________________ Paraffin blocks: Tissue block(s)________ Cell block(s) _________ Formalin Bouin’s B5 Prefer Michel’s (skin IF TM) Other Slides: Unstained_______ Stained_______ Smears: Air-dried_______ Fixed_______ Stained_______Blood Bone marrow aspirate Bone marrow core bx Body fluid/CSF

(NOTE: Fresh specimens: EDTA preferred, Heparin ok)Multiple specimens submitted: Test all Select best block

L A B O R A T O R I E SPhenoPath

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

CLINICAL HISTORY

DIAGNOSIS UNDER CONSIDERATION / REQUEST

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BILLING INFO (Must be provided or Institution will be billed)Please complete or attach copy of insurance card

BILL: Ins Medicare Medicaid (WA DSHS only) Institution Pt

Referral/Authorization # _______________ ICD-9 # ______________

Medicare # _______________________________________________

Advance Beneficiary Notice Yes (provide copy) No

Healthplan _______________________________________________

Address _________________________________________________

Policy/Cert # ______________________ Group/Plan # ___________

Name of Insured ___________________ Relationship ___________

Secondary Insurance Yes (Please attach separate sheet) No

MOLECULAR ONLY REQUISITION FORMTHIS SECTION FOR PHENOPATH USE ONLY

MOLECULAR ONLY

Name (Last, First, MI) ________________________________________

SSN # ____________________ DOB____________ Male Female

Inpatient Outpatient Non-Hospital Patient

Address __________________________________________________

_________________________________ Phone __________________

Medical Record # __________________________ Pt # _____________

PATIENT INFORMATION

REQUIRED

Person completing form __________________________________________

Date ______________ Phone _____________________________________

REQUESTING INSTITUTION NAME & ADDRESS

TREATING PHYSICIAN

Phone _______________________ FAX ______________________

Ordering Pathologist/Physician

Name ___________________________________________________

NPI # ______________________________ UPIN # ________________

_________________________________________________________Name NPI # UPIN #

Phone _______________________ Fax ________________________Institution _______________________________________________Address _________________________________________________

City, State Zip _____________________________________________

Mail/Fax add’l copy of report to treating physician Complete information REQUIRED BELOW

Rev. 05/08/2009

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VISIT US AT THE FOLLOWING MEETINGS:For up-to-date information, visit our website: www.phenopath.com

South Bay Pathology Society Presents:Immunohistochemistry in Surgical Pathology: Challenges and Diagnostic PitfallsMay 9, 2009, Monterey Conference Center, Monterey, CAAllen M. Gown, MD presented as follows:

9:00 – 10:30 AM: General Issues – Allen Gown, MDThyroid – Saul Suster, MDBreast – Allen Gown, MD11:00 AM to Noon: Lung & Pleura – Saul Suster, MDGastrointestinal Tract – Allen Gown, MD1:30 – 3:00 PM: Soft Tissue – Saul Suster, MDLiver – Allen Gown, MDSkin Neoplasms – Saul Suster, MD3:30 – 4:30 PM: Male Genitourinary – Allen Gown, MDMiscellaneous Lesions – Saul Suster, MD

Pritzker Memorial LectureJune 15, 2009, 5:00-6:00 pm, Mount Sinai Hospital, TorontoAllen M. Gown, MD is a featured speaker and will present a talk entitled “Immunohistochemistry: The Past as Prologue to the Future”

Oregon Pathologists AssociationSeptember 11-12, 2009, Allen M. Gown, MD presents as follows:

September 11, 2009, 7:30 PM, Oregon Medical Association Building“Seeing the World on a Bicycle’September 12, 2009, 9:00 AM to Noon, St. Vincent Hospital, Portland, OR“Applications of Immunohistochemistry to Problems in Surgical Pathology”

CAP Virtual Management ConferenceOctober 13, 2009,Allen M. Gown, MD is a featured speaker and will present a talk entitled“Antibody & Test Validation in IHC”

24th Annual Clinical Cytometry Society Meeting & CourseOctober16-21, 2009, Hyatt Regency Jacksonville Riverfront, Jacksonville, FL

California Society of Pathologists 62nd Annual Convention:Seminars in PathologyDecember 2-5, 2009, Hyatt Regency San Francisco, Embarcadero Center

San Antonio Breast Cancer SymposiumDecember 9-13, 2009, Henry B. Gonzalez Convention Center, San Antonio, TX

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PhenoPathL A B O R A T O R I E SExpertise, Innovation and Excellence

At Our Spring Conference

FeaturingDr. Philip LeBoit

Philip LeBoit, M.D., of the University of California, San Francisco, CA, will present “Difficult Diagnoses in Dermatopathology made Easier with Molecular and Immunopathologic Techniques” at The PhenoPath Spring Conference on Thursday, June 4, 2009. The format of the confer-ence is a social hour commencing at 6:30 PM, followed by Dr. LeBoit’s lecture at 7:30 PM. A light catered dinner will be served during the social hour.

Dr. LeBoit is a Professor of Pathology and Dermatology, and Co-Director of the UCSF Dermatopathology Service. He received an MD degree from Albany Medical College, and completed his residency at UCSF and at Mt. Sinai School of Medicine, followed by a Dermatopathology Fel-lowship at Cornell University Medical Center. Dr. LeBoit founded the UCSF Dermatopathology Service in 1987, which is the largest university-based outreach dermatopathology laboratory in the western United States. The service sees over 90,000 cases/year, including over 20,000 cases in which pathologists, dermatologists or dermatopathologists seek a second opinion.

Dr. LeBoit’s main research interest in dermatopathology is how does one make a more accurate diagnosis based on a skin biopsy. To this end, he studies both inflammatory and neoplastic diseases, applying both high- and low-tech methods as the situation warrants. Of particular interest are entities whose essential natures are unclear, such as Spitz nevus, a tumor of melanocytes in which large cells occur, sometimes simulating melanoma.

Dr. LeBoit is one of the leading figures in contemporary dermatopathology, as a consultant, author and clinical investigator. He has written over 150 articles, edited or co-authored 8 books, including the two-volume text “Cutaneous Medicine and Surgery” (1996), “Histopathology of Nevi and Melanoma” (2004), and “Skin Tumors” in the WHO Tumors and Genetics Series (2006). He served as editor-in-chief of the American Journal of Dermatopathology from 1997-2007 and as co-editor of Seminars in Cutaneous Medicine and Surgery. He is currently President of the International Society of Dermatopathology. His contributions to the medical literature include the first comprehensive descriptions of bacillary angiomatosis, primitive polypoid granular cell tumor and nephrogenic fibrosing dermopathy, and many studies of Spitz nevus. A renowned speaker, Dr. LeBoit has given over 250 invited lectures, on every continent except Antarctica.