algorithmic immunohistochemical diagnosis of undifferentiated ...

1. ALGORITHMIC IMMUNOHISTOCHEMICAL DIAGNOSIS OF UNDIFFERENTIATED TUMORS & METASTATIC CARCINOMAS Mark R. Wick, M.D.

2. CURRENT DILEMMAS INDIAGNOSTIC IMMUNOHISTOLOGY Ever-increasing number of commercial reagents for use in evaluation of human specimens; as of 12/02, there were over 2000 such antibodies listed in the Linscott catalogue Many confusing entries in the pathology literature, vis-a-vis the “specificity” (and implied usefulness) of a great many antibody reagents

3. “SPECIFICITY” OFIMMUNOHISTOLOGIC REAGENTS Related, but not identical, to classical definition given by Galen & Gambino: Spec = True negatives/True negatives + False positives In reality, “specificity” of immunohisto-logical reagents must be evaluated in many restricted and well-defined contexts Hence, “specificity” is a relative term in this applied clinical setting

4. THE INTEGRATED APPROACH TO IMMUNOHISTOLOGY: USING “NON-SPECIFIC” REAGENTS TO ADVANTAGE One is faced with an “either-or” decision in diagnostic immuno-histology; either use no reagents at all (since none is “absolutely” specific in the most stringent terms), or use them in particular ways that are dictated by relative antibody specificities The latter approaches do not “waste” information that is potentially gleaned from “non-specific” antibodies, and at the same time safeguard the user against erroneous interpretations

5. INTEGRATED ALGORITHMIC IMMUNOHISTOLOGY OF MALIGNANT TUMORS:NECESSARY CONDITIONS The user must control and standardize the processing of all tissues in his or her laboratory (e.g., fixative; fixation times; microwave-mediated antigen “retrieval”) The user must personally define optimal antibody concentrations rather than accept the recommendations of manufacturers Data must be accrued on spectra of reactivity for all antibodies used in the laboratory, either “in-house” or by adopting the EXACT methods of someone else who has already done this type of reagent analysis

6. INTEGRATED ALGORITHMIC IMMUNOHISTOLOGY OF MALIGNANT TUMORS:NECESSARY CONDITIONS (CONT.) Data on antibody performance must be used as the substrate for formal statistical analysis of specificity & sensitivity in well-defined diagnostic settings, producing a “relative values” system The sequence of interpretation of panels of immunostains is governed by their relative statistical values within each predefined setting, moving from highest to lowest predictive values Discrete morphological categories must be established within which the statistical data are applied; e.g., small round-cell tumors, large polygonal-cell tumors, etc.

7. POTENTIALLY MORPHOLOGICALLY-INDETERMINATE TUMORS: GENERIC CATEGORIES Small round-cell tumors Large polygonal-cell tumors Spindle-cell tumors Pleomorphic tumors Hematopoietic tumors (in subsets of the first two categories)

8. ALGORITHMIC IMMUNOHISTOLOGY: CHOICE OF REAGENTS Panels of antibodies in each algorithm are dependent upon the generic classification of the tumor under study Several antibodies appear in more than one algorithm, but their places in the relative sequences of interpretation (or the diagnoses that positivity yields) differs from one setting to another New antibodies may be substituted for old ones or used to supplement existing reagents in all algorithms

9. KEY POINTS IN ALGORITHMIC IMMUNOHISTOLOGY Consistency of results is totally dependent upon consistency of METHODOLOGY In order to adopt the algorithms of another investigator, his or her methods MUST be followed down to the last detail or differences in antibody performance will result

10. POTENTIALLY UNDIFFERENTIATED SMALL CELL TUMORS Small cell squamous carcinoma Small cell neuroendocrine carcinoma Malignant melanoma Lymphoma Granulocytic sarcoma Neuroblastoma Primitive neuroectodermal tumor Rhabdomyosarcoma

12. POTENTIALLY UNDIFFERENTIATED LARGE-CELL TUMORS Poorly-differentiated squamous cell carcinoma Adenocarcinoma Malignant melanoma Malignant lymphoma Granulocytic sarcoma Epithelioid-cell sarcomas Epithelioid sarcoma Clear cell sarcoma Alveolar soft part sarcoma Epithelioid malignant peripheral nerve sheath tumor Epithelioid leiomyosarcoma

14. POTENTIALLY UNDIFFERENTIATED SPINDLE-CELL TUMORS Sarcomatoid squamous cell carcinoma Spindle-cell malignant melanoma Leiomyosarcoma Fibrosarcoma Malignant fibrous histiocytoma Malignant peripheral nerve sheath tumor Angiosarcoma

16. POTENTIALLY UNDIFFERENTIATED PLEOMORPHIC TUMORS Sarcomatoid squamous cell carcinoma Pleomorphic malignant melanoma “Dedifferentiated” sarcomas Malignant fibrous histiocytoma Malignant peripheral nerve sheath tumor Angiosarcoma

18. ANTIBODIES USED IN EVALUATION OF METASTATIC CARCINOMAS OF UNKNOWN ORIGIN Keratin & Epithelial membrane antigen Prostate-specific antigen Thyroglobulin Thyroid transcription factor-1 Gross cystic disease fluid protein-15 S100 protein Carcinoembryonic antigen CA19.9 CA-125 Placental alkaline phosphatase CD15 Estrogen & Progesterone receptor proteins

19. ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS KERATIN S100 EMA HMB45 CD45 MART-1 CD15 FLI-1 VIMENTIN SYN DES CD56 MSA CD99 MYOGENIN

20. ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS KERATIN S100 VIMENTIN HMB45 BER-EP4 MART-1 CD45 CEA CD15 THROMBOMODULIN MOC31 CALRETININ CD117

21. REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS KERATIN S100 PROTEIN EMA CD56 VIMENTIN CD31 DESMIN CD34 MSA CD57 THROMBOMODULIN CD99

22. REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS KERATIN HMB45 EMA MART-1 VIMENTIN THROMBOMODULIN DESMIN CD31 MSA CD34 S100 PROTEIN CD99

23. REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS CD15 CD79A CD20 LYSOZYME CD30 MYELOPEROXIDASE CD43 CD3 CD45 CD5 CD68 CD163

24. DETERMINATION OF ORIGIN FOR CARCINOMAS OF OCCULT OR CLOSELY-APPOSED SITES Use of immunohistochemistry in this setting relies on use of antigen “catalogues” for a variety of human carcinoma types Antigens are grouped into “inclusionary” & “exclusionary” categories with respect to each tumor type, and arranged algorithmically in order of relative predictive values Resulting antigen profiles constitute protein “fingerprints” of individual carcinoma types, which allow for their discrimination from one another Few tumor-specific markers are available; hence, broad antibody panels are used

25. EXAMPLES OF CHARACTERISTIC IMMUNOPHENOTYPES OF SELECTED CARCINOMAS Breast: CK+; GCDFP15+; S100+/-; CEA-; ER+/-; CA125-; CA19.9-; PLAP-; EMA+; TTF-1- Lung: CK+; GCDFP15-; S100-; CEA+; ER-; CA125-; CA19.9+/-; PLAP+/-; EMA+; TTF-1+ Kidney: CK+; GCDFP15-; S100-; CEA-; ER-; CA125-; CA19.9-; PLAP+/-; EMA+; TTF-1- Adrenal: CK-; GCDFP15-; S100-; CEA-; ER-; CA125-; CA19.9-; PLAP-; EMA-; TTF-1- Ovary: CK+; GCDFP15-; S100+/-; CEA-; ER+/-; CA125+; CA19.9+/-; PLAP+/-; EMA+; TTF-1- Germ Cell: CK+/-; GCDFP15-; S100-; CEA-; ER-; CA125-; CA19.9-; PLAP+; EMA-; TTF-1-