Safety, Feasibility, and Merits of Longitudinal Molecular Testing of Multiple Metastatic Sites to Inform mTNBC Patient Treatment in the Intensive Trial of Omics in Cancer.

PURPOSE: Patients with metastatic triple-negative breast cancer (mTNBC) have poor outcomes. The Intensive Trial of Omics in Cancer (ITOMIC) sought to determine the feasibility and potential efficacy of informing treatment decisions through multiple biopsies of mTNBC deposits longitudinally over time, accompanied by analysis using a distributed network of experts. METHODS: Thirty-one subjects were enrolled and 432 postenrollment biopsies performed (clinical and study-directed) of which 332 were study-directed. Molecular profiling included whole-genome sequencing or whole-exome sequencing, cancer-associated gene panel sequencing, RNA-sequencing, and immunohistochemistry. To afford time for analysis, subjects were initially treated with cisplatin (19 subjects), or another treatment they had not received previously. The results were discussed at a multi-institutional ITOMIC Tumor Board, and a report transmitted to the subject's oncologist who arrived at the final treatment decision in conjunction with the subject. Assistance was provided to access treatments that were predicted to be effective. RESULTS: Multiple biopsies in single settings and over time were safe, and comprehensive analysis was feasible. Two subjects were found to have lung cancer, one had carcinoma of unknown primary site, tumor samples from three subjects were estrogen receptor-positive and from two others, human epidermal growth factor receptor 2-positive. Two subjects withdrew. Thirty-four of 112 recommended treatments were accessed using approved drugs, clinical trials, and single-patient investigational new drugs. After excluding the three subjects with nonbreast cancers and the two subjects who withdrew, 22 of 26 subjects (84.6%) received at least one ITOMIC Tumor Board-recommended treatment. CONCLUSION: Further exploration of this approach in patients with mTNBC is merited.

Differential outcomes of patients with thyroid FNA diagnoses of AUS/FLUS with and without nuclear atypia: The potential need for separation in the Bethesda System.

BACKGROUND: In the current version of The Bethesda System (TBS) for thyroid cytopathology, the atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) category has an estimated risk of malignancy of 10% to 30%. Diagnostic criteria include presence of nuclear atypia, suggestive of papillary thyroid carcinoma (PTC), as well as other types of atypia, which can be seen with non-malignant entities. Aim of this study was to investigate differential outcomes of AUS/FLUS, based on specific morphologic criteria, and assess their respective malignancy risks. METHODS: From a total of 1233 patients undergoing thyroid FNAs between 2010 and 2014 at the University of Washington, 119 had AUS/FLUS without nuclear atypia, and 64 with nuclear atypia. Outcomes for patients with and without nuclear atypia (with the exception of 24 patients lost to follow-up) were evaluated and results were compared. RESULTS: 16/57 (28.1%) patients with AUS/FLUS and nuclear atypia subsequently had carcinomas on thyroidectomy, statistically higher than the 8/102 patients (7.8%, P = .001) without nuclear atypia. When comparing only patients who underwent surgery (n = 63), again those with AUS/FLUS and nuclear atypia had statistically higher rates of carcinoma (16/31, 51.6%), compared to those without (8/32, 25%; P = .0394). Overall, 24/159 (15.1%) of patients with AUS/FLUS had carcinoma on subsequent histology. CONCLUSION: Malignancy rates for AUS/FLUS were in line with TBS estimated risks. However, our data demonstrate that the presence or absence of nuclear atypia is associated with different malignancy rates, suggesting the possibility that the AUS/FLUS category may best be split into two subcategories with different implied risks of malignancy.

Signature maps for automatic identification of prostate cancer from colorimetric analysis of H&E- and IHC-stained histopathological specimens.

Prostate cancer (PCa) is a major cause of cancer death among men. The histopathological examination of post-surgical prostate specimens and manual annotation of PCa not only allow for detailed assessment of disease characteristics and extent, but also supply the ground truth for developing of computer-aided diagnosis (CAD) systems for PCa detection before definitive treatment. As manual cancer annotation is tedious and subjective, there have been a number of publications describing methods for automating the procedure via the analysis of digitized whole-slide images (WSIs). However, these studies have focused only on the analysis of WSIs stained with hematoxylin and eosin (H&E), even though there is additional information that could be obtained from immunohistochemical (IHC) staining. In this work, we propose a framework for automating the annotation of PCa that is based on automated colorimetric analysis of both H&E and IHC WSIs stained with a triple-antibody cocktail against high-molecular weight cytokeratin (HMWCK), p63, and α-methylacyl CoA racemase (AMACR). The analysis outputs were then used to train a regression model to estimate the distribution of cancerous epithelium within slides. The approach yielded an AUC of 0.951, sensitivity of 87.1%, and specificity of 90.7% as compared to slide-level annotations, and generalized well to cancers of all grades.

Sleeping Beauty Insertional Mutagenesis Reveals Important Genetic Drivers of Central Nervous System Embryonal Tumors.

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

Quantitative comparison and reproducibility of pathologist scoring and digital image analysis of estrogen receptor ß2 immunohistochemistry in prostate cancer.

BACKGROUND: Digital image analysis offers advantages over traditional pathologist visual scoring of immunohistochemistry, although few studies examining the correlation and reproducibility of these methods have been performed in prostate cancer. We evaluated the correlation between digital image analysis (continuous variable data) and pathologist visual scoring (quasi-continuous variable data), reproducibility of each method, and association of digital image analysis methods with outcomes using prostate cancer tissue microarrays (TMAs) stained for estrogen receptor-ß2 (ERß2). METHODS: Prostate cancer TMAs were digitized and evaluated by pathologist visual scoring versus digital image analysis for ERß2 staining within tumor epithelium. Two independent analysis runs were performed to evaluate reproducibility. Image analysis data were evaluated for associations with recurrence-free survival and disease specific survival following radical prostatectomy. RESULTS: We observed weak/moderate Spearman correlation between digital image analysis and pathologist visual scores of tumor nuclei (Analysis Run A: 0.42, Analysis Run B: 0.41), and moderate/strong correlation between digital image analysis and pathologist visual scores of tumor cytoplasm (Analysis Run A: 0.70, Analysis Run B: 0.69). For the reproducibility analysis, there was high Spearman correlation between pathologist visual scores generated for individual TMA spots across Analysis Runs A and B (Nuclei: 0.84, Cytoplasm: 0.83), and very high correlation between digital image analysis for individual TMA spots across Analysis Runs A and B (Nuclei: 0.99, Cytoplasm: 0.99). Further, ERß2 staining was significantly associated with increased risk of prostate cancer-specific mortality (PCSM) when quantified by cytoplasmic digital image analysis (HR 2.16, 95 % CI 1.02-4.57, p = 0.045), nuclear image analysis (HR 2.67, 95 % CI 1.20-5.96, p = 0.016), and total malignant epithelial area analysis (HR 5.10, 95 % CI 1.70-15.34, p = 0.004). After adjusting for clinicopathologic factors, only total malignant epithelial area ERß2 staining was significantly associated with PCSM (HR 4.08, 95 % CI 1.37-12.15, p = 0.012). CONCLUSIONS: Digital methods of immunohistochemical quantification are more reproducible than pathologist visual scoring in prostate cancer, suggesting that digital methods are preferable and especially warranted for studies involving large sample sizes.

A Distributed Network for Intensive Longitudinal Monitoring in Metastatic Triple-Negative Breast Cancer.

Accelerating cancer research is expected to require new types of clinical trials. This report describes the Intensive Trial of OMics in Cancer (ITOMIC) and a participant with triple-negative breast cancer metastatic to bone, who had markedly elevated circulating tumor cells (CTCs) that were monitored 48 times over 9 months. A total of 32 researchers from 14 institutions were engaged in the patient's evaluation; 20 researchers had no prior involvement in patient care and 18 were recruited specifically for this patient. Whole-exome sequencing of 3 bone marrow samples demonstrated a novel ROS1 variant that was estimated to be present in most or all tumor cells. After an initial response to cisplatin, a hypothesis of crizotinib sensitivity was disproven. Leukapheresis followed by partial CTC enrichment allowed for the development of a differential high-throughput drug screen and demonstrated sensitivity to investigational BH3-mimetic inhibitors of BCL-2 that could not be tested in the patient because requests to the pharmaceutical sponsors were denied. The number and size of CTC clusters correlated with clinical status and eventually death. Focusing the expertise of a distributed network of investigators on an intensively monitored patient with cancer can generate high-resolution views of the natural history of cancer and suggest new opportunities for therapy. Optimization requires access to investigational drugs.

Detection of Prostate Cancer: Quantitative Multiparametric MR Imaging Models Developed Using Registered Correlative Histopathology.

Purpose To develop multiparametric magnetic resonance (MR) imaging models to generate a quantitative, user-independent, voxel-wise composite biomarker score (CBS) for detection of prostate cancer by using coregistered correlative histopathologic results, and to compare performance of CBS-based detection with that of single quantitative MR imaging parameters. Materials and Methods Institutional review board approval and informed consent were obtained. Patients with a diagnosis of prostate cancer underwent multiparametric MR imaging before surgery for treatment. All MR imaging voxels in the prostate were classified as cancer or noncancer on the basis of coregistered histopathologic data. Predictive models were developed by using more than one quantitative MR imaging parameter to generate CBS maps. Model development and evaluation of quantitative MR imaging parameters and CBS were performed separately for the peripheral zone and the whole gland. Model accuracy was evaluated by using the area under the receiver operating characteristic curve (AUC), and confidence intervals were calculated with the bootstrap procedure. The improvement in classification accuracy was evaluated by comparing the AUC for the multiparametric model and the single best-performing quantitative MR imaging parameter at the individual level and in aggregate. Results Quantitative T2, apparent diffusion coefficient (ADC), volume transfer constant (K(trans)), reflux rate constant (kep), and area under the gadolinium concentration curve at 90 seconds (AUGC90) were significantly different between cancer and noncancer voxels (P < .001), with ADC showing the best accuracy (peripheral zone AUC, 0.82; whole gland AUC, 0.74). Four-parameter models demonstrated the best performance in both the peripheral zone (AUC, 0.85; P = .010 vs ADC alone) and whole gland (AUC, 0.77; P = .043 vs ADC alone). Individual-level analysis showed statistically significant improvement in AUC in 82% (23 of 28) and 71% (24 of 34) of patients with peripheral-zone and whole-gland models, respectively, compared with ADC alone. Model-based CBS maps for cancer detection showed improved visualization of cancer location and extent. Conclusion Quantitative multiparametric MR imaging models developed by using coregistered correlative histopathologic data yielded a voxel-wise CBS that outperformed single quantitative MR imaging parameters for detection of prostate cancer, especially when the models were assessed at the individual level. (©) RSNA, 2016 Online supplemental material is available for this article.

Registration of in vivo prostate MRI and pseudo-whole mount histology using Local Affine Transformations guided by Internal Structures (LATIS).

PURPOSE: To present a novel registration approach called LATIS (Local Affine Transformation guided by Internal Structures) for coregistering post prostatectomy pseudo-whole mount (PWM) pathological sections with in vivo MRI (magnetic resonance imaging) images. MATERIALS AND METHODS: Thirty-five patients with biopsy-proven prostate cancer were imaged at 3T with an endorectal coil. Excised prostate specimens underwent quarter mount step-section pathologic processing, digitization, annotation, and assembly into a PWM. Manually annotated macro-structures on both pathology and MRI were used to assist registration using a relaxed local affine transformation approximation. Registration accuracy was assessed by calculation of the Dice similarity coefficient (DSC) between transformed and target capsule masks and least-square distance between transformed and target landmark positions. RESULTS: LATIS registration resulted in a DSC value of 0.991 ± 0.004 and registration accuracy of 1.54 ± 0.64 mm based on identified landmarks common to both datasets. Image registration performed without the use of internal structures led to an 87% increase in landmark-based registration error. Derived transformation matrices were used to map regions of pathologically defined disease to MRI. CONCLUSION: LATIS was used to successfully coregister digital pathology with in vivo MRI to facilitate improved correlative studies between pathologically identified features of prostate cancer and multiparametric MRI.

Trp53 haploinsufficiency modifies EGFR-driven peripheral nerve sheath tumorigenesis.

Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1λ cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.

Evaluation of protein biomarkers of prostate cancer aggressiveness.

BACKGROUND: Prognostic multibiomarker signatures in prostate cancer (PCa) may improve patient management and provide a bridge for developing novel therapeutics and imaging methods. Our objective was to evaluate the association between expression of 33 candidate protein biomarkers and time to biochemical failure (BF) after prostatectomy. METHODS: PCa tissue microarrays were constructed representing 160 patients for whom clinicopathologic features and follow-up data after surgery were available. Immunohistochemistry for each of 33 proteins was quantified using automated digital pathology techniques. Relationships between clinicopathologic features, staining intensity, and time to BF were assessed. Predictive modeling using multiple imputed datasets was performed to identify the top biomarker candidates. RESULTS: In univariate analyses, lymph node positivity, surgical margin positivity, non-localized tumor, age at prostatectomy, and biomarkers CCND1, HMMR, IGF1, MKI67, SIAH2, and SMAD4 in malignant epithelium were significantly associated with time to BF. HMMR, IGF1, and SMAD4 remained significantly associated with BF after adjusting for clinicopathologic features while additional associations were observed for HOXC6 and MAP4K4 following adjustment. In multibiomarker predictive models, 3 proteins including HMMR, SIAH2, and SMAD4 were consistently represented among the top 2, 3, 4, and 5 most predictive biomarkers, and a signature comprised of these proteins best predicted BF at 3 and 5 years. CONCLUSIONS: This study provides rationale for investigation of HMMR, HOXC6, IGF1, MAP4K4, SIAH2, and SMAD4 as biomarkers of PCa aggressiveness in larger cohorts.

Elevated hyaluronan and hyaluronan-mediated motility receptor are associated with biochemical failure in patients with intermediate-grade prostate tumors.

BACKGROUND: The clinical course of prostate cancer (PCa) measured by biochemical failure (BF) after prostatectomy remains unpredictable in many patients, particularly in intermediate Gleason score (GS) 7 tumors, suggesting that identification of molecular mechanisms associated with aggressive PCa biology may be exploited for improved prognostication or therapy. Hyaluronan (HA) is a high molecular weight polyanionic carbohydrate produced by synthases (HAS1 through HAS3) and fragmented by oxidative/nitrosative stress and hyaluronidases (HYAL1 through HYAL4, SPAM1) common in PCa microenvironments. HA and HA fragments interact with receptors CD44 and hyaluronan-mediated motility receptor (HMMR), resulting in increased tumor aggressiveness in experimental PCa models. This study evaluated the association of HA-related molecules with BF after prostatectomy in GS7 tumors. METHODS: Tissue microarrays were constructed from a 96-patient cohort. HA histochemistry and HAS2, HYAL1, CD44, CD44v6, and HMMR immunohistochemistry were quantified using digital pathology techniques. RESULTS: HA in tumor-associated stroma and HMMR in malignant epithelium were significantly and marginally significantly associated with time to BF in univariate analysis, respectively. After adjusting for clinicopathologic features, both HA in tumor-associated stroma and HMMR in malignant epithelium were significantly associated with time to BF. Although not significantly associated with BF, HAS2 and HYAL1 positively correlated with HMMR in malignant epithelium. Cell culture assays demonstrated that HMMR bound native and fragmented HA, promoted HA uptake, and was required for a promigratory response to fragmented HA. CONCLUSIONS: HA and HMMR are factors associated with time to BF in GS7 tumors, suggesting that increased HA synthesis and fragmentation within the tumor microenvironment stimulates aggressive PCa behavior through HA-HMMR signaling.

CBP loss cooperates with PTEN haploinsufficiency to drive prostate cancer: implications for epigenetic therapy.

Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbp(pc-/-);Pten(pc+/-) mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp(-/-); Pten(+/-) and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27(KIP1), and p21(CIP1). Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbp(pc-/-);Pten(pc+/-) mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic-targeted therapy in patients with prostate cancer.

Expression of FAP, ADAM12, WISP1, and SOX11 is heterogeneous in aggressive fibromatosis and spatially relates to the histologic features of tumor activity.

Aggressive fibromatosis (AF) represents a group of tumors with a variable and unpredictable clinical course, characterized by a monoclonal proliferation of myofibroblastic cells. The optimal treatment for AF remains unclear. Identification and validation of genes whose expression patterns are associated with AF may elucidate biological mechanisms in AF, and aid treatment selection. This study was designed to examine the protein expression by immunohistochemistry (IHC) of four genes, ADAM12, FAP, SOX11, and WISP1, that were found in an earlier study to be uniquely overexpressed in AF compared with normal tissues. Digital image analysis was performed to evaluate inter- and intratumor heterogeneity, and correlate protein expression with histologic features, including a histopathologic assessment of tumor activity, defined by nuclear chromatin density ratio (CDR). AF tumors exhibited marked inter- and intratumor histologic heterogeneity. Pathologic assessment of tumor activity and digital assessment of average nuclear size and CDR were all significantly correlated. IHC revealed protein expression of all four genes. IHC staining for ADAM12, FAP, and WISP1 correlated with CDR and was higher, whereas SOX11 staining was lower in tumors with earlier recurrence following excision. All four proteins were expressed, and the regional variation in tumor activity within and among AF cases was demonstrated. A spatial correlation between protein expression and nuclear morphology was observed. IHC also correlated with the probability of recurrence following excision. These proteins may be involved in AF pathogenesis and the corresponding pathways could serve as potential targets of therapy.

Activation of the FGFR-STAT3 pathway in breast cancer cells induces a hyaluronan-rich microenvironment that licenses tumor formation.

Aberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth, progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blocking hyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-mediated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a protumorigenic microenvironment.

Effects of topical copper tripeptide complex on wound healing in an irradiated rat model.

OBJECTIVES: To evaluate clinical and immunohistopathological effects of topical glycyl-histidyl-lysine-copper (GHK-Cu) on in vivo irradiated rat wounds. DESIGN: Animal model. SETTING: Academic institution. SUBJECTS AND METHODS: After dorsal irradiation and a 28-day recovery period, 2 × 8 cm cranially based dorsal flaps were created in Sprague-Dawley rats. Twice daily GHK-Cu gel (test) or aquaphilic ointment (control) was applied for 10 days. Animals were euthanized, digital images of flaps were taken, and harvested tissues were immunohistochemically stained for a vascular endothelium marker, caveolin-1, and vascular endothelial growth factor (VEGF). Digital image analysis was used for outcome measures. Unpaired t-tests were used for statistical analyses; significance of P < .01 accounted for multiple comparisons. RESULTS: By digital analysis of clinical images, 13 test and 10 control animals showed mean ischemic areas of 5.0 cm(2) (SD = 0.9) for tests and 3.8 cm(2) (SD = 1.1; P = .011) for controls. Whole slide digitized images allowed quantification of caveolin-1-stained blood vessels and VEGF expression in fibroblasts at the interface of healing flaps. Caveolin-1 analyses showed a mean of 209.0 vessels (SD = 111.1) and a mean vessel luminal area of 525.7 um(2) (SD = 191.0) in tests and 207.4 vessels (SD = 109.4; P = .973) and 422.8 um(2) (SD = 109.7; P = .118) in controls. VEGF quantified as the percentage of pixels exceeding a colorimetric threshold, with higher fractions of positive pixels indicating more intense staining, showed a mean intensity score of 0.34 (SD = 0.19) in tests and 0.54 (SD = 0.41; P = .169) in controls. CONCLUSIONS: Irradiated dorsal rat flaps treated with topical GHK-Cu gel demonstrated no difference in flap ischemia, blood vessel number or area, or VEGF expression compared to controls.

Expression of FGFR3 and FGFR4 and clinical risk factors associated with progression-free survival in synovial sarcoma.

Although rare, synovial sarcoma (SS) is one of the most common soft tissue sarcomas affecting young adults. To investigate potential tumor markers related to synovial sarcoma prognosis, we carried out a single-institution retrospective analysis of 103 patients diagnosed with SS between 1980 and 2009. Clinical outcome data were obtained from medical records, and archived tissue samples were used to evaluate the relationship between progression-free survival (PFS) and several prognostic factors, including tumor expression of FGFR3 and FGFR4. No associations were found between PFS and gender, body mass index, tumor site, SS18-SSX translocation, or FGFR4 expression. As seen in previous studies, age at diagnosis (<35, 63% versus ≥35 years, 31% 10-year PFS; P = .033), histologic subtype (biphasic, 75% versus monophasic 34% 10-year PFS; P = .034), and tumor size (≤5 cm, 70% versus >5 cm, 22% 10-year PFS; P < .0001) were associated with PFS in SS patients. In addition, in a subset of patients with available archived tumor samples taken prior to chemotherapy or radiation (n = 34), higher FGFR3 expression was associated with improved PFS (P = .030). To the best of our knowledge, this is the largest study of SS to date to suggest a potential clinical role for FGFR3. While small numbers make this investigation somewhat exploratory, the findings merit future investigation on a larger scale.

Canonical Wnt/ß-catenin signaling drives human schwann cell transformation, progression, and tumor maintenance.

Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of ß-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.