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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
Journal Reading

醫學系七年級 (Intern):陳姿璇(B8701059)

指導教授:鄭建睿 醫師

journal reading1
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
journal reading2
Journal Reading
  • Recurrent chromosomal translocations
  • Gene fusions
  • Novel chimeric proteins
  • Tumor-specific diagnostic immunohistochemistry (IHC) assays
  • Falini and Mason
immunohistochemistry ihc assays
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
alveolar soft part sarcoma asps
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
renal cell carcinomas
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
material and methods
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
material and methods1
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
material and methods3
Material and Methods
  • Test cases: Archival pediatric renal carcinoma cases
  • Lack of frozen tissue, molecular analysis
  • TFE3 antibody:
  • P-16 polyclonal antibody
material and methods5
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)
material and methods6
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)
results
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+)
results1
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
results3
Results
  • Screening cases: Table 1
  • 6/1476 TMA: negative
results6
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)
discussion
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%
discussion2
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
discussion3
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
discussion4
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
reference
Reference
  • Atlas of Genetics and Cytogenetics in Oncology and Haematology
  • http://www.infobiogen.fr/services/chromcancer/Genes/TFE3ID86.html
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