Search

Supplementary Data - PDF file 1112K, Supplementary Figures 1-6 and Supplementary Tables 1&2

Soft-tissue sarcomas are a heterogeneous group of tumors arising from connective tissue. Recently, mutations in the neurofibromin 1 (NF1) tumor suppressor gene were identified in multiple subtypes of human soft-tissue sarcomas. To study the effect of NF1 inactivation in the initiation and progression of distinct sarcoma subtypes, we have developed a novel mouse model of temporally and spatially restricted NF1-deleted sarcoma. To generate primary sarcomas, we inject adenovirus containing Cre recombinase into NF1flox/flox; Ink4a/Arfflox/flox mice at two distinct orthotopic sites: intramuscularly or in the sciatic nerve. The mice develop either high-grade myogenic sarcomas or malignant peripheral nerve sheath tumor (MPNST)-like tumors, respectively. These tumors reflect the histologic properties and spectrum of sarcomas found in patients. To explore the use of this model for preclinical studies, we conducted a study of mitogen-activated protein kinase (MAPK) pathway inhibition with the MEK inhibitor PD325901. Treatment with PD325901 delays tumor growth through decreased cyclin D1 mRNA and cell proliferation. We also examined the effects of MEK inhibition on the native tumor stroma and find that PD325901 decreases VEGFα expression in tumor cells with a corresponding decrease in microvessel density. Taken together, our results use a primary tumor model to show that sarcomas can be generated by loss of NF1 and Ink4a/Arf, and that these tumors are sensitive to MEK inhibition by direct effects on tumor cells and the surrounding microenvironment. These studies suggest that MEK inhibitors should be further explored as potential sarcoma therapies in patients with tumors containing NF1 deletion. Mol Cancer Ther; 12(9); 1906–17. ©2013 AACR.

CCR Translation on this Article from C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

Purpose: Tumor regression has been observed in some patients with metastatic renal cell carcinoma (RCC) after nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Cellular and molecular characterization of antigens recognized by tumor-reactive T cells isolated from responding patients could potentially provide insight into the mechanisms of tumor regression.Experimental Design: CD8+ CTL clones that recognized a novel RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201 were isolated from two patients with metastatic RCC who experienced tumor regression or stable disease following nonmyeloablative allogeneic HCT. These clones were used to screen a cDNA library and isolate the unique cDNA encoding the antigen.Results: An alternative open reading frame in the C19orf48 gene located on chromosome 19q13 encodes the HLA-A*0201–restricted minor H antigen recognized by the RCC-reactive T cells. The differential T-cell recognition of donor- and recipient-derived target cells is attributable to a nonsynonymous single-nucleotide polymorphism within the nucleotide interval that encodes the antigenic peptide. Assays for gene expression and CTL recognition showed that the C19orf48-encoded peptide is widely expressed in renal tumors and solid tumors of other histologies. The antigenic peptide can be processed for CTL recognition via both TAP-dependent and TAP-independent pathways.Conclusions: Donor T-cell responses against the HLA-A*0201–restricted minor H antigen encoded by C19orf48 may contribute to RCC regression after MHC-matched allogeneic HCT.

Supplementary Figures S1-S2 from C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

A progressive increase in copy number variation (CNV) characterizes the natural history of cutaneous melanoma progression toward later disease stages, but our understanding of genetic drivers underlying chromosomal arm-level CNVs remains limited. To identify candidate progression drivers, we mined the TCGA SKCM dataset and identified HDGF as a recurrently amplified gene whose high mRNA expression correlates with poor patient survival. Using melanocyte-specific overexpression in the zebrafish BRAF(V600E)-driven MiniCoopR melanoma model, we show that HDGF accelerates melanoma development in vivo. Transcriptional analysis of HDGF compared to control EGFP tumors showed the activation of endothelial/angiogenic pathways. We validated this observation using an endothelial kdrl:mCherry reporter line which showed HDGF to increases tumor vasculature. HDGF is frequently co-altered with the established melanoma driver SETDB1. Both genes are located on chromosome 1q, and their co-amplification is observed in up to 13% of metastatic melanoma. TCGA patients with both genes amplified and/or overexpressed have a worse melanoma specific survival. We tested co-expression of HDGF and SETDB1 in the MiniCoopR model, which resulted in faster and more aggressive melanoma development than either gene individually. Our work identifies the co-amplification of HDGF and SETDB1 as a functional driver of melanoma progression and poor patient prognosis.

Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.

Recent genomic and scRNA-seq analyses of melanoma identified common transcriptional states correlating with invasion or drug resistance, but failed to find recurrent drivers of metastasis. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhlgh and AQP1+NGFR1high drug resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. Here we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in endogenous tumors.

Background The characterization of poorly differentiated neoplasms in fine-needle aspiration (FNA) and small biopsy specimens usually requires immunohistochemistry (IHC) with a panel of markers. Because of an increasing need to preserve limited diagnostic material for tumor genotyping and a mounting demand for cost containment, the authors investigated the usefulness of dual-color IHC with antibodies directed against broad-spectrum keratins and SOX10, a neuroectodermal transcription factor consistently expressed in melanoma, in the workup of epithelioid malignant neoplasms. Methods A total of 107 cases of FNA cell blocks (49 cases) and small biopsies (58 cases) were selected, including 34 melanomas, 31 epithelioid/pleomorphic sarcomas, and 42 carcinomas. IHC was performed on all specimens using a peroxidase-based brown chromogen for SOX10 and an alkaline phosphatase-based red chromogen for keratins AE1/AE3. The presence or absence of staining in lesional cells was scored. Results The majority of tumors demonstrated 1 of 3 distinct patterns: 1) malignant melanomas with nuclear SOX10 (sensitivity of 94% and specificity of 95%); 2) epithelioid/pleomorphic sarcomas negative for both SOX10 and AE1/AE3 (sensitivity of 84% and specificity of 88%); and 3) carcinomas with cytoplasmic AE1/AE3 (sensitivity of 76% and specificity of 98%). In addition, a fourth pattern with cytoplasmic AE1/AE3 and nuclear SOX10 was observed in a subset of carcinomas, most notably triple-negative breast cancers. Conclusions SOX10/keratin dual-color IHC appears to be an effective, sensitive, and specific test to distinguish between melanoma, sarcoma, and carcinoma. This approach can identify melanoma, prioritize additional studies, and limit the number of markers needed to workup an epithelioid malignant neoplasm, thereby potentially reducing costs and preserving valuable tissue for ancillary studies with which to guide therapy. Cancer Cytopathol 2018;126:179-89. © 2018 American Cancer Society.

BACKGROUND The reclassification of noninvasive follicular variant of papillary thyroid carcinoma as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) has created diagnostic and management issues for thyroid fine-needle aspiration (FNA). In response to these challenges, the authors' laboratory adopted a NIFTP policy including 1) stringent criteria (requiring pseudo-inclusions, papillae, and/or psammoma bodies) for a malignant diagnosis of papillary carcinoma to limit false-positive results due to NIFTP and 2) the use of explanatory notes in cases with cytomorphologic features suggestive of possible NIFTP to encourage lobectomy over thyroidectomy. This study examined the effects of this policy on FNA classification and subsequent surgical management. METHODS All thyroid FNAs performed at Brigham and Women's Hospital (n = 1300) during a 1-year period were evaluated for changes in the use of diagnostic categories, explanatory NIFTP notes, and surgical follow-up in comparison with historical controls. RESULTS The use of specific Bethesda categories did not significantly change. Only a single case of NIFTP was mistakenly classified as malignant. NIFTP was seldom suspected prospectively (17 of 1300; 1.3%); when NIFTP was suspected, cases were reported as suspicious for a follicular neoplasm/follicular neoplasm (n = 10) or suspicious for malignancy (SUS; n = 7). Five of the 7 SUS cases (71%) underwent partial thyroidectomy, compared to 19% of those classified as SUS without an explanatory NIFTP note (P

Purpose: Recurrent or metastatic adenoid cystic carcinoma (R/M ACC) is a rare cancer often arising from the salivary glands of the head and neck. While effective therapies are lacking, preclinical models have suggested that retinoic acid agonists may inhibit ACC growth by blocking MYB binding at translocated gene enhancers. We conducted a phase II trial evaluating retinoic acid as a potential novel therapy for R/M ACC. Patients and methods: Patients with histologically confirmed R/M ACC of any primary site with measurable disease (RECIST v1.1), clinical or radiographic progression within 12 months prior to enrollment, and any number of prior lines of therapy were eligible. Cohort 1 (CH1) received ATRA 45 mg/m2 split oral daily dosing on days 1-14 of a 28-day cycle; cohort 2 (CH2) received the same dosing continuously; treatment continued until disease progression. The primary endpoint was best overall response: ≥5 patients in CH1 with disease in response (CR+PR) among N=25 (assuming ≥2 of N=14 accrued in first stage of a two-stage design had disease in response) provided 85% power to target a >10% response rate (one-sided 9% binomial test). Secondary endpoints: safety, progression-free survival (PFS), overall survival. Exploratory analyses: ATRA impact on MYB expression, define resistance mechanisms, and monitor circulating tumor DNA. All had targeted tumor sequencing prior to enrollment. Results: Between 8/2019 and 2/2020, N=14 enrolled in stage 1 CH1. Primary endpoint of response to continue accrual into stage 2 in CH1 was not met; by 5/2020, N=4 enrolled in CH2 when the trial closed to accrual (budget constraints). Among 18 patients, median age: 58, 61% (11/18) women; each patient had a median of 3 organs (range, 1-4) with metastatic disease. 39% had 2+ prior lines of therapy. Best overall response: CR+PR 0%; SD 61% (11/18); PD 28% (5/18); unevaluable 11% (2/18). Median duration of stability 3.5 months (range, 1-12.3+). One patient (CH1) remains on drug with SD >1 year. Half of those who received prior VEGFR therapy achieved SD (4/8). At a median follow-up of 7.9 months, median PFS was 3.2 months (95% CI, 1.8-3.9). N=1 required dose adjustment; N=1 came off drug for toxicity. There were no grade 3-4 adverse events (headache, dry skin were common); no deaths due to treatment. Median tumor mutational burden was 0 (range, 0-5). NOTCH1 was the most frequent alteration (4, 22%) with 2 evaluable NOTCH1-mutant patients exhibiting SD. Mutations in the PI3K pathway, TP53, and TERT promoter were common. Low MYB protein expression was associated with longer duration of stability (p Citation Format: Glenn J. Hanna, Anne O'Neill, Jennifer M. Cutler, Michelle Flynn, Tushara Vijaykumar, John R. Clark, Lori J. Wirth, Jochen H. Lorch, Jong C. Park, Jeffrey Mito, Jens G. Lohr, Jeffrey Kaufman, Leonard I. Zon, Robert I. Haddad. A phase II trial of all-trans retinoic acid (ATRA) in advanced adenoid cystic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT165.

Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The SPRED1 gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with SPRED1 loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that SPRED1 functions as a tumor suppressor, particularly in the context of KIT mutations. SPRED1 knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer.

The goal of resection of soft tissue sarcomas located in the extremity is to preserve limb function while completely excising the tumor with a margin of normal tissue. With surgery alone, one-third of patients with soft tissue sarcoma of the extremity will have local recurrence due to microscopic residual disease in the tumor bed. Currently, a limited number of intraoperative pathology-based techniques are used to assess margin status; however, few have been widely adopted due to sampling error and time constraints. To aid in intraoperative diagnosis, we developed a quantitative optical microscopy toolbox, which includes acriflavine staining, fluorescence microscopy, and analytic techniques called sparse component analysis and circle transform to yield quantitative diagnosis of tumor margins. A series of variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82 and 75%. The utility of this approach was tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78 and 82%. For comparison, if pathology was used to predict local recurrence in this data set, it would achieve a sensitivity of 29% and a specificity of 71%. These results indicate a robust approach for detecting microscopic residual disease, which is an effective predictor of local recurrence.

Objectives Accurate diagnosis of malignant peripheral nerve sheath tumor (MPNST) is often challenging on fine-needle aspiration (FNA) or core needle biopsy. Recurrent mutations in EED and SUZ12, which encode subunits of polycomb repressive complex 2 (PRC2), have been identified in 70% to 92% of MPNSTs; PRC2 inactivation leads to loss of trimethylation of lysine 27 of histone H3 (H3K27me3). We evaluated the utility of H3K27me3 immunohistochemistry for distinguishing MPNST from its cytomorphologic mimics. Methods H3K27me3 immunohistochemistry was performed on 180 cases of spindle cell neoplasms sampled by FNA (n = 66) and needle biopsy (n = 114), and loss of nuclear staining was scored. Tumor types included MPNST, dedifferentiated liposarcoma, schwannoma, solitary fibrous tumor, leiomyosarcoma, melanoma, synovial sarcoma, sarcomatoid carcinoma, gastrointestinal stromal tumor, desmoid fibromatosis, low-grade fibromyxoid sarcoma, and unclassified spindle cell sarcoma/undifferentiated pleomorphic sarcoma. Results Complete loss of H3K27me3 was observed in 54% (13/24) of MPNSTs. In contrast, only two (of 156) histologic mimics showed complete loss of H3K27me3. Partial loss of H3K27me3 was present in a subset of cases (26/180), including both MPNST and non-MPNSTs. Conclusions Complete loss of H3K27me3 is a highly specific (98.7%) marker of MPNST that can distinguish MPNST from cytomorphologic mimics in FNA cell block and small biopsy specimens.

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes.

Soft-tissue sarcomas are a heterogeneous group of tumors arising from connective tissue. Recently, mutations in the neurofibromin 1 (NF1) tumor suppressor gene were identified in multiple subtypes of human soft-tissue sarcomas. To study the effect of NF1 inactivation in the initiation and progression of distinct sarcoma subtypes, we have developed a novel mouse model of temporally and spatially restricted NF1-deleted sarcoma. To generate primary sarcomas, we inject adenovirus containing Cre recombinase into NF1flox/flox; Ink4a/Arfflox/flox mice at two distinct orthotopic sites: intramuscularly or in the sciatic nerve. The mice develop either high-grade myogenic sarcomas or malignant peripheral nerve sheath tumor (MPNST)-like tumors, respectively. These tumors reflect the histologic properties and spectrum of sarcomas found in patients. To explore the use of this model for preclinical studies, we conducted a study of mitogen-activated protein kinase (MAPK) pathway inhibition with the MEK inhibitor PD325901. Treatment with PD325901 delays tumor growth through decreased cyclin D1 mRNA and cell proliferation. We also examined the effects of MEK inhibition on the native tumor stroma and find that PD325901 decreases VEGFα expression in tumor cells with a corresponding decrease in microvessel density. Taken together, our results use a primary tumor model to show that sarcomas can be generated by loss of NF1 and Ink4a/Arf, and that these tumors are sensitive to MEK inhibition by direct effects on tumor cells and the surrounding microenvironment. These studies suggest that MEK inhibitors should be further explored as potential sarcoma therapies in patients with tumors containing NF1 deletion. Mol Cancer Ther; 12(9); 1906–17. ©2013 AACR.

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes., Damon Runyon Cancer Research Foundation (Damon Runyon-Rachleff Innovation Award)

Treatment of soft tissue sarcoma (STS) includes complete tumor excision. However, in some patients, residual sarcoma cells remain in the tumor bed. We previously described a novel hand-held imaging device prototype that uses molecular imaging to detect microscopic residual cancer in mice during surgery.To test this device in a clinical trial of dogs with naturally occurring sarcomas, we asked: (1) Are any adverse clinical or laboratory effects observed after intravenous administration of the fluorescent probes? (2) Do canine sarcomas exhibit fluorescence after administration of the cathepsin-activated probe? (3) Is the tumor-to-background ratio sufficient to distinguish tumor from tumor bed? And (4) can residual fluorescence be detected in the tumor bed during surgery and does this correlate with a positive margin?We studied nine dogs undergoing treatment for 10 STS or mast cell tumors. Dogs received an intravenous injection of VM249, a fluorescent probe that becomes optically active in the presence of cathepsin proteases. After injection, tumors were removed by wide resection. The tumor bed was imaged using the novel imaging device to search for residual fluorescence. We determined correlations between tissue fluorescence and histopathology, cathepsin protease expression, and development of recurrent disease. Minimum followup was 9 months (mean, 12 months; range, 9-15 months).Fluorescence was apparent from all 10 tumors and ranged from 3 × 10(7) to 1 × 10(9) counts/millisecond/cm(2). During intraoperative imaging, normal skeletal muscle showed no residual fluorescence. Histopathologic assessment of surgical margins correlated with intraoperative imaging in nine of 10 cases; in the other case, there was no residual fluorescence, but tumor was found at the margin on histologic examination. No animals had recurrent disease at 9 to 15 months.These initial findings suggest this imaging system might be useful to intraoperatively detect residual tumor after wide resections.The ability to assess the tumor bed intraoperatively for residual disease has the potential to improve local control.

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue malignancies. Patients with large, high grade sarcomas often develop fatal lung metastases. Understanding the mechanisms underlying sarcoma metastasis are needed to improve treatment of these patients. Micro-RNAs (miRNAs) are a class of small RNAs that post-transcriptionally regulate gene expression. Global alterations in miRNAs are frequently observed in a number of disease states including cancer. The signaling pathways that regulate miRNA biogenesis are beginning to emerge. To test the relevance of specific oncogenic mutations on miRNA biogenesis in sarcoma, we used primary soft tissue sarcomas expressing either BrafV600E or KrasG12D. We find that BrafV600E mutant tumors, which have increased MAPK signaling, have higher levels of mature miRNAs and enhanced miRNA processing. To investigate the relevance of oncogene dependent alterations in miRNA biogenesis, we introduce conditional mutations in Dicer and show that Dicer haploinsufficiency promotes the development of distant metastases in an oncogene dependent manner. These results demonstrate that a specific oncogenic mutation can cooperate with mutation in Dicer to promote tumor progression in vivo.

BACKGROUND: The goal of limb-sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization. METHODS: A primary mouse model of soft tissue sarcoma and a wide field-of-view imaging device were used to investigate a series of exogenously administered, near-infrared (NIR) fluorescent probes activated by cathepsin proteases for real-time intraoperative imaging. RESULTS: The authors demonstrated that exogenously administered cathepsin-activated probes can be used for image-guided surgery to identify microscopic residual NIR fluorescence in the tumor beds of mice. The presence of residual NIR fluorescence was correlated with microscopic residual sarcoma and local recurrence. The removal of residual NIR fluorescence improved local control. CONCLUSIONS: The authors concluded that their technique has the potential to be used for intraoperative image-guided surgery to identify microscopic residual disease in patients with cancer. Cancer 2012. © 2012 American Cancer Society.

The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8+ cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705–restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)–identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705+ patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8+ T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.

Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

CD8-positive T lymphocytes recognize peptides that are usually derived from the degradation of cellular proteins and are presented by class I molecules of the major histocompatibility complex. Here we describe a human minor histocompatibility antigen created by a polymorphism in theSP110nuclear phosphoprotein gene. The antigenic peptide comprises two noncontiguous SP110 peptide segments spliced together in reverse order to that in which they occur in the predicted SP110 protein. The antigenic peptide could be produced in vitro by incubation of precursor peptides with highly purified 20Sproteasomes. Cutting and splicing probably occur within the proteasome by transpeptidation.

Approximately thirty percent of patients with soft-tissue sarcoma die from pulmonary metastases. The mechanisms that drive sarcoma metastasis are not well understood. Recently, we identified miR-182 as a driver of sarcoma metastasis in a primary mouse model of soft-tissue sarcoma. We also observed elevated miR-182 in a subset of primary human sarcomas that metastasized to the lungs. Here, we show that myogenic differentiation factors regulate miR-182 levels to contribute to metastasis in mouse models. We find that MyoD directly binds the miR-182 promoter to increase miR-182 expression. Furthermore, mechanistic studies revealed that Pax7 can promote sarcoma metastasis in vivo through MyoD-dependent regulation of pro-metastatic miR-182. Taken together, these results suggest that sarcoma metastasis can be partially controlled through Pax7/MyoD-dependent activation of miR-182 and provide insight into the role that myogenic transcription factors play in sarcoma progression.

A novel method is proposed for the production of long-chain polyunsaturated fatty acids (LCPUFA) by labyrinthulids. The method comprises a monoxenic culture with Psychlobacter phenylpyruvicus, using agar medium in which oil was dispersed. Soybean oil (SBO) was selected as the optimum material for an oil-dispersed agar medium. The labyrinthulids showed three-dimensional growth and an anastomosing ectoplasmic network in the SBO-dispersed agar medium. The oil plate changed from an opaque culture to a more transparent culture, due to growth of the labyrinthulids. The optimum culture conditions were 25-30 degrees C, an initial pH of 6-10 and artificial seawater with a salt concentration of 50-100%. These conditions are close to those where these strains were isolated. The maximum LCPUFA production (0.59 g/l) and dry cell weight (4.93 g/l) was obtained using strain S3-2 (isolated from Ishigaki Island) with 1.5% SBO at 14 days. This value was about 30 times more than that using glucose instead of SBO. The method proposed is promising in terms of the production of LCPUFA from reproducible oils.

SummaryRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7+ and MyoD+ myogenic progenitors by expressing oncogenic KrasG12D and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

Purpose To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. Materials and Methods Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Results Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. Conclusion The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue malignancies. Patients with large, high-grade sarcomas often develop fatal lung metastases. Understanding the mechanisms underlying sarcoma metastasis is needed to improve treatment of these patients. Micro-RNAs (miRNAs) are a class of small RNAs that post-transcriptionally regulate gene expression. Global alterations in miRNAs are frequently observed in a number of disease states including cancer. The signalling pathways that regulate miRNA biogenesis are beginning to emerge. To test the relevance of specific oncogenic mutations in miRNA biogenesis in sarcoma, we used primary soft tissue sarcomas expressing either Braf(V600E) or Kras(G12D). We found that Braf(V600E) mutant tumours, which have increased MAPK signalling, have higher levels of mature miRNAs and enhanced miRNA processing. To investigate the relevance of oncogene-dependent alterations in miRNA biogenesis, we introduced conditional mutations in Dicer and showed that Dicer haploinsufficiency promotes the development of distant metastases in an oncogene-dependent manner. These results demonstrate that a specific oncogenic mutation can cooperate with mutation in Dicer to promote tumour progression in vivo.

Recent advances in sarcoma genomics have identified novel mutations in the PI3K pathway in human sarcomas. Here, we use a mouse model of primary soft-tissue sarcoma for preclinical testing of doxorubicin and inhibitors of the PI3K pathway: BKM120 (PI3K inhibitor) and BEZ235 (a dual PI3K/mTOR inhibitor). Doxorubicin-treated tumors (n=15) showed a partial response rate of 6.6%, just as the majority of human sarcomas do not respond to doxorubicin. Treatment with BKM120 elicited a partial response in 50% of tumors (n=10), which was also seen in combination with doxorubicin (n=10). Additionally, BKM120 treatment produced a robust delay in tumor growth kinetics. BEZ235-treated tumors (n=9) showed a complete response rate of 11.1%. Combining BEZ235 with doxorubicin (n=10) increased the complete response rate to 50% (P=0.035). These studies demonstrate that PI3K pathway inhibition is a viable and attractive target for soft-tissue sarcomas.

Soft-tissue sarcomas (STSs) are rare mesenchymal tumors that arise from muscle, fat and connective tissue. Currently, over 75 subtypes of STS are recognized. The rarity and heterogeneity of patient samples complicate clinical investigations into sarcoma biology. Model organisms might provide traction to our understanding and treatment of the disease. Over the past 10 years, many successful animal models of STS have been developed, primarily genetically engineered mice and zebrafish. These models are useful for studying the relevant oncogenes, signaling pathways and other cell changes involved in generating STSs. Recently, these model systems have become preclinical platforms in which to evaluate new drugs and treatment regimens. Thus, animal models are useful surrogates for understanding STS disease susceptibility and pathogenesis as well as for testing potential therapeutic strategies.

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.

The adoptive transfer of donor T cells that recognize recipient minor histocompatibility antigens (mHAgs) is a potential strategy for preventing or treating leukemic relapse after allogeneic hematopoietic cell transplantation (HCT). A total of 7 patients with recurrent leukemia after major histocompatibility complex (MHC)–matched allogeneic HCT were treated with infusions of donor-derived, ex vivo–expanded CD8+ cytotoxic T lymphocyte (CTL) clones specific for tissue-restricted recipient mHAgs. The safety of T-cell therapy, in vivo persistence of transferred CTLs, and disease response were assessed. Molecular characterization of the mHAgs recognized by CTL clones administered to 3 patients was performed to provide insight into the antileukemic activity and safety of T-cell therapy. Pulmonary toxicity of CTL infusion was seen in 3 patients, was severe in 1 patient, and correlated with the level of expression of the mHAg-encoding genes in lung tissue. Adoptively transferred CTLs persisted in the blood up to 21 days after infusion, and 5 patients achieved complete but transient remissions after therapy. The results of these studies illustrate the potential to selectively enhance graft-versus-leukemia activity by the adoptive transfer of mHAg-specific T-cell clones and the challenges for the broad application of this approach in allogeneic HCT. This study has been registered at as [NCT00107354][1]. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00107354&atom=%2Fbloodjournal%2F115%2F19%2F3869.atom

Undifferentiated pleomorphic sarcoma/Malignant Fibrous Histiocytoma (MFH) is one of the most common subtypes of human soft tissue sarcoma. Using cross species genomic analysis, we define a geneset from the LSL-Kras[superscript G12D]; Trp53[superscript Flox/Flox] mouse model of soft tissue sarcoma that is highly enriched in human MFH. With this mouse geneset as a filter, we identify expression of the RAS target FOXM1 in human MFH. Expression of Foxm1 is elevated in mouse sarcomas that metastasize to the lung and tissue microarray analysis of human MFH correlates overexpression of FOXM1 with metastasis. These results suggest that genomic alterations present in human MFH are conserved in the LSL-Kras[superscript G12D]; p53[superscript Flox/Flox] mouse model of soft tissue sarcoma and demonstrate the utility of this pre-clinical model.

Purpose: Tumor regression has been observed in some patients with metastatic renal cell carcinoma (RCC) after nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Cellular and molecular characterization of antigens recognized by tumor-reactive T cells isolated from responding patients could potentially provide insight into the mechanisms of tumor regression.Experimental Design: CD8+ CTL clones that recognized a novel RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201 were isolated from two patients with metastatic RCC who experienced tumor regression or stable disease following nonmyeloablative allogeneic HCT. These clones were used to screen a cDNA library and isolate the unique cDNA encoding the antigen.Results: An alternative open reading frame in the C19orf48 gene located on chromosome 19q13 encodes the HLA-A*0201–restricted minor H antigen recognized by the RCC-reactive T cells. The differential T-cell recognition of donor- and recipient-derived target cells is attributable to a nonsynonymous single-nucleotide polymorphism within the nucleotide interval that encodes the antigenic peptide. Assays for gene expression and CTL recognition showed that the C19orf48-encoded peptide is widely expressed in renal tumors and solid tumors of other histologies. The antigenic peptide can be processed for CTL recognition via both TAP-dependent and TAP-independent pathways.Conclusions: Donor T-cell responses against the HLA-A*0201–restricted minor H antigen encoded by C19orf48 may contribute to RCC regression after MHC-matched allogeneic HCT.

Intra-operative detection of residual cancer in the tumor bed can be used to decrease the risk of a positive surgical margin, reduce the rate of re-excision, and tailor adjuvant therapy. LUM015 is a pegylated protease-activated imaging agent containing a near infrared fluorophore and quencher attached by a polypeptide linker. Using mouse models of soft tissue sarcoma (STS), we showed that LUM015 selectively localizes to the tumor and upon cleavage of the linker by proteases in the tissue, the quencher is released, allowing fluorescence to be detected. We recently completed a phase I clinical trial to test the safety of LUM015 in human patients with cancer. This open-label nonrandomized trial compared 3 dose cohorts (0.5, 1.0, and 1.5 mg/kg) of LUM015 in order to determine a safe dose of LUM015 that labels tumors in humans. A total of 15 subjects, 12 with STS and 3 with breast cancer, received IV LUM015 prior to surgical resection without any adverse pharmacological activity (APA). Quantitative fluorescence imaging of the resected tissues revealed that tumor fluorescence was significantly higher than corresponding normal tissue from the same patient (p We conducted comparative analyses of LUM015 pharmacodynamics in mouse and human subjects showing that biodistribution into tumors and activation by proteases is conserved across species. These studies revealed the presence of a novel, small molecule metabolite that correlates strongly with tissue fluorescence. Additional in vivo studies in mice and in vitro studies with mouse and human tissues showed that LUM015 is selectively distributed to and accumulates in tumors to result in increased fluorescence when compared to normal tissues. Citation Format: Melodi J. Whitley, Diana M. Cardona, Dan G. Blazer, Shelley Hwang, Rachel A. Greenup, Paul J. Mosca, Joan Cahill, Jeffrey K. Mito, Kyle C. Cuneo, Nicole Larrier, Erin O'Reilly, Ivan Spasojevic, Richard F. Riedel, William C. Eward, Linda G. Griffith, Moungi G. Bawendi, Jorge Ferrer, David B. Strasfeld, W. David Lee, Brian Brigman, David G. Kirsch. Intraoperative molecular imaging with protease-activated fluorescent imaging agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY36-03. doi:10.1158/1538-7445.AM2015-SY36-03

Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A(*)0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19(+) B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19(+) cells, and identify PANE1 as a potential therapeutic target in B-CLL.

MicroRNAs (miRNAs) can regulate tumor cell invasion and metastasis in a tumor-specific manner. We recently demonstrated that global downregulation of miRNAs after deleting dicer can promote development of distant metastases in a mouse model of primary soft tissue sarcoma (STS). In this study, we identified miRNAs that are differentially downregulated in metastatic STS in both human and mouse, and investigated the role of these miRNAs in metastasis. miRNA- TaqMan PCR arrays showed a global downregulation of miRNAs in metastatic human sarcomas. Similar analysis in mouse metastatic sarcomas revealed overlap for several downregulated miRNAs including miR-16, miR-103, miR-146a, miR-223, miR-342 and miR-511. Restoration of these downregulated miRNAs in mouse primary sarcoma cell lines showed that miR-16, but not other downregulated miRNAs, was able to significantly suppress both migration and invasion in vitro, without altering cell proliferation. In addition, orthotopic transplantation of a sarcoma cell line stably expressing miR-16 into the muscle of immunocompromised mice revealed that restoration of miR-16 can significantly decrease lung metastasis in vivo. However, no change in the rate of lung metastasis was observed when miR-16 was deleted in mouse primary sarcomas at sarcoma initiation. Taken together, these results indicate that miR-16 can have metastasis-suppressing properties both in vitro and in vivo. However, the loss-of-function experiments in autochthonous tumors indicate that loss of miR-16 is not sufficient to promote metastasis in vivo., Summary: This work underscores the importance of studying cancer metastasis using in vivo model systems in addition to in vitro and transplant model systems.

Limb-sparing surgery for extremity soft tissue sarcoma removes all cancer cells at the primary tumor site in the majority of patients. However, without radiation therapy, microscopic residual sarcoma cells left behind in the tumor bed will cause a tumor recurrence in approximately one-third of patients. Therefore, adjuvant radiation therapy is delivered to most patients, even when the tumor bed lacks residual cancer. Here, we present an imaging system for residual cancer assessment, consisting of a novel wide field-of-view imaging device and a protease-activated fluorescent probe. We demonstrate that this system directly images microscopic residual sarcoma cells in the tumor bed of mice when primary soft tissue sarcomas are resected. Moreover, this system can detect single tumor cells that have activated the fluorescent probe in vivo. This technology has the potential to be used as an intra-operative tool to identify microscopic residual disease for soft tissue sarcoma and other cancers with the goal of reducing rates of local recurrence, re-operation for positive margins, and adjuvant radiation therapy to tumor beds that lack residual cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4316.