Journal Reading The American Journal of Surgical Pathology

Journal Reading ¡ The American Journal of Surgical Pathology 27: 750 -761, 2003 ¡ Aberrant nuclear immunoreactivity for TFE 3 (Transcription Factor E 3) in neoplasms with TFE 3 gene fusions ¡ A sensitive and specific immunohistochemical (IHC)assay ¡ The department of pathology , Johns Hopkins Medical Institutions, Baltimore, Maryland the department of pathology , Memorial Sloan. Kettering Cancer Center, New. York

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 ¡ ALK 1, anaplastic large cell lymphoma; inflammatory myofibroblastic tumor ¡ The C-terminus of WT 1 desmoplastic small round cell tumor

Breakpoints

Alveolar Soft Part Sarcoma (ASPS) ¡ t(X; 17), (p 11. 2; q 25) ¡ TFE 3 (Transcription Factor E 3), chromosome X p 11. 2 Renal cell carcinomas, children and young adults ¡ ASPL (Alveolar Soft Part Sarcoma Locus) gene, chromosome 17 q 25 ¡ unknown

Renal cell carcinomas ¡ t (X; 1)(p 11. 2; q 21) ¡ TFE 3 -PRCC(Papillary Renal Cell Carcinoma) gene fusion ¡ t (X; 17)(p 11. 2; q 25) ¡ TFE 3 -ASPL (Alveolar Soft Part Sarcoma Locus) gene fusion ¡ Xp 11. 2, TFE 3 gene, 3’ end ¡ IHC, useful diagnostic marker

Material and Methods Positive control cases: Formalin-fixed, paraffin-embedded tissue sections Xp 11. 2, TFE 3 ASPS, three types of renal carcinoma ASPS-positive control group: 19 Renal carcinoma-positive control group: 21 t(X; 17)(p 11. 2; q 25)/ ASPL (Alveolar Soft Part Sarcoma Locus) -TFE 3: 7 ¡ t(X; 1)(p 11. 2; q 21)/ PRCC (Papillary Renal Cell Carcinoma) -TFE 3: 11 ¡ t(X; 1)(p 11. 2; p 34)/ PSF(PTB-associated splicing factor) -TFE 3: 3 ¡ ¡ ¡ ¡

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. 0 mm ¡ Low-density TMA: 27 -35 cores, single core per tumor , 3 mm in diameter ¡ The Johns Hopkins Hospital: ¡ TMA: 99 spots, 2 mm in diameter ¡ Positive/weak positive-donor blocks- whole sections ¡ -immunostained for TFE 3

Material and Methods

Material and Methods ¡ Test cases: Archival pediatric renal carcinoma cases ¡ Lack of frozen tissue, molecular analysis ¡ TFE 3 antibody: ¡ P-16 polyclonal antibody

Material and Methods

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

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

Material and Methods

Results ¡ Normal tissues: lung, thyroid, lymph node, breast, colon, liver, gallbladder, pancreas, ut erus, ovary, bone, kidney, bladder, adrenal, prostate and skin ¡ No detectable TFE 3 nuclear protein ¡ 1/18 chronic pancreatitis, weak(1+)

Results ¡ Positive control cases: tumors with known. Xp 11. 2 translocationsor. TFE 3 gene fusions: ¡ 39/40: moderate or strong ¡ 19 ASPS: 18 strong ; 1 moderate ¡ 7 t(X; 17)(p 11. 2; q 25) (ASPL-TFE 3): strong; 5 moderate ¡ 3 t(X; 1)(p 11. 2; p 34)(PSF-TFE 3): strong ¡ 11 t(X; 1)(p 11. 2; q 21)(PRCC-TFE 3): weak (negative); 2 strong; 8 moderate 2 1

Results

Results ¡ Screening cases: Table 1 ¡ 6/1476 TMA: negative

Results

Results

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

Results

Discussion ¡ TFE 3(Transcription Factor E 3): a basic helix-loop-helix ¡ ¡ ¡ ¡ DNA binding domain and a leucine zipper dimerization domain Nuclear localization signal Native TFE 3 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%

TFE 3(Transcription Factor E 3)

Discussion

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

Discussion ¡ False-positive: ¡ upregulated native TFE 3 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 ¡ Xp 11. 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-TFE 3 ¡ Adult: rare

Reference ¡ Atlas of Genetics and Cytogenetics in Oncology and Haematology ¡ http: //www. infobiogen. fr/services/chromcancer /Genes/TFE 3 ID 86. html