Molecular Cytogenetic Analysis of JAZF1, PHF1, and YWHAE in Endometrial Stromal Tumors  Jennelle C. Hodge, Patrick P. Bedroske, Kathryn E. Pearce, William R. Sukov  The Journal of Molecular Diagnostics  Volume 18, Issue 4, Pages 516-526 (July 2016) DOI: 10.1016/j.jmoldx.2016.02.001 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Fluorescence in situ hybridization probe design schematic (not drawn to scale). A: The JAZF1 break-apart probe set is composed of clones flanking JAZF1, including those centromeric (cen) to 5′JAZF1 (green) and telomeric (telo) to 3′JAZF1 (red). B: The PHF1 break-apart probe set includes clones telomeric to 5′PHF1 (green) and a centromeric clone flanking 3′PHF1 (red). C: The YWHAE break-apart probe set includes clones centromeric to 5′YWHAE (green) and a telomeric clone flanking 3′YWHAE (red). D: The MEAF6 break-apart probe set is composed of clones centromeric to 5′MEAF6 (red) and telomeric to 3′MEAF6 (green). Disruption of any break-apart probe set separates the normal yellow fusion signal into red and green, indicating a gene rearrangement. The Journal of Molecular Diagnostics 2016 18, 516-526DOI: (10.1016/j.jmoldx.2016.02.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Analysis of a uterine endometrial stromal nodule (ESN) from a 59-year-old woman retrospectively collected on the basis of karyotype and fluorescence in situ hybridization (FISH) probe patterns suggestive of atypical JAZF1 rearrangements. A: Chromosome studies demonstrate an apparently balanced t(7;17)(p15;q11.2) as the sole abnormality. B: Metaphase FISH analysis using the JAZF1 break-apart probe set shows gene rearrangement with the 5′JAZF1 green signal remaining on the der(7) and the 3′JAZF1 red signal moving to the der(17). The normal chromosome 7 is not disrupted at JAZF1 and retains a yellow fusion signal. C: Interphase FISH studies confirm JAZF1 rearrangement with the signal pattern 1R1G1F. D: The most commonly observed atypical pattern, as occurred in case 5, is consistent with rearrangement of one JAZF1 gene copy and loss of the normal JAZF1 copy (1R1G). Other patterns include JAZF1 rearrangement with two 5′JAZF1, one 3′JAZF1, and one intact JAZF1 gene (1R2G1F) in case 8 (E), JAZF1 rearrangement with one 5′JAZF1 and one to two 3′JAZF1 with loss of the normal JAZF1 copy (1-2R1G) in case 9 (F), and a single intact JAZF1 copy and a single JAZF1 rearrangement with loss of 3′JAZF1 (1G1F) in case 26 (G). Red, green, and yellow arrows highlight the corresponding red, green, and fusion probe signals in example nuclei. Original magnification, ×1000 (E–G). The Journal of Molecular Diagnostics 2016 18, 516-526DOI: (10.1016/j.jmoldx.2016.02.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 PHF1 rearrangement-positive endometrial stromal sarcoma (ESS) involving a partner gene other than JAZF1 or MEAF6. A: This tumor (case 23) shows typical pathological characteristics of a primary ESS, but no features consistent with sex cord differentiation were observed. B: Although fluorescence in situ hybridization for both the JAZF1 and YWHAE genes is negative, a typical rearrangement pattern involving one copy of the PHF1 gene is identified. C: Because MEAF6 has recently been described as another potential partner gene for PHF1, an MEAF6 break-apart (BAP) probe set was applied to this case and no rearrangement was identified. Red, green, and yellow arrows highlight the corresponding red, green, and yellow fusion probe signals in example nuclei. Original magnification, ×1000 (B and C) The Journal of Molecular Diagnostics 2016 18, 516-526DOI: (10.1016/j.jmoldx.2016.02.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Histological features of primary and metastatic endometrial stromal sarcomas (ESSs) and undifferentiated uterine sarcomas (UUSs) with abnormalities of JAZF1, PHF1, and YWHAE. A: A primary ESS with no rearrangement of JAZF1, PHF1, or YWHAE (case 20) is well differentiated and composed of sheets of oval to spindle-shaped cells with scant cytoplasm, which concentrically proliferate around an extensive vascular network without necrosis. B: In comparison, a primary UUS with no JAZF1, PHF1, or YWHAE disruption (case 29) is poorly differentiated and characterized by large cells with marked cytologic atypia and necrosis. C: A YWHAE-disrupted metastatic ESS involving a fallopian tube and ovary (case 25) presents as a high-grade tumor with solid growth of cells that have eosinophilic cytoplasm and large, irregular, round nuclei with visible nucleoli and granular chromatin. D: A metastatic ESS involving the bowel (case 24) with JAZF1 and PHF1 disruption that likely represents a JAZF1/PHF1 fusion shows dense growth of uniformly sized cells with ovoid to round nuclei. E: A metastatic tumor classified as UUS involving the chest wall with YWHAE disruption and three intact copies of JAZF1 (case 33) also has a high-grade round cell component. The Journal of Molecular Diagnostics 2016 18, 516-526DOI: (10.1016/j.jmoldx.2016.02.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Probe signal patterns and interpretations. The signal pattern for a normal result (ie, no separation) with the break-apart probe sets for JAZF1 (A), PHF1 (B), or YWHAE (C) is two yellow (fusion or F) signals, designated 2F. D: The typical JAZF1 rearrangement results in a separation of the 5′JAZF1 (green or G) signal, which remains on the derivative chromosome 7, from the 3′JAZF1 (red/orange or R) signal, which moves to the translocation partner chromosome, thereby producing the signal pattern 1R1G1F. Typical rearrangements of the PHF1 break-apart probe set (E) and the YWHAE break-apart probe set (F) display a similar signal pattern. Red, green, and yellow arrows highlight the corresponding red, green, and fusion probe signals in example nuclei. The Journal of Molecular Diagnostics 2016 18, 516-526DOI: (10.1016/j.jmoldx.2016.02.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions