Patient Priorities Concerning Treatment Decisions for Advanced Neuroendocrine Tumors Identified by Discrete Choice Experiments.

BACKGROUND: Patients with advanced neuroendocrine tumors (NETs) have multiple treatment options. Ideally, treatment decisions are shared between physician and patient; however, previous studies suggest that oncologists and patients place different value on treatment attributes such as adverse event (AE) rates. High-quality information on NET patient treatment preferences may facilitate patient-centered decision making by helping clinicians understand patient priorities. METHODS: This study used 2 discrete choice experiments (DCE) to elicit preferences of NET patients regarding advanced midgut and pancreatic NET (pNET) treatments. The DCEs used the "potentially all pairwise rankings of all possible alternatives" (PAPRIKA) method. The primary objective was to determine relative utility rankings for treatment attributes, including progression-free survival (PFS), treatment modality, and AE rates. Ranking of attribute profiles matching specific treatments was also determined. Levels for treatment attributes were obtained from randomized clinical trial data of NET treatments. RESULTS: One hundred and 10 participants completed the midgut NET DCE, and 132 completed the pNET DCE. Longer PFS was the highest ranked treatment attribute in 64.5% of participants in the midgut NET DCE, and in 59% in the pNET DCE. Approximately, 40% of participants in both scenarios prioritized lower AE rates or less invasive treatment modalities over PFS. Ranking of treatment profiles in the midgut NET scenario identified 60.9% of participants favoring peptide receptor radionuclide therapy (PRRT), and 30.0% somatostatin analogue dose escalation. CONCLUSION: NET patients have heterogeneous priorities when choosing between treatment options based on the results of 2 independent DCEs. These results highlight the importance of shared decision making for NET patients.

A Case of Primary Insular Ovarian Carcinoid Tumor with Hyperandrogenism and Carcinoid Heart Disease.

BACKGROUND Carcinoid heart disease typically occurs in the presence of metastatic carcinoid tumor deposits in the liver, as vasoactive substances access the systemic circulation through the hepatic vein. Primary ovarian carcinoid tumors are rare neuroendocrine tumors, and can be associated with carcinoid syndrome and carcinoid heart disease. CASE REPORT We describe the case of a 40-year-old woman who presented with secondary amenorrhea, acne, hirsutism, and diarrhea. She was found to have a heart murmur on exam in the absence of severe symptoms of heart failure. Her investigations demonstrated elevated urinary 5-hydroxyindoleacetic acid (5-HIAA), chromogranin A, and free testosterone. Abdominal computed tomography enterography showed a large and hypervascular pelvic mass. Octreotide scintigraphy confirmed the diagnosis of primary ovarian carcinoid tumor in the setting of an intensely octreotide-avid mass with no evidence of distant metastases. Transesophageal echocardiography showed severe tricuspid regurgitation with severe dilation of the right heart chambers. She underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy. The pathology demonstrated a 14-cm carcinoid tumor of 'insular' type confined to the ovary, pT1apNX, grade 1, positive for chromogranin and synaptophysin (neuroendocrine markers) and positive mib-1 (Ki-67). Postoperatively, clinical and biochemical parameters improved significantly but her cardiac function regressed over time, resulting in a tricuspid valve replacement 6 years later. CONCLUSIONS Primary ovarian carcinoid tumors can result in carcinoid heart disease, even in the absence of liver metastases. Early diagnosis and treatment contribute to favorable outcomes.

Changing Rates of Goals of Care Designations in Patients With Advanced Pancreatic Cancer During a Multifactorial Advanced Care Planning Initiative: A Real-World Evidence Study.

PURPOSE: Goals of care (GoC) designations are an important part of advanced care planning (ACP) for patients with incurable cancers. Studies of outpatient oncology records show that most patients do not have GoC documented. We performed a retrospective analysis of changes in GoC designations in patients with advanced pancreatic cancer in Northern Alberta, Canada, during a system-wide ACP quality improvement initiative. METHODS: Four hundred seventy-one patients with newly diagnosis of advanced, non-neuroendocrine pancreatic cancer between 2010 and 2015 in Northern Alberta, Canada, were included. The ACP initiation launched April 2014, and included educational materials for patients and families, and a coded system of GoC designations describing care philosophies and preferences for resuscitation and medical interventions. Data sources included the Alberta Cancer Registry and oncology-specific electronic medical records. RESULTS: 25.5% of patients had a documented GoC, which increased over the study period (Mantel-Haenszel test-of-trend P < .001; increased from 7.8% in 2010 to 50.0% in 2015). GoC designations occurred later in patients who received palliative chemotherapy versus those who did not (median 130 days from diagnosis [95% CI, 76.019 to 183.981] v 36 days [95% CI, 28.107 to 43.893]; P < .001), and coincided with the end of treatment (median 4.5 days from last treatment). 64.8% of GoC designations were documented by palliative care physicians, but the proportion documented by medical oncologists increased with time (Mantel-Haenszel test-of-trend P = .020; increased from 0% in 2010 to 52.1% in 2015). CONCLUSION: GoC documentation increased in the outpatient records of patients with advanced pancreatic cancer during the system-wide, multifactorial ACP initiative. GoC documentation by medical oncologists also increased. These data provide real-world evidence supporting the impact of a specific ACP initiative to improve rates of GoC designation in patients with advanced cancer.

Managing 5FU Cardiotoxicity in Colorectal Cancer Treatment.

Fluorouracil (5FU) is the backbone chemotherapy agent in the treatment of colorectal cancer (CRC). Cardiotoxicity represents an uncommon but serious side effect of treatment with 5FU. Here, we review the current literature on 5FU-cardiotoxicity in the setting of CRC specifically, with a focus on data from the modern era of combination chemotherapy. Despite decades of study, there is little consensus on risk factors and biomarkers for 5FU-cardiotoxicity, nor how patients with CRC should be managed following a cardiotoxicity event. Given the elevated risk of recurrent cardiotoxicity on rechallenge, the use of alternative regimens that do not contain 5FU is a critical aspect of management. Data on the cardiotoxicity risk and efficacy of non-5FU regimens in CRC are therefore reviewed in detail.

Prognostic Role of Immune Checkpoint Regulators in Cholangiocarcinoma: A Pilot Study.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-ß and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes.

Proposal for 'segmented peer review' of multidisciplinary papers.

We propose a new process for peer review of multidisciplinary journal submissions called 'segmented peer review'. The current translational research environment increasingly requires complex and multidisciplinary studies that span multiple distinct specialties within a single paper. Such papers present logistic and practical barriers to effective peer review. To address these barriers, papers undergoing segmented peer review require authors to explicitly i) identify each of the areas of expertise required to review the paper, ii) direct each reviewer to the relevant portions of the manuscript, and iii) suggest in-field reviewers. This segmentation of the paper is then followed by a 'segmented peer review request' tailored to the expertise of each potential reviewer, with a request to confine his / her review to those 'in-scope' aspects of the paper, while de-emphasizing any optional 'out-of-scope' comments. Each reviewer indicates the fitness for publication, or suitability for revision, of their particular segment of the manuscript. The segmented peer review process is completed when the editors integrate the segmented peer reviews. We propose segmented peer review as a fit-for-purpose process with tangible advantages for authors, reviewers, and journal editors. It should reduce the specific barriers to publication inherent in the evaluation of multidisciplinary research efforts.

Aberrant pregnancy-associated plasma protein-A expression in breast cancers prognosticates clinical outcomes.

Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.

Rearrangement bursts generate canonical gene fusions in bone and soft tissue tumors.

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between EWSR1, a gene encoding an RNA binding protein, and E26 transformation-specific (ETS) transcription factors. We explored how and when EWSR1-ETS fusions arise by studying the whole genomes of Ewing sarcomas. In 52 of 124 (42%) of tumors, the fusion gene arises by a sudden burst of complex, loop-like rearrangements, a process called chromoplexy, rather than by simple reciprocal translocations. These loops always contained the disease-defining fusion at the center, but they disrupted multiple additional genes. The loops occurred preferentially in early replicating and transcriptionally active genomic regions. Similar loops forming canonical fusions were found in three other sarcoma types. Chromoplexy-generated fusions appear to be associated with an aggressive form of Ewing sarcoma. These loops arise early, giving rise to both primary and relapse Ewing sarcoma tumors, which can continue to evolve in parallel.

Monitoring Immune Checkpoint Regulators as Predictive Biomarkers in Hepatocellular Carcinoma.

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3, and VISTA were independently associated with worse prognosis, while B7-H4, CD73, and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73, and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB.

The developmental programme for genesis of the entire kidney is recapitulated in Wilms tumour.

Wilms tumour (WT) is an embryonal tumour that recapitulates kidney development. The normal kidney is formed from two distinct embryological origins: the metanephric mesenchyme (MM) and the ureteric bud (UB). It is generally accepted that WT arises from precursor cells in the MM; however whether UB-equivalent structures participate in tumorigenesis is uncertain. To address the question of the involvement of UB, we assessed 55 Wilms tumours for the molecular features of MM and UB using gene expression profiling, immunohistochemsitry and immunofluorescence. Expression profiling primarily based on the Genitourinary Molecular Anatomy Project data identified molecular signatures of the UB and collecting duct as well as those of the proximal and distal tubules in the triphasic histology group. We performed immunolabeling for fetal kidneys and WTs. We focused on a central epithelial blastema pattern which is the characteristic of triphasic histology characterized by UB-like epithelial structures surrounded by MM and MM-derived epithelial structures, evoking the induction/aggregation phase of the developing kidney. The UB-like epithelial structures and surrounding MM and epithelial structures resembling early glomerular epithelium, proximal and distal tubules showed similar expression patterns to those of the developing kidney. These observations indicate WTs can arise from a precursor cell capable of generating the entire kidney, such as the cells of the intermediate mesoderm from which both the MM and UB are derived. Moreover, this provides an explanation for the variable histological features of mesenchymal to epithelial differentiation seen in WT.

Impact of Reflex EGFR/ ALK Testing on Time to Treatment of Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer.

PURPOSE: Optimal first-line systemic therapy for patients with advanced nonsquamous (nonsq) non-small-cell lung cancer (NSCLC) requires confirmation of EGFR/ ALK status, which can delay treatment. We evaluated the impact of reflex testing, defined as pathologists initiating EGFR/ ALK testing at the time of diagnosis of nonsq NSCLC, on time to treatment (TTT). METHODS: We conducted a retrospective review of patients with nonsq NSCLC with medical oncology consultation at Sunnybrook Odette Cancer Centre between March 18, 2010 and April 30, 2014. Data were compared during routine and reflex testing. TTT was defined as the interval between the first medical oncology visit with advanced NSCLC and the initiation of systemic therapy. RESULTS: A total of 306 patients were included (n = 232 for routine testing, n = 74 for reflex testing). There was a trend to improvement in median TTT with reflex testing (36 days [interquartile range {IQR}, 16 to 71 days v 26 days [IQR, 8 to 41 days], P = .071). Omitting patients with intentional delays in systemic therapy for low-volume disease, poor performance status, comorbidity management, and/or radiation therapy, median TTT improved (34 days [IQR, 15 to 67 days] v 22 days [IQR, 8 to 42 days], P = .049). Time to optimal first-line systemic therapy according to published guidelines improved (median, 36 days [IQR, 16 to 91 days] v 24 days [IQR, 8 to 43 days], P = .036). There was no impact on receipt of any first-line systemic therapy (55% v 59%, P = .66). The quality of biomarker testing improved, with fewer unsuccessful tests ( EGFR, 14% v 4%, P = .039; and ALK, 17% v 3%, P = .037). CONCLUSION: Reflex testing of EGFR/ ALK improved the time to optimal systemic therapy and the quality of biomarker testing for patients with advanced nonsq NSCLC.

Systems analysis identifies miR-29b regulation of invasiveness in melanoma.

BACKGROUND: In many cancers, microRNAs (miRs) contribute to metastatic progression by modulating phenotypic reprogramming processes such as epithelial-mesenchymal plasticity. This can be driven by miRs targeting multiple mRNA transcripts, inducing regulated changes across large sets of genes. The miR-target databases TargetScan and DIANA-microT predict putative relationships by examining sequence complementarity between miRs and mRNAs. However, it remains a challenge to identify which miR-mRNA interactions are active at endogenous expression levels, and of biological consequence. METHODS: We developed a workflow to integrate TargetScan and DIANA-microT predictions into the analysis of data-driven associations calculated from transcript abundance (RNASeq) data, specifically the mutual information and Pearson's correlation metrics. We use this workflow to identify putative relationships of miR-mediated mRNA repression with strong support from both lines of evidence. Applying this approach systematically to a large, published collection of unique melanoma cell lines - the Ludwig Melbourne melanoma (LM-MEL) cell line panel - we identified putative miR-mRNA interactions that may contribute to invasiveness. This guided the selection of interactions of interest for further in vitro validation studies. RESULTS: Several miR-mRNA regulatory relationships supported by TargetScan and DIANA-microT demonstrated differential activity across cell lines of varying matrigel invasiveness. Strong negative statistical associations for these putative regulatory relationships were consistent with target mRNA inhibition by the miR, and suggest that differential activity of such miR-mRNA relationships contribute to differences in melanoma invasiveness. Many of these relationships were reflected across the skin cutaneous melanoma TCGA dataset, indicating that these observations also show graded activity across clinical samples. Several of these miRs are implicated in cancer progression (miR-211, -340, -125b, -221, and -29b). The specific role for miR-29b-3p in melanoma has not been well studied. We experimentally validated the predicted miR-29b-3p regulation of LAMC1 and PPIC and LASP1, and show that dysregulation of miR-29b-3p or these mRNA targets can influence cellular invasiveness in vitro. CONCLUSIONS: This analytic strategy provides a comprehensive, systems-level approach to identify miR-mRNA regulation in high-throughput cancer data, identifies novel putative interactions with functional phenotypic relevance, and can be used to direct experimental resources for subsequent experimental validation. Computational scripts are available: http://github.com/uomsystemsbiology/LMMEL-miR-miner.

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

BACKGROUND: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. METHODS: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. RESULTS: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. CONCLUSION: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

The Distribution and Cellular Lineages of XX and XY Cells in Gonads Associated with Ovotesticular Disorder of Sexual Development.

Individuals with a 46,XX/46,XY karyotype are categorized as ovotesticular disorder of sexual development (ODSD) and have gonads with either an ovary on one side and a testis on the other side or a mixed ovotestis. To examine the distribution of 46,XX and 46,XY cells in gonads of 3 patients with ODSD, FISH for X and Y chromosomes and immunohistochemistry for SOX9 and FOXL2 were carried out. FISH analysis showed that XX signals were present in Sertoli cells in the seminiferous tubules, while cells containing Y signals were seen in epithelia of ovarian follicles. The immunolabeling of SOX9 and FOXL2 in the seminiferous tubules and ovarian follicles was mutually exclusive, irrespective of the presence of reversed sex chromosomes. We therefore suggest that the fate of individual gonadal epithelial cells is determined not only by the sex chromosomes but also by local environmental factors.

Identifying and targeting determinants of melanoma cellular invasion.

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells.

BACKGROUND: The metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell's phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype. METHODS: The expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2'-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells. RESULTS: Expression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells. CONCLUSIONS: Our data provide evidence for an important role of TKTL1 in aerobic glycolysis and tumor promotion in melanoma that may result from defective promoter methylation. This epigenetic change may enable the natural selection of tumor cells with a metabolic phenotype and thereby provide a potential therapeutic target for a subset of melanoma tumors with elevated TKTL1 expression.

Mismatch in epitope specificities between IFNγ inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.

BACKGROUND: A current focus in cancer treatment is to broaden responses to immunotherapy. One reason these therapies may prove inadequate is that T lymphocytes fail to recognize the tumor due to differences in immunogenic epitopes presented by the cancer cells under inflammatory or non-inflammatory conditions. The antigen processing machinery of the cell, the proteasome, cleaves proteins into peptide epitopes for presentation on MHC complexes. Immunoproteasomes in inflammatory melanomas, and in antigen presenting cells of the immune system, are enzymatically different to standard proteasomes expressed by tumors with no inflammation. This corresponds to alterations in protein cleavage between proteasome subtypes, and a disparate repertoire of MHC-presented epitopes. METHODS: We assessed steady state and IFNγ-induced immunoproteasome expression in melanoma cells. Using epitope specific T-lymphocyte clones, we studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Our experimental model allowed comparison of the processing of three distinct epitopes from a single antigen presented on the same HLA complex. We further investigated processing of these epitopes by direct inhibition, or siRNA mediated knockdown, of the immunoproteasome catalytic subunit LMP7. RESULTS: Our data demonstrated a profound difference in the way in which immunogenic T-lymphocyte epitopes are presented by melanoma cells under IFNγ inflammatory versus non-inflammatory conditions. These alterations led to significant changes in the ability of T-lymphocytes to recognize and target melanoma cells. CONCLUSIONS: Our results illustrate a little-studied mechanism of immune escape by tumor cells which, with appropriate understanding and treatment, may be reversible. These data have implications for the design of cancer vaccines and adoptive T cell therapies.

Mycoplasma Infection Alters Cancer Stem Cell Properties in Vitro.

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

Pregnancy associated plasma protein-A links pregnancy and melanoma progression by promoting cellular migration and invasion.

Melanoma is the most common cancer diagnosed in pregnant women and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. Here, we report that melanoma migration, invasiveness and progression are promoted by Pregnancy-Associated Plasma Protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of IGF1 by cleaving it from a circulating complex formed with IGFBP4. We show that PAPPA is widely expressed by metastatic melanoma tumors and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in vivo within the embryonic chicken neural tube. PAPPA-enriched pregnancy serum treatment enhanced melanoma motility in vitro. Furthermore, we report that IGF1 can induce the phenotypic and functional effects of epithelial-to-mesenchymal transition (EMT) in melanoma cells. In this study, we establish a clear relationship between a pregnancy-associated protein PAPPA, melanoma and functional effects mediated through IGF1 that provides a plausible mechanism for accelerated melanoma progression during pregnancy. This opens the possibility of targeting the PAPPA/IGF1 axis therapeutically.

Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma.

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.