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Journal Reading

Journal Reading. 醫學系七年級 (Intern): 陳姿璇 (B8701059) 指導教授 : 鄭建睿 醫師 . Journal Reading. The American Journal of Surgical Pathology 27: 750-761, 2003 Aberrant nuclear immunoreactivity for TFE3 (Transcription Factor E3) in neoplasms with TFE3 gene fusions

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Journal Reading

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  1. Journal Reading 醫學系七年級 (Intern):陳姿璇(B8701059) 指導教授:鄭建睿 醫師

  2. Journal Reading • The American Journal of Surgical Pathology 27: 750-761, 2003 • Aberrant nuclear immunoreactivity for TFE3 (Transcription Factor E3) in neoplasms with TFE3 gene fusions • A sensitive and specific immunohistochemical (IHC)assay • The department of pathology , Johns Hopkins Medical Institutions, Baltimore,Maryland and the department of pathology , Memorial Sloan-Kettering Cancer Center,NewYork

  3. Journal Reading • Recurrent chromosomal translocations • Gene fusions • Novel chimeric proteins • Tumor-specific diagnostic immunohistochemistry (IHC) assays • Falini and Mason

  4. Immunohistochemistry (IHC) assays • Antibody-fusion protein breakpoint; • Difficulty in raising specific antibodies • Robust antibodies-a portion of one ofthe proteins; • Successful, overexpression • ALK1, anaplastic large cell lymphoma; inflammatory myofibroblastic tumor • The C-terminus of WT1desmoplastic small round cell tumor

  5. Breakpoints

  6. Alveolar Soft Part Sarcoma (ASPS) • t(X;17),(p11.2;q25) • TFE3 (Transcription Factor E3), chromosome X p 11.2 Renal cell carcinomas, children and young adults • ASPL (Alveolar Soft Part Sarcoma Locus) gene, chromosome 17q25 • unknown

  7. Renal cell carcinomas • t (X;1)(p11.2;q21) • TFE3-PRCC(Papillary Renal Cell Carcinoma) gene fusion • t (X;17)(p11.2;q25) • TFE3-ASPL (Alveolar Soft Part Sarcoma Locus) gene fusion • Xp11.2, TFE3 gene, 3’ end • IHC, useful diagnostic marker

  8. Material and Methods • Positive control cases: • Formalin-fixed, paraffin-embedded tissue sections • Xp11.2,TFE3 • ASPS, three types of renal carcinoma • ASPS-positive control group: 19 • Renal carcinoma-positive control group:21 • t(X;17)(p11.2;q25)/ ASPL (Alveolar Soft Part Sarcoma Locus) -TFE3: 7 • t(X;1)(p11.2;q21)/ PRCC (Papillary Renal Cell Carcinoma) -TFE3: 11 • t(X;1)(p11.2;p34)/ PSF(PTB-associated splicing factor)-TFE3: 3

  9. Material and Methods • Screening cases: • Organ-specific tissue microarray (TMAs) • Memorial Sloan Kettering Cancer Center: • High-density TMA: 7-270 cores, ranging from 3 to 6 cores per tumor, with a core diameter of 0.4-1.0mm • Low-density TMA: 27-35 cores, single core per tumor , 3mm in diameter • The Johns Hopkins Hospital: • TMA: 99 spots,2mm in diameter • Positive/weak positive-donor blocks- whole sections • -immunostained for TFE3

  10. Material and Methods

  11. Material and Methods • Test cases: Archival pediatric renal carcinoma cases • Lack of frozen tissue, molecular analysis • TFE3 antibody: • P-16 polyclonal antibody

  12. Material and Methods

  13. Material and Methods • IHC method: • 4um sections mounted onto positively charged slides • Xylene- deparaffinized-30 minutes • Ethanol-rehydrated • Steamed for 30 minutes at 98-99C in EDTA buffer in a vegetable steamer • Quenching: Hydrogen peroidase • Biotin blocking: Avidin • Incubation overnight:1:600dilution polyclonal antibody in phosphate-buffered saline • Detection: biotinylated secondary antibody, horseradish peroxidase-conjugated streptavidin, 3’-3’-diaminobenzidine (Chromogen)

  14. Material and Methods • Scoring of TFE3 nuclear immunoreactivity: • 0-3+, Nucleus • 1+: weak/equivocal (negative) • 2+: moderate • 3+ strong • readily apparent at low-power magnification (4X objective)

  15. Material and Methods

  16. Results • Normal tissues: lung, thyroid, lymph node, breast,colon,liver,gallbladder,pancreas,uterus,ovary,bone,kidney,bladder,adrenal, prostate and skin • No detectable TFE3 nuclear protein • 1/18 chronic pancreatitis,weak(1+)

  17. Results • Positive control cases: tumors with knownXp11.2 translocationsorTFE3 gene fusions: • 39/40: moderate or strong • 19 ASPS: 18 strong ;1 moderate • 7 t(X;17)(p11.2;q25) (ASPL-TFE3): 2 strong; 5 moderate • 3 t(X;1)(p11.2;p34)(PSF-TFE3): strong • 11 t(X;1)(p11.2;q21)(PRCC-TFE3): 1 weak (negative); 2 strong; 8 moderate

  18. Results

  19. Results • Screening cases: Table 1 • 6/1476 TMA: negative

  20. Results

  21. Results

  22. Results • Test cases: relationship between TFE3 immunoreactivity and morphology in pediatric renal carcinoma: • 11 cases • 8 TFE3: 5 ASPL-TFE3; 3PRCC-TFE3 • 7/8: TFE3 staining (5ASPL-FE; 2 PRCC-TFE3)

  23. Results

  24. Discussion • TFE3(Transcription Factor E3):a basic helix-loop-helix DNA binding domain and a leucine zipper dimerization domain • Nuclear localization signal • Native TFE3 protein: not detectable • Half-life short, tightly regulated • Strong nuclear immunoreactivity: highly sensitive, highly specific • Sensitivity:97.5% (39/40)---100% • Bouin’s acidic fixative: render specific antigen less detecbale by IHC • Specificity: 99.6% (6/1476)---99.1%

  25. TFE3(Transcription Factor E3)

  26. Discussion

  27. Discussion • 3 ASPS blocks: >20 years of age, strong • time • 1 PSF-TFE3 block: unstained over one year, stored at room temperature • oxidation • Immunoreactivity: edges> center • complete fixation

  28. Discussion • False-positive: • upregulated native TFE3 protein • artifacts of fixation • ASPS: PAS (Periodic acid-Schiff) stain: needle-shaped cytoplasmic deposits • Clear cell sarcoma of kidney (CCSK): fine chromatin of CCSK nuclei , chemotherapy: doxorubicin • Xp11.2-related renal cell carcinoma: interferon

  29. Discussion • The prevalence of these tumors in adults and children: • One 68-year-old woman, one 38-year-old man, one 39-year-old woman: ASPL-TFE3 • Adult: rare

  30. Reference • Atlas of Genetics and Cytogenetics in Oncology and Haematology • http://www.infobiogen.fr/services/chromcancer/Genes/TFE3ID86.html

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