Christina V. Angeles

Frequent Alterations and Epigenetic Silencing of Differentiation Pathway Genes in Structurally Rearranged Liposarcomas

UNLABELLED We explored diverse alterations contributing to liposarcomagenesis by sequencing the genome, exome, transcriptome, and cytosine methylome of a primary and recurrent dedifferentiated liposarcoma (DLPS) from distinct chemotherapy/radiotherapy-naïve patients. The liposarcoma genomes had complex structural rearrangements, but in different patterns, and with varied effects on the structure and expression of affected genes. While the point mutation rate was modest, integrative analyses and additional screening identified somatic mutations in HDAC1 in 8.3% of DLPS. Liposarcoma methylomes revealed alterations in differentiation pathway genes, including CEBPA methylation in 24% of DLPS. Treatment with demethylating agents, which restored CEBPA expression in DLPS cells, was anti-proliferative and pro-apoptotic in vitro and reduced tumor growth in vivo. Both genetic and epigenetic abnormalities established a role for small RNAs in liposarcomagenesis, typified by methylation-induced silencing of microRNA-193b in DLPS but not its well-differentiated counterpart. These findings reveal an unanticipated role for epigenetic abnormalities in DLPS tumors and suggest demethylating agents as potential therapeutics. SIGNIFICANCE Multimodality sequence analysis of DLPS revealed recurrent mutations and epigenetic abnormalities critical to liposarcomagenesis and to the suppression of adipocyte differentiation. Pharmacologic inhibition of DNA methylation promoted apoptosis and differentiated DLPS cells in vitro and inhibited tumor growth in vivo, providing a rationale for investigating methylation inhibitors in this disease.

Expression Profiling of Liposarcoma Yields a Multigene Predictor of Patient Outcome and Identifies Genes That Contribute to Liposarcomagenesis

Liposarcomas are the most common type of soft tissue sarcoma but their genetics are poorly defined. To identify genes that contribute to liposarcomagenesis and serve as prognostic candidates, we undertook expression profiling of 140 primary liposarcoma samples, which were randomly split into training set (n = 95) and test set (n = 45). A multigene predictor for distant recurrence-free survival (DRFS) was developed by the supervised principal component method. Expression levels of the 588 genes in the predictor were used to calculate a risk score for each patient. In validation of the predictor in the test set, patients with low risk score had a 3-year DRFS of 83% versus 45% for high risk score patients (P = 0.001). The HR for high versus low score, adjusted for histologic subtype, was 4.42 (95% CI, 1.26-15.55; P = 0.021). The concordance probability for risk score was 0.732. In contrast, the concordance probability for histologic subtype, which had been considered the best predictor of outcome in liposarcoma, was 0.669. Genes related to adipogenesis, DNA replication, mitosis, and spindle assembly checkpoint control were all highly represented in the multigene predictor. Three genes from the predictor, TOP2A, PTK7, and CHEK1, were found to be overexpressed in liposarcoma samples of all five subtypes and in liposarcoma cell lines. RNAi-mediated knockdown of these genes in liposarcoma cell lines reduced proliferation and invasiveness and increased apoptosis. Taken together, our findings identify genes that seem to be involved in liposarcomagenesis and have promise as therapeutic targets, and support the use of this multigene predictor to improve risk stratification for individual patients with liposarcoma.

Resident memory T cells in the skin mediate durable immunity to melanoma

Resident memory CD8 T cells maintained in vitiligo-affected skin mediate long-lived protection against melanoma. Resident memory to cancer Melanoma patients with vitiligo are more likely to have a positive outcome, but the mechanism behind this association has remained unclear. Now, Malik et al. report that skin-resident memory T (TRM) cells specific to melanoma antigens are maintained in vitiligo-affected skin. These cells persist and function independently of the lymphoid compartment, suggesting that the vitiligo lesions provide a niche for the TRM cells. The TRM cells provide durable memory to the tumor, even in pigmented skin. These data suggest that skin TRM cells are critical for maintaining antitumor immunity. Tissue-resident memory T (TRM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen–specific TRM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein–1) or LAG-3 (lymphocyte activation gene–3), and were capable of making IFN-γ (interferon-γ). CD103 expression on CD8 T cells was required for the establishment of TRM cells in the skin but was dispensable for vitiligo development. CD103+ CD8 TRM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating antitumor immunity.

ZIC1 Overexpression Is Oncogenic in Liposarcoma

Liposarcomas are aggressive mesenchymal cancers with poor outcomes that exhibit remarkable histologic diversity (there are five recognized subtypes). Currently, the mainstay of therapy for liposarcoma is surgical excision because liposarcomas are often resistant to traditional chemotherapy. In light of the high mortality associated with liposarcoma and the lack of effective systemic therapy, we sought novel genomic alterations driving liposarcomagenesis that might serve as therapeutic targets. ZIC1, a critical transcription factor for neuronal development, is overexpressed in all five subtypes of liposarcoma compared with normal fat, and in liposarcoma cell lines compared with adipose-derived stem cells. Here, we show that ZIC1 contributes to the pathogenesis of liposarcoma. ZIC1 knockdown inhibits proliferation, reduces invasion, and induces apoptosis in dedifferentiated and myxoid/round cell liposarcoma cell lines, but not in either adipose-derived stem cells or in a lung cancer cell line with low ZIC1 expression. ZIC1 knockdown is associated with increased nuclear expression of p27 proteins and the downregulation of prosurvival target genes BCL2L13, JunD, Fam57A, and EIF3M. Our results show that ZIC1 expression is essential for liposarcomagenesis and that targeting ZIC1 or its downstream targets might lead to novel therapy for liposarcoma.

Myeloid Cells That Impair Immunotherapy Are Restored in Melanomas with Acquired Resistance to BRAF Inhibitors

Acquired resistance to BRAFV600E inhibitors (BRAFi) in melanoma remains a common clinical obstacle, as is the case for any targeted drug therapy that can be developed given the plastic nature of cancers. Although there has been significant focus on the cancer cell-intrinsic properties of BRAFi resistance, the impact of BRAFi resistance on host immunity has not been explored. Here we provide preclinical evidence that resistance to BRAFi in an autochthonous mouse model of melanoma is associated with restoration of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, initially reduced by BRAFi treatment. In contrast to restoration of MDSCs, levels of T regulatory cells remained reduced in BRAFi-resistant tumors. Accordingly, tumor gene expression signatures specific for myeloid cell chemotaxis and homeostasis reappeared in BRAFi-resistant tumors. Notably, MDSC restoration relied upon MAPK pathway reactivation and downstream production of the myeloid attractant CCL2 in BRAFi-resistant melanoma cells. Strikingly, although combination checkpoint blockade (anti-CTLA-4 + anti-PD-1) was ineffective against BRAFi-resistant melanomas, the addition of MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist) prevented outgrowth of BRAFi-resistant tumors. Our results illustrate how extrinsic pathways of immunosuppression elaborated by melanoma cells dominate the tumor microenvironment and highlight the need to target extrinsic as well as intrinsic mechanisms of drug resistance. Cancer Res; 77(7); 1599-610. ©2017 AACR.

VISTA expression on tumor-infiltrating inflammatory cells in primary cutaneous melanoma correlates with poor disease-specific survival

Adaptive immune responses contribute to the pathogenesis of melanoma by facilitating immune evasion. V-domain Ig suppressor of T-cell activation (VISTA) is a potent negative regulator of T-cell function and is expressed at high levels on monocytes, granulocytes, and macrophages, and at lower densities on T-cell populations within the tumor microenvironment. In this study, 85 primary melanoma specimens were selected from pathology tissue archives and immunohistochemically stained for CD3, PD-1, PD-L1, and VISTA. Pearson’s correlation coefficients identified associations in expression between VISTA and myeloid infiltrate (r = 0.28, p = 0.009) and the density of PD-1+ inflammatory cells (r = 0.31, p = 0.005). The presence of VISTA was associated with a significantly worse disease-specific survival in univariate analysis (hazard ratio = 3.57, p = 0.005) and multivariate analysis (hazard ratio = 3.02, p = 0.02). Our findings show that VISTA expression is an independent negative prognostic factor in primary cutaneous melanoma and suggests its potential as an adjuvant immunotherapeutic intervention in the future.

A novel case of an aggressive superficial spindle cell sarcoma in an adult resembling fibrosarcomatous dermatofibrosarcoma protuberans and harboring an EML4-NTRK3 fusion

A subset of soft tissue sarcomas often harbors recurrent fusions involving protein kinases. While some of these fusion events have shown utility in arriving at a precise diagnosis, novel fusions in otherwise difficult to classify sarcomas continue to be identified. We present a case of a 40‐year‐old female who noted a lower back nodule in 2010 that was initially labeled as a dermatofibrosarcoma protuberans with fibrosarcomatous transformation. The lesion recurred the following year and metastasized to the groin 6 years later. Because of some morphologic peculiarities, molecular characterization was pursued in the metastatic focus, which revealed the neoplasm was negative for the COL1A1‐PDGFB fusion. However, anchored multiplex polymerase chain reaction for targeted next‐generation sequencing (Archer Dx) detected an EML4‐NTRK3 fusion, which was confirmed by reverse transcription‐PCR, Sanger sequencing and RNA sequencing analysis of the recurrent and metastatic specimens. Although various soft tissue neoplasms involving fusions with NTRK genes are well‐reported, the current case could not be easily classified in any of the established entities. Nevertheless, it raises interesting questions regarding the importance of classification, prognosis, and treatment for some of these tyrosine kinase fusion‐driven sarcomas.

Oncogenic BRAFV600E Governs Regulatory T-cell Recruitment during Melanoma Tumorigenesis

Regulatory T cells (Treg) are critical mediators of immunosuppression in established tumors, although little is known about their role in restraining immunosurveillance during tumorigenesis. Here, we employ an inducible autochthonous model of melanoma to investigate the earliest Treg and CD8 effector T-cell responses during oncogene-driven tumorigenesis. Induction of oncogenic BRAFV600E and loss of Pten in melanocytes led to localized accumulation of FoxP3+ Tregs, but not CD8 T cells, within 1 week of detectable increases in melanocyte differentiation antigen expression. Melanoma tumorigenesis elicited early expansion of shared tumor/self-antigen-specific, thymically derived Tregs in draining lymph nodes, and induced their subsequent recruitment to sites of tumorigenesis in the skin. Lymph node egress of tumor-activated Tregs was required for their C-C chemokine receptor 4 (Ccr4)-dependent homing to nascent tumor sites. Notably, BRAFV600E signaling controlled expression of Ccr4-cognate chemokines and governed recruitment of Tregs to tumor-induced skin sites. BRAFV600E expression alone in melanocytes resulted in nevus formation and associated Treg recruitment, indicating that BRAFV600E signaling is sufficient to recruit Tregs. Treg depletion liberated immunosurveillance, evidenced by CD8 T-cell responses against the tumor/self-antigen gp100, which was concurrent with the formation of microscopic neoplasia. These studies establish a novel role for BRAFV600E as a tumor cell-intrinsic mediator of immune evasion and underscore the critical early role of Treg-mediated suppression during autochthonous tumorigenesis.Significance: This work provides new insights into the mechanisms by which oncogenic pathways impact immune regulation in the nascent tumor microenvironment. Cancer Res; 78(17); 5038-49. ©2018 AACR.

Collagen quantification in breast tissue using a 12-wavelength near infrared spectral tomography (NIRST) system

A portable near infrared spectral tomography (NIRST) system was adapted for breast cancer detection and treatment monitoring with improved speed of acquisition for parallel 12 wavelengths of parallel frequency-domain (FD) and continuous-wavelength (CW) measurement. Using a novel gain adjustment scheme in the Photomultiplier Tube detectors (PMTs), the data acquisition time for simultaneous acquisition involving three FD and three CW wavelengths, has been reduced from 90 to 55 seconds, while signal variation was also reduced from 2.1% to 1.1%. Tomographic images of breast collagen content have been recovered for the first time, and image reconstruction approaches with and without collagen content included have been validated in simulation studies and normal subject exams. Simulations indicate that including collagen content into the reconstruction procedure can significantly reduce the overestimation in total hemoglobin, water and lipid by 8.9μM, 1.8% and 15.8%, respectively, and underestimates in oxygen saturation by 9.5%, given an average 10% background collagen content. A breast cancer patient with invasive ductal carcinoma was imaged and the reconstructed images show that the recovered tumor/background contrast in total hemoglobin increased from 1.5 to 1.7 when collagen was included in reconstruction.