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1.  The Role of Erythropoietin and Bone Marrow Concentrate in the Treatment of Osteochondral Defects in Mini-Pigs 
PLoS ONE  2014;9(3):e92766.
Background
All available treatment options for osteochondral and chondral defects do not restore hyaline cartilage and are limited to decreasing associated pain, and maintaining or improving joint function. The purpose of this study was to evaluate the potential of erythropoietin (EPO) in combination with bone marrow aspiration concentrate (BMAC) in the treatment of osteochondral defects of mini-pigs.
Methods
14 Goettinger mini-pigs, in which a 6×10 mm osteochondral defect in the medial femoral condyle of both knee joints was created, were randomized into four groups: biphasic scaffold alone, scaffold with EPO, scaffold with BMAC and scaffold in combination with EPO and BMAC. After 26 weeks all animals were euthanized and histological slides were evaluated using a modified ÓDriscoll Score.
Results
In the therapy groups, areas of chondrogenic tissue that contained collagen II were present. Adding EPO (p = 0.245) or BMAC (p = 0.099) alone to the scaffold led to a non-significant increase in the score compared to the control group. However, the combination of EPO and BMAC in the implanted scaffold showed a significant improvement (p = 0.02) in the histological score.
Conclusion
The results of our study show that in mini-pigs, the combination of EPO and BMAC leads to an enhanced osteochondral healing. However, additional research is necessary to further improve the repair tissue and to define the role of MSCs and EPO in cartilage repair.
doi:10.1371/journal.pone.0092766
PMCID: PMC3968023  PMID: 24676029
2.  Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B along the Juan de Fuca Ridge flank 
To expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.
doi:10.3389/fmicb.2014.00119
PMCID: PMC3971187  PMID: 24723917
deep subsurface; microorganisms; diversity; Juan de Fuca Ridge; SSU ribosomal RNA gene; Ocean Drilling Program
3.  NY-ESO-1 antibody as a novel tumour marker of gastric cancer 
British Journal of Cancer  2013;108(5):1119-1125.
Background:
NY-ESO-1 antibodies are specifically observed in patients with NY-ESO-1-expressing tumours. We analysed whether the NY-ESO-1 humoral immune response is a useful tumour marker of gastric cancer.
Methods:
Sera from 363 gastric cancer patients were screened by enzyme-linked immunosorbent assay (ELISA) to detect NY-ESO-1 antibodies. Serial serum samples were obtained from 25 NY-ESO-1 antibody-positive patients, including 16 patients with curative resection and 9 patients who received chemotherapy alone.
Results:
NY-ESO-1 antibodies were detected in 3.4% of stage I, 4.4% of stage II, 25.3% of stage III, and 20.0% of stage IV patients. The frequency of antibody positivity increased with disease progression. When the NY-ESO-1 antibody was used in combination with carcinoembryonic antigen and CA19-9 to detect gastric cancer, information gains of 11.2% in stages III and IV, and 5.8% in all patients were observed. The NY-ESO-1 immune response levels of the patients without recurrence fell below the cutoff level after surgery. Two of the patients with recurrence displayed incomplete decreases. The nine patients who received chemotherapy alone continued to display NY-ESO-1 immune responses.
Conclusion:
When combined with conventional tumour markers, the NY-ESO-1 humoral immune response could be a useful tumour marker for detecting advanced gastric cancer and inferring the post-treatment tumour load in seropositive patients.
doi:10.1038/bjc.2013.51
PMCID: PMC3619069  PMID: 23403818
surgical treatment; detection marker; follow-up marker; recurrence; prognosis
4.  Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies 
Brain  2013;136(2):508-521.
The β-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and β-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of β-tropomyosin-related myopathies through the identification of a novel β-tropomyosin mutation in two clinical contexts not previously associated with β-tropomyosin. The first clinical phenotype is core-rod myopathy, with a β-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of β-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric β-tropomyosin, a change predicted to alter protein–protein binding between β-tropomyosin and other molecules and to disturb head-to-tail polymerization of β-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del β-tropomyosin in the developing zebrafish. p.K7del β-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail β-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel β-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with β-tropomyosin and pathogenic data from the first animal model of β-tropomyosin-related myopathies.
doi:10.1093/brain/aws344
PMCID: PMC3572924  PMID: 23413262
nemaline; myopathies; muscle and nerve pathology; mutation; neuromuscular disorders
5.  Prevalence and Type of BRCA Mutations in Hispanics Undergoing Genetic Cancer Risk Assessment in the Southwestern United States: A Report From the Clinical Cancer Genetics Community Research Network 
Journal of Clinical Oncology  2012;31(2):210-216.
Purpose
To determine the prevalence and type of BRCA1 and BRCA2 (BRCA) mutations among Hispanics in the Southwestern United States and their potential impact on genetic cancer risk assessment (GCRA).
Patients and Methods
Hispanics (n = 746) with a personal or family history of breast and/or ovarian cancer were enrolled in an institutional review board–approved registry and received GCRA and BRCA testing within a consortium of 14 clinics. Population-based Hispanic breast cancer cases (n = 492) enrolled in the Northern California Breast Cancer Family Registry, negative by sequencing for BRCA mutations, were analyzed for the presence of the BRCA1 ex9-12del large rearrangement.
Results
Deleterious BRCA mutations were detected in 189 (25%) of 746 familial clinic patients (124 BRCA1, 65 BRCA2); 21 (11%) of 189 were large rearrangement mutations, of which 62% (13 of 21) were BRCA1 ex9-12del. Nine recurrent mutations accounted for 53% of the total. Among these, BRCA1 ex9-12del seems to be a Mexican founder mutation and represents 10% to 12% of all BRCA1 mutations in clinic- and population-based cohorts in the United States.
Conclusion
BRCA mutations were prevalent in the largest study of Hispanic breast and/or ovarian cancer families in the United States to date, and a significant proportion were large rearrangement mutations. The high frequency of large rearrangement mutations warrants screening in every case. We document the first Mexican founder mutation (BRCA1 ex9-12del), which, along with other recurrent mutations, suggests the potential for a cost-effective panel approach to ancestry-informed GCRA.
doi:10.1200/JCO.2011.41.0027
PMCID: PMC3532393  PMID: 23233716
6.  Microbial diversity within basement fluids of the sediment-buried Juan de Fuca Ridge flank 
The ISME Journal  2012;7(1):161-172.
Despite its immense size, logistical and methodological constraints have largely limited microbiological investigations of the subseafloor basement biosphere. In this study, a unique sampling system was used to collect fluids from the subseafloor basaltic crust via a Circulation Obviation Retrofit Kit (CORK) observatory at Integrated Ocean Drilling Program borehole 1301A, located at a depth of 2667 m in the Pacific Ocean on the eastern flank of the Juan de Fuca Ridge. Here, a fluid delivery line directly accesses a 3.5 million years old basalt-hosted basement aquifer, overlaid by 262 m of sediment, which serves as a barrier to direct exchange with bottom seawater. At an average of 1.2 × 104 cells ml−1, microorganisms in borehole fluids were nearly an order of magnitude less abundant than in surrounding bottom seawater. Ribosomal RNA genes were characterized from basement fluids, providing the first snapshots of microbial community structure using a high-integrity fluid delivery line. Interestingly, microbial communities retrieved from different CORKs (1026B and 1301A) nearly a decade apart shared major community members, consistent with hydrogeological connectivity. However, over three sampling years, the dominant gene clone lineage changed from relatives of Candidatus Desulforudis audaxviator within the bacterial phylum Firmicutes in 2008 to the Miscellaneous Crenarchaeotic Group in 2009 and a lineage within the JTB35 group of Gammaproteobacteria in 2010, and statistically significant variation in microbial community structure was observed. The enumeration of different phylogenetic groups of cells within borehole 1301A fluids supported our observation that the deep subsurface microbial community was temporally dynamic.
doi:10.1038/ismej.2012.73
PMCID: PMC3526168  PMID: 22791235
deep subsurface; marine microorganisms; diversity; Juan de Fuca Ridge; SSU ribosomal RNA gene; IODP
7.  Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2 
Brain  2013;137(1):44-56.
Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.
doi:10.1093/brain/awt315
PMCID: PMC3891447  PMID: 24253200
childhood neuronopathy; Brown-Vialetto-Van Laere syndrome; riboflavin therapy; RFVT2; SLC52A2
9.  NY-ESO-1 is a Ubiquitous Immunotherapeutic Target Antigen for Patients with Myxoid/ Round Cell Liposarcoma 
Cancer  2012;118(18):4564-4570.
Background
Myxoid/ round cell liposarcoma (MRCL) is the second most common liposarcoma subtype, accounting for more than one third of liposarcomas and approximately 10% of all soft tissue sarcomas. Although MRCL is a chemosensitive subtype, patients with metastatic disease have a poor outcome. NY-ESO-1 is a cancer-testis antigen (also known as cancer germ cell antigen) that has been successfully targeted in vaccine and also adoptive T cell therapy trials for the treatment of several solid tumors.
Methods
We examined the feasibility of targeting NY-ESO-1 in patients with MRCL by evaluating the prevalence of NY-ESO-1 expression among tumors using immunohistochemistry and qRT-PCR. We also analyzed NY-ESO-1 specific tumor recognition by NY-ESO-1 specific T cells using chromium release assay.
Results
A search of the University of Washington Sarcoma Tissue Bank revealed paraffin embedded tumor samples from 25 patients with MRCL. NY-ESO-1 expression was observed in every MRCL tumor assessed (100%); in 18 (72%), staining was homogenous. In all but 2 cases, staining was sufficiently robust (2+) that such patients would be eligible for clinical trials of NY-ESO-1 directed therapy. Using NY-ESO-1 specific CD8+ T cells, we demonstrate in vitro sensitivity of myxoid liposarcoma cell lines to antigen-specific lysis.
Conclusions
These results establish NY-ESO-1 as an important target antigen for the treatment of patients with MRCL.
doi:10.1002/cncr.27446
PMCID: PMC3361576  PMID: 22359263
NY-ESO-1; Sarcoma; Myxoid; immunotherapy; cancer testis antigens
10.  Bone Marrow Aspiration Concentrate and Platelet Rich Plasma for Osteochondral Repair in a Porcine Osteochondral Defect Model 
PLoS ONE  2013;8(8):e71602.
Background
Bone marrow aspiration concentrate (BMAC) may possess a high potency for cartilage and osseous defect healing because it contains stem cells and multiple growth factors. Alternatively, platelet rich plasma (PRP), which contains a cocktail of multiple growth factors released from enriched activated thrombocytes may potentially stimulate the mesenchymal stem cells (MSCs) in bone marrow to proliferate and differentiate.
Methods
A critical size osteochondral defect (10×6 mm) in both medial femoral condyles was created in 14 Goettinger mini-pigs. All animals were randomized into the following four groups: biphasic scaffold alone (TRUFIT BGS, Smith & Nephew, USA), scaffold with PRP, scaffold with BMAC and scaffold in combination with BMAC and PRP. After 26 weeks all animals were euthanized and histological slides were cut, stained and evaluated using a histological score and immunohistochemistry.
Results
The thrombocyte number was significantly increased (p = 0.049) in PRP compared to whole blood. In addition the concentration of the measured growth factors in PRP such as BMP-2, BMP-7, VEGF, TGF-β1 and PDGF were significantly increased when compared to whole blood (p<0.05). In the defects of the therapy groups areas of chondrogenic tissue were present, which stained blue with toluidine blue and positively for collagen type II. Adding BMAC or PRP in a biphasic scaffold led to a significant improvement of the histological score compared to the control group, but the combination of BMAC and PRP did not further enhance the histological score.
Conclusions
The clinical application of BMAC or PRP in osteochondral defect healing is attractive because of their autologous origin and cost-effectiveness. Adding either PRP or BMAC to a biphasic scaffold led to a significantly better healing of osteochondral defects compared with the control group. However, the combination of both therapies did not further enhance healing.
doi:10.1371/journal.pone.0071602
PMCID: PMC3741121  PMID: 23951201
11.  Evaluation of a Novel Spine and Surface Topography System for Dynamic Spinal Curvature Analysis during Gait 
PLoS ONE  2013;8(7):e70581.
Introduction
The assessment of spinal deformities with rasterstereography can enhance the understanding, as well as can reduce the number of x-rays needed. However, to date this technique only allows measurements under static conditions. Since it would be of great value to be able to also analyze the spine in dynamic conditions, the present study evaluated a novel rasterstereographic system.
Materials and Methods
A new rasterstereographic device was evaluated in a comparison with the gold standard in motion analysis, the VICON system. After initial testing using 12 flat infrared markers adhered to a solid plate, the two systems were evaluated with the markers adhered onto the backs of 8 test subjects. Four triangles were defined using the markers, and the sides of each triangle were measured under static and dynamic conditions.
Results
On the solid plate, the sides of the 4 triangles were measured with a measuring tape and then by the two optical systems. Rasterstereography showed a high accuracy in marker detection on the solid plate. Under dynamic conditions, with the subjects walking on a treadmill, the rasterstereographically-measured side lengths were compared with the lengths measured by the VICON system as an assessment of marker detection. No significant differences (p>0.05) were found between the systems, differing only 0.07–1.1% for all sides of the four triangles with both systems.
Discussion
A novel rasterstereographic measurement device that allows surface and spine topography under dynamic conditions was assessed. The accuracy of this system was with one millimeter on a solid plate and during dynamic measurements, to the gold standard for motion detection. The advantage of rasterstereography is that it can be used to determine a three-dimensional surface map and also allows the analysis of the underlying spine.
doi:10.1371/journal.pone.0070581
PMCID: PMC3720906  PMID: 23894674
12.  Expression of cancer testis antigens in human BRCA-associated breast cancers: potential targets for immunoprevention? 
Introduction
Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets that are expressed in early cancers, but absent in normal breast epithelium of these high-risk individuals, could provide the basis for the development of effective immunoprophylactic strategies. Cancer testis (CT) antigens are potential candidates because their expression is restricted to tumors, and accumulating data suggest that they play important roles in cellular proliferation, stem cell function, and carcinogenesis. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers.
Methods
Archived breast cancer tissues (n = 26) as well as morphologically normal breast tissues (n = 7) from women carrying deleterious BRCA 1 and/or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1. CT7, NY-ESO-1, MAGE-C2/CT10, and GAGE.
Results
CT antigens were expressed in 16/26 (61.5%, 95% CI 43–80%) of BRCA-associated cancers, including in situ tumors. Thirteen of twenty-six (50%) breast cancers expressed two or more CT antigens; three cancers expressed all seven CT antigens. MAGE-A was expressed in 13/26 (50%) of cancers, NY-ESO-1 was expressed in 10/26 (38%) of tumors. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (P = 0.003).
Conclusions
We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.
doi:10.1007/s00262-011-1005-7
PMCID: PMC3678385  PMID: 21465317
Cancer testis antigen; NY-ESO-1; MAGE-A; Breast cancer; BRCA1/2; Vaccine; Prevention
13.  Cancer testis antigens and NY-BR-1 expression in primary breast cancer: prognostic and therapeutic implications 
BMC Cancer  2013;13:271.
Background
Cancer–testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors.
Methods
The reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival.
Results
Expression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively).
Conclusions
Our findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies.
doi:10.1186/1471-2407-13-271
PMCID: PMC3700769  PMID: 23731661
Breast Cancer; Cancer-testis Antigen; NY-BR-1; Immunotherapy; Prognosis
14.  CTLA-4 blockade increases antigen-specific CD8+ T-cells in prevaccinated patients with melanoma: three cases 
Background
Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector memory T-cell response.
Methods
To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100209–217 and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100209–217 (ITDQVPFSV), tyrosinase369–377 (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.
Results
Following vaccination, patients generated weak to no CD4+ or CD8+ T-cell response specific to the vaccine antigen but demonstrated increases in effector memory (CCR7loCD45RAlo) tetramer+CD8+ T-cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.
Conclusion
Vaccination induced a measurable antigen-specific T-cell response which increased following CTLA-4 blockade, potentially “boosting” the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.
doi:10.1007/s00262-011-1011-9
PMCID: PMC3654853  PMID: 21465316
melanoma; cytotoxic T-lymphocyte antigen-4; ipilimumab; T-cell response; vaccines
15.  Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase 
British Journal of Cancer  2012;106(9):1481-1485.
Background:
Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I–II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown.
Methods:
Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20.
Results:
Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS−), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS− patients receiving at least four doses at 320 IU m−2 was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024.
Conclusion:
ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.
doi:10.1038/bjc.2012.106
PMCID: PMC3341859  PMID: 22472884
melanoma; arginine; arginine deiminase
16.  Neuropilin-2: a novel biomarker for malignant melanoma? 
Human Pathology  2011;43(3):381-389.
Neuropilin-2 (NRP2), a cell surface receptor involved in angiogenesis and axonal guidance, has recently been shown to be a critical mediator of tumor-associated lymphangiogenesis. Given that lymphangiogenesis is a major conduit of metastasis in melanomas and that blocking NRP2 function in vivo is effective in inhibiting tumor cell metastasis, we sought to determine the clinical relevance of NRP2 expression in cutaneous melanoma. Immunohistochemical analysis of NRP2 expression was evaluated in nevomelanocytic proliferations that included a tissue microarray (TMA) and histologic sections (HS) from samples of primary melanomas (n=42; 40 TMA, 2 HS), metastatic melanomas (n=30; 22TMA, 8 HS) and nevi (n=30; 5 TMA, 25HS), as well as a panel of normal human tissues and select non-melanocytic tumors. Staining for grading and intensity of NRP2 expression was estimated semi-quantitatively as follows for the former: <20%, 20-60% and >60% of tissue present and, for the latter from 0-3 with 3 being the highest and 0 the lowest intensity. In nevomelanocytic proliferations, >20% staining for NRP2 was noted in 36/42 cases (86%) of primary melanoma, in 27/30 cases (90%) of metastatic melanoma and in 9/30 cases (30%) of nevi with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (p<0.0001). For staining intensity, an intensity of 2 or more was noted in 36/42 cases (86%) of primary melanoma, in 17/30 cases (57%) of metastatic melanoma and in 7/23 (30%) of nevi with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (p<0.0001). In normal human tissue, consistently strong NRP2 staining was noted in kidney (glomerular endothelial cells, collecting tubules and collecting ducts), skin (epidermal keratinocytes) and testes (epithelium of the seminiferous tubules), while in tumoral tissue, consistently strong staining was noted only in renal cell carcinoma but not in any of the other tumors studied. More recently, using a heterotypic co-culture methodology with melanoma and endothelial cells, we have demonstrated successful up-regulation of NRP2 and confirmed the critical role of NRP2 in melanoma-endothelial interactions. Since these co-culture methods were developed to model melanoma metastasis, the significantly increased and enhanced expression of NRP2 staining in primary and metastatic melanoma versus nevi in the current study suggests that it is also relevant in vivo.
doi:10.1016/j.humpath.2011.05.008
PMCID: PMC3246122  PMID: 21840568
17.  A Pilot Study of Anti-CTLA4 Antibody Ipilimumab in Patients with Synovial Sarcoma 
Sarcoma  2013;2013:168145.
Background. Patients with recurrent synovial sarcomas have few options for systemic therapy. Since they express large amounts of endogenous CT (cancer testis) antigens such as NY-ESO-1, we investigated the clinical activity of single agent anti-CTLA4 antibody ipilimumab in patients with advanced or metastatic synovial sarcoma. Methods. A Simon two-stage phase II design was used to determine if there was sufficient activity to pursue further. The primary endpoint was tumor response rate by RECIST 1.0. Patients were treated with ipilimumab 3 mg/kg intravenously every 3 weeks for three cycles and then restaged. Retreatment was possible for patients receiving an extra three-week break from therapy. Sera and peripheral blood mononuclear cells were collected before and during therapy to assess NY-ESO-1-specific immunity. Results. Six patients were enrolled and received 1–3 cycles of ipilimumab. All patients showed clinical or radiological evidence of disease progression after no more than three cycles of therapy, for a RECIST response rate of 0%. The study was stopped for slow accrual, lack of activity, and lack of immune response. There was no evidence of clinically significant either serologic or delayed type hypersensitivity responses to NY-ESO-1 before or after therapy. Conclusion. Despite high expression of CT antigens by synovial sarcomas of patients treated in this study, there was neither clinical benefit nor evidence of anti-CT antigen serological responses. Assessment of the ability of synovial sarcoma cell lines to present cancer-germ cell antigens may be useful in determining the reason for the observed lack of immunological or clinical activity.
doi:10.1155/2013/168145
PMCID: PMC3608267  PMID: 23554566
20.  Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae 
Eukaryotic Cell  2012;11(8):1021-1032.
In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.
doi:10.1128/EC.05240-11
PMCID: PMC3416059  PMID: 22660623
21.  Arginine deiminase PEG20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase 
British Journal of Cancer  2011;106(2):324-332.
Background:
Some cancers have been shown to lack expression of argininosuccinate synthetase (ASS), an enzyme required for the synthesis of arginine and a possible biomarker of sensitivity to arginine deprivation. Arginine deiminase (ADI) is a microbial enzyme capable of efficiently depleting peripheral blood arginine.
Methods:
Argininosuccinate synthetase expression was assessed in human small cell lung cancer (SCLC) by immunohistochemistry (IHC), with expression also assessed in a panel of 10 human SCLC by qRT-PCR and western blot. Proliferation assays and analyses of apoptosis and autophagy assessed the effect of pegylated ADI (ADI-PEG20) in vitro. The in vivo efficacy of ADI-PEG20 was determined in mice bearing SCLC xenografts.
Results:
Approximately 45% of SCLC tumours and 50% of cell lines assessed were negative for ASS. Argininosuccinate synthetase-deficient SCLC cells demonstrated sensitivity to ADI-PEG20, which was associated with the induction of autophagy and caspase-independent cell death. Arginine deiminase-PEG20 treatment of ASS-negative SCLC xenografts caused significant, dose-dependent inhibition of tumour growth of both small and established tumours.
Conclusion:
These results suggest a role for ADI-PEG20 in the treatment of SCLC, and a clinical trial exploring this therapeutic approach in patients with ASS-negative SCLC by IHC has now been initiated.
doi:10.1038/bjc.2011.524
PMCID: PMC3261683  PMID: 22134507
arginine deiminase; SCLC; argininosuccinate synthetase; in vivo; autophagy
22.  Aging-related episodic memory decline: are emotions the key? 
Episodic memory refers to the recollection of personal experiences that contain information on what has happened and also where and when these events took place. Episodic memory function is extremely sensitive to cerebral aging and neurodegerative diseases. We examined episodic memory performance with a novel test in young (N = 17, age: 21–45), middle-aged (N = 16, age: 48–62) and aged but otherwise healthy participants (N = 8, age: 71–83) along with measurements of trait and state anxiety. As expected we found significantly impaired episodic memory performance in the aged group as compared to the young group. The aged group also showed impaired working memory performance as well as significantly decreased levels of trait anxiety. No significant correlation between the total episodic memory and trait or state anxiety scores was found. The present results show an age-dependent episodic memory decline along with lower trait anxiety in the aged group. Yet, it still remains to be determined whether this difference in anxiety is related to the impaired episodic memory performance in the aged group.
doi:10.3389/fnbeh.2013.00002
PMCID: PMC3561617  PMID: 23378831
aging; anxiety; episodic memory; depression; working memory
23.  Immunologic Correlates of the Abscopal Effect in a Patient with Melanoma 
The New England Journal of Medicine  2012;366(10):925-931.
Summary
The abscopal effect is a phenomenon in which local radiotherapy is associated with the regression of metastatic cancer at a distance from the irradiated site. The abscopal effect may be mediated by activation of the immune system. Ipilimumab is a monoclonal antibody that inhibits an immunologic checkpoint on T cells, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). We report a case of the abscopal effect in a patient with melanoma treated with ipilimumab and radiotherapy. Temporal associations were noted: tumor shrinkage with antibody responses to the cancer–testis antigen NY-ESO-1, changes in peripheral-blood immune cells, and increases in antibody responses to other antigens after radiotherapy. (Funded by the National Institutes of Health and others.)
doi:10.1056/NEJMoa1112824
PMCID: PMC3345206  PMID: 22397654
24.  Cancer-testis antigen expression and immunogenicity in AL amyloidosis 
Blood Cancer Journal  2012;2(9):e90-.
Light-chain amyloidosis (AL) is a plasma cell dyscrasia closely related to multiple myeloma. In multiple myeloma, the cancer-testis antigens (CTAs) CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A CTAs are expressed in up to 80% of cases. In this study, we investigated the expression and immunogenicity of several CTAs in patients with AL amyloidosis in a total of 38 bone marrow specimens by employing standard immunohistochemistry techniques on paraffin-embedded archival tissues. Plasma samples from 35 patients (27 with matched bone marrow samples) were also analyzed by ELISA for sero reactivity to a group of full-length CTA proteins. CT7 was present in 25/38 (66%) while CT10 was demonstrated in 3/38 and GAGE in 1/38 AL amyloid cases. The expression pattern was mostly focal. There were no significant differences with regard to organ involvement, response to treatment, or prognosis in CTA positive compared to negative cases. None of the specimens showed spontaneous humoral immunity to CT7, but sero reactivity was observed in individual patients to other CTAs. This study identifies CT7 as the prevalent CTA in plasma cells of patients with AL amyloidosis. Further analyses determining the biology of CTAs in AL amyloidosis and their value as potential targets for immunotherapy are warranted.
doi:10.1038/bcj.2012.32
PMCID: PMC3461704  PMID: 22983433
AL amyloidosis; cancer-testis antigens; stem cell transplantation
25.  T cell Surveillance of Oncogene-induced Prostate Cancer is Impeded by T Cell-derived TGF-β1 Cytokine 
Immunity  2011;35(1):123-134.
Summary
Tolerance induction in T cells takes place in most tumors and is thought to account for tumor evasion from immune eradication. Production of the cytokine TGF-β is implicated in immunosuppression, however the cellular mechanism by which TGF-β induces T cell dysfunction remains unclear. Using a transgenic model of prostate cancer, we showed that tumor development was not suppressed by the adaptive immune system, which was associated with heightened TGF-β signaling in T cells from the tumor-draining lymph nodes. Blockade of TGF-β signaling in T cells enhanced tumor antigen-specific T cell responses, and inhibited tumor development. Surprisingly, T cell- but not Treg cell-specific ablation of TGF-β1 was sufficient to augment T cell cytotoxic activity and blocked tumor growth and metastases. These findings reveal that T cell production of TGF-β1 is an essential requirement for tumors to evade immunosurveillance independent of TGF-β produced by tumors.
doi:10.1016/j.immuni.2011.04.019
PMCID: PMC3430371  PMID: 21757379

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