Brendan C. Dickson

CIC::DUX4 sarcoma (CDS) is a lethal cancer driven by a fusion between tumor suppressor Capicua (CIC) and pioneer transcription factor double homeobox 4 (DUX4). We previously generated three genetically engineered mouse models (GEMMs) of CDS with CIC::DUX4 regulated by loxP-STOP-loxP cassettes, however, all three models developed spontaneous tumors without Cre recombinase. Here, we established a next-generation GEMM of CDS (dFLEx CDS) that employs a dual recombinase (Cre + FLPE) FLEx-switch design to activate CIC::DUX4 expression and initiate sarcomagenesis in a spatially and temporally-controlled manner. Because CIC::DUX4 drives sarcoma development by activating a oncogenic transcriptional program, we performed a drug screen on human-derived CDS cell lines using a library of compounds that modulate transcription. This screen identified Minnelide, an inhibitor of RNA polymerase II-mediated transcription, as a selective inhibitor of CDS. Mechanistically, Minnelide acted through xeroderma pigmentosum type B to alter phosphorylation of RPB1, the largest subunit of RNA polymerase II. Subsequently, RPB1 underwent degradation leading to apoptosis of CDS cells. Minnelide demonstrated in vivo efficacy in dFLEx CDS GEMMs and in human CDS xenografts. As Minnelide has already been demonstrated to be safe in clinical trials, these findings nominate Minnelide as a potential therapeutic option to test in CDS patients.

The classification of mesenchymal neoplasms is undergoing a period of punctuated equilibrium thanks to the increasing availability and routine application of next-generation sequencing. This is especially true for the characterization of undifferentiated/ unclassified mesenchymal tumors, which have historically been grouped morphologically as spindle cell, epithelioid cell, round cell and pleomorphic neoplasms. Herein, we report four undifferentiated epithelioid mesenchymal neoplasms harboring a novel GLI2::CREBBP gene fusion. All patients were adult males who presented with a small superficial mass. Histologically, the tumors were composed of nests or sheets of bland epithelioid cells often embedded within prominent collagenous stroma. One case contained metaplastic bone, and dystrophic calcification in a region of prior ischemia. Immunohistochemistry showed variable keratin expression in three cases, but was otherwise non-contributory. Targeted next generation sequencing revealed a GLI2::CREBBP fusion in all cases. Clinically, three of the tumors followed an indolent clinical course; however, one patient ultimately died as a result of metastatic disease, suggesting GLI2::CREBBP -rearranged nested-hyalinizing epithelioid neoplasms may behave potentially aggressive. Further study is necessary to characterize the biological potential and molecular-pathologic spectrum of this potentially novel entity.

Background and objectives: GREB1 ‐rearranged uterine tumours encompass a group of uterine mesenchymal tumours with varied histologic appearances. The fusion partners to GREB1 include NCOA1‐3, SS18 and NR4A3 . Given that some GREB1 ‐rearranged uterine tumours exhibit histologic features of uterine tumours resembling ovarian sex cord tumour (UTROSCT), there is a general belief that GREB1 ‐rearranged uterine mesenchymal tumours are part of the UTROSCT family. Methods: In this study, we applied global DNA methylation and copy number analyses to a series of 10 GREB1‐ rearranged uterine tumours and 21 classic UTROSCTs (7 of which were molecularly confirmed to harbour ESR1::NCOA2/3 fusions). Results: We found that GREB1 ‐rearranged uterine tumors show an overlap in their global methylation profiles with UTROSCT, including ESR1::NCOA2/3 positive cases. Together, these tumours form a DNA methylation cluster separate from uterine smooth muscle tumours (leiomyomas and leiomyosarcomas), endometrial stromal sarcomas (low‐grade and high‐grade), embryonal rhabdomyosarcoma and SMARCA4‐deficient uterine sarcomas. However, despite their epigenetic similarity, there were two notable differences. First, GREB1 ‐rearranged uterine tumours as a group displayed a greater degree of genomic complexity with more extensive copy number alterations than conventional UTROSCTs, including those harbouring ESR1::NCOA2/3 . Second, GREB1 ‐rearranged uterine tumours frequently lacked overt sex cord morphology: while all 7 ESR1::NCOA2/3 UTROSCTs demonstrated corded, nested, trabecular and/or tubular/sertoliform patterns, only 1 GREB1 ‐rearranged uterine tumour displayed a prominent trabecular pattern, with the remaining cases showing exclusively or predominantly diffuse/solid growth. Conclusions: Overall, our findings confirm that GREB1 ‐rearranged uterine tumours are part of the UTROSCT spectrum, though they frequently exhibit a more diffuse growth pattern and a higher degree of genomic instability.

Supplementary Table 1 includes the metadata on all subjects, including basic demographics and medical history. Supplementary Table 2 provides statistics on chrom 8 gene expression among tumors with H3K27me3 loss. Note: no changes have been made to this file since our last resubmission.

Supplementary figures provide additional details on the genomic landscape of MPNST and interesting cases within the cohort.

Demonstration of MDM2 amplification is currently recommended to confirm the diagnosis of well-differentiated adipocytic tumors arising in the retroperitoneum, abdomen, and pelvis; recurrent “lipomas”; those with equivocal cytologic atypia; deep extremity tumors >10 cm which lack cytologic atypia and occur in patients >50 years old, and in special clinical circumstances (Clay et al., 2015 [Clay criteria]). In our practice, fluorescence in situ hybridization studies (FISH) for MDM2 were historically performed for all referred-in testing requests, for all well-differentiated fatty tumors >10 cm, and to confirm the diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) in cases with definite atypia. As part of a test utilization review, after exclusion of dedifferentiated liposarcoma, we reviewed 1151 ostensibly fatty tumors on which FISH was successfully performed. After exclusion of 203 cases for which testing was inappropriate or indicated for other reasons, 233/948 (24.6%) fatty tumors met at least one Clay criterion, and 222 (23.4%) had definite nuclear atypia. Among tumors with definite atypia, 152 (68.5%) were ALT/WDL, and 48 (21.6%) were spindle cell/pleomorphic lipoma arising in unusual locations or atypical spindle cell/pleomorphic lipomatous tumors. Thirty (12.9%) cases meeting any Clay criteria were ALT/WDL, and 4/453 (0.9%) tumors not meeting Clay criteria or having atypia were ALT/WDL. The sensitivity of Clay criteria plus nuclear atypia for ALT/WDL was 97.8%, while specificity was 62.7%. We conclude that it is safe to defer MDM2 FISH in well-differentiated fatty tumors not meeting Clay criteria and lacking nuclear atypia.

Supplementary Figure 1. A Kaplan-Meier overall survival (OS) curve based on STS type for 136 patients who gave primary specimens and had available follow up information