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 of the 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(P apillary 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 (P apillary 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.  Test cases: Archival pediatric renal carcinoma cases  Lack of frozen tissue, molecular analysis  TFE3 antibody:  P-16 polyclonal antibody

12. Material and Methods

13.  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,uteru s,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.  Screening cases: Table 1  6/1476 TMA: negative

20. Results

22.  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.  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/Ge nes/TFE3ID86.html http://www.infobiogen.fr/services/chromcancer/Ge nes/TFE3ID86.html