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
 A sensitive and specific immunohistochemical
(IHC)assay
 The department of pathology , Johns Hopkins
Medical Institutions, Baltimore,Maryland and the
department of pathology , Memorial SloanKettering Cancer Center,NewYork
Journal Reading
 Recurrent chromosomal translocations
 Gene fusions
 Novel chimeric proteins
 Tumor-specific diagnostic
immunohistochemistry (IHC) assays
 Falini and Mason
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
Breakpoints
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
 t (X;1)(p11.2;q21)
 TFE3-PRCC(Papillary Renal Cell
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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
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
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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
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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 Methods
Material and Methods
 Test cases: Archival pediatric renal
carcinoma cases
 Lack of frozen tissue, molecular analysis
 TFE3 antibody:
 P-16 polyclonal antibody
Material and Methods
Material and Methods
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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 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)
Material and Methods
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+)
Results
 Positive control cases: tumors with knownXp11.2
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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
Results
Results
 Screening cases: Table 1
 6/1476 TMA: negative
Results
Results
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 PRCCTFE3)
Results
Discussion
 TFE3(Transcription Factor E3):a basic helix-loop-helix
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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%
TFE3(Transcription Factor E3)
Discussion
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
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
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
 Atlas of Genetics and Cytogenetics in Oncology
and Haematology
 http://www.infobiogen.fr/services/chromcancer/Ge
nes/TFE3ID86.html