Effect of dermatological consultation on survival in patients with checkpoint inhibitor-associated cutaneous toxicity.

BACKGROUND: Cutaneous immune-related adverse events (cirAEs) are a common side-effect of immune checkpoint inhibitors (ICIs). However, prior work examining these toxicities in detail has considered only the fraction of events evaluated by dermatologists. Associations between dermatology referral, cirAE treatment and survival outcomes remain underexplored across care settings. OBJECTIVES: To comprehensively categorize cirAE patterns among all patients treated with immunotherapy at our institution, and to evaluate: (i) the effect of dermatology referral on cirAE treatment and (ii) the impact of cirAE treatment on survival. METHODS: This was a retrospective cohort analysis of patients with cancer who initiated ICI therapy between 1 January 2016 and 8 March 2019 and developed one or more cirAEs, as screened for using International Classification of Diseases 10th revision codes and confirmed via manual chart review (n = 358). All relevant information documented prior to 31 March 2020 was included. RESULTS: CirAEs evaluated by dermatologists were significantly more likely to be treated than cirAEs that were not referred (odds ratio 6·08, P < 0·001). Patients who received any cirAE treatment had improved progression-free survival [hazard ratio (HR) 0·59, P = 0·001] and overall survival (HR 0·58, P = 0·007) compared with those who did not. CONCLUSIONS: CirAEs evaluated by dermatologists were significantly more likely to be treated than cirAEs that were not referred, and patients who received any treatment for a cirAE had improved survival outcomes.

Heparin/collagen surface coatings modulate the growth, secretome, and morphology of human mesenchymal stromal cell response to interferon-gamma.

The therapeutic potential of human mesenchymal stromal cells (h-MSC) is dependent on the viability and secretory capacity of cells both modulated by the culture environment. Our previous studies introduced heparin and collagen I (HEP/COL) alternating stacked layers as a potential substrate to enhance the secretion of immunosuppressive factors of h-MSCs. Herein, we examined the impact of HEP/COL multilayers on the growth, morphology, and secretome of bone marrow and adipose-derived h-MSCs. The physicochemical properties and stability of the HEP/COL coatings were confirmed at 0 and 30 days. Cell growth was examined using cell culture media supplemented with 2 and 10% serum for 5 days. Results showed that HEP/COL multilayers supported h-MSC growth in 2% serum at levels equivalent to 10% serum. COL and HEP as single component coatings had limited impact on cell growth. Senescent studies performed over three sequential passages showed that HEP/COL multilayers did not impair the replicative capacity of h-MSCs. Examination of 27 cytokines showed significant enhancements in eight factors, including intracellular indoleamine 2, 3-dioxygenase, on HEP/COL multilayers when stimulated with interferon-gamma (IFN-γ). Image-based analysis of cell micrographs showed that serum influences h-MSC morphology; however, HEP-ended multilayers generated distinct morphological changes in response to IFN-γ, suggesting an optical detectable assessment of h-MSCs immunosuppressive potency. This study supports HEP/COL multilayers as a culture substrate for undifferentiated h-MSCs cultured in reduced serum conditions.

Current Views on Clinical Oncology Training from the 2015 Oncology Registrars' Forum Survey.

The major role of the Oncology Registrars' Forum (ORF) of the Royal College of Radiologists is to voice the opinions of the clinical oncology trainee body and work towards improving all aspects of clinical oncology training in the UK. In order to provide data to support these efforts, the ORF undertakes a biennial survey of all trainees. As with the previous surveys, this year's ORF survey produced data that highlight areas of good training as well as new and ongoing areas of concern. This summary highlights the key survey results and provides recommendations for improving the delivery of clinical oncology training in the UK.

LNK (SH2B3): paradoxical effects in ovarian cancer.

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography-mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.

PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells.

BACKGROUND: The PIAS4 protein belongs to the family of protein inhibitors of activated STAT, but has since been implicated in various biological activities including the post-translational modification known as sumoylation. In this study, we explored the roles of PIAS4 in pancreatic tumourigenesis. METHODS: The expression levels of PIAS4 in pancreatic cancer cells were examined. Cell proliferation and invasion was studied after overexpression and gene silencing of PIAS4. The effect of PIAS4 on hypoxia signalling was investigated. RESULTS: The protein was overexpressed in pancreatic cancer cells compared with the normal pancreas. Gene silencing by PIAS4 small interfering RNA (siRNA) suppressed pancreatic cancer cell growth and overexpression of PIAS4 induced expression of genes related to cell growth. The overexpression of PIAS4 is essential for the regulation of the hypoxia signalling pathway. PIAS4 interacts with the tumour suppressor von Hippel-Lindau (VHL) and leads to VHL sumoylation, oligomerization, and impaired function. Pancreatic cancer cells (Panc0327, MiaPaCa2) treated with PIAS4 siRNA suppressed expression of the hypoxia-inducible factor hypoxia-inducible factor 1 alpha and its target genes JMJD1A, VEGF, and STAT3. CONCLUSION: Our study elucidates the role of PIAS4 in the regulation of pancreatic cancer cell growth, where the suppression of its activity represents a novel therapeutic target for pancreatic cancers.

Overexpression of miR-26a-2 in human liposarcoma is correlated with poor patient survival.

Approximately 90% of well-differentiated/de-differentiated liposarcomas (WDLPS/DDLPS), the most common LPS subtype, have chromosomal amplification at 12q13-q22. Many protein-coding genes in the region, such as MDM2 and , have been studied as potential therapeutic targets for LPS treatment, with minimal success. In the amplified region near the MDM2 gene, our single nucleotide polymorphism (SNP) array analysis of 75 LPS samples identified frequent amplification of miR-26a-2. Besides being in the amplicon, miR-26a-2 was overexpressed significantly in WDLPS/DDLPS (P<0.001), as well as in myxoid/round cell LPS (MRC) (P<0.05). Furthermore, Kaplan-Meier survival analysis showed that overexpression of miR-26a-2 significantly correlated with poor patient survival in both types of LPS (P<0.05 for WDLPS/DDLPS; P<0.001 for MRC). Based on these findings, we hypothesized that miR-26a-2 has an important role in LPS tumorigenesis, regardless of LPS subtypes. Overexpression of miR-26a-2 in three LPS cell lines (SW872, LPS141 and LP6) enhanced the growth and survival of these cells, including faster cell proliferation and migration, enhanced clonogenicity, suppressed adipocyte differentiation and/or resistance to apoptosis. Inhibition of miR-26a-2 in LPS cells using anti-miR-26a-2 resulted in the opposite responses. To explain further the effect of miR-26a-2 overexpression in LPS cells, we performed in silico analysis and identified 93 candidate targets of miR-26a-2. Among these genes, RCBTB1 (regulator of chromosome condensation and BTB domain-containing protein 1) is located at 13q12.3-q14.3, a region of recurrent loss of heterozygosity (LOH) in LPS. Indeed, either overexpression or inhibition of RCBTB1 made LPS cells more susceptible or resistant to apoptosis, respectively. In conclusion, our study for the first time reveals the contribution of miR-26a-2 to LPS tumorigenesis, partly through inhibiting RCBTB1, suggesting that miR-26a-2 is a novel therapeutic target for human LPS.

miR-34a functions as a tumor suppressor modulating EGFR in glioblastoma multiforme.

Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1, -B1, -D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.

Effect of dasatinib against thyroid cancer cell lines in vitro and a xenograft model in vivo.

Tyrosine kinase inhibitors (TKIs) have emerged as a promising class of agents against thyroid cancer. The aim of the present study was to investigate the in vitro and in vivo activity of dasatinib against a panel of thyroid cancer cell lines and explore possible mechanisms of action, using various assays and western blotting. Our results showed that dasatinib exhibits prominent cytostatic activity both in vitro and in vivo against thyroid cancer cell lines with RET/PTC rearrangement (BHP2-7) and KRAS mutation (Cal62). Although dasatinib has primarily been described as an ABL/SRCfamily kinase inhibitor, the cytostatic activity observed in the present study is mediated by several off-target effects of dasatinib, some of which have not previously been reported. These effects include a reduction in phospho-FAK, FAK, RAS, Caveolin and SYK protein levels and an increase in ß-catenin protein expression, which leads to the induction of senescence, an increase in the adhesiveness of the cells, a decrease in reactive oxygen species level, and changes in the expression profile of molecules involved in cellular adhesion such as integrins. Therefore, we propose that dasatinib is an effective therapeutic agent for certain patients with thyroid cancer, and these candidate patients may be identifiable on the basis of standard genotypic analyses.

Perinatal outcomes following the ultrasound diagnosis of echogenic bowel: an Australian perspective.

INTRODUCTION: The aim of this study was to describe the association between fetal echogenic bowel (FEB) diagnosed during the second trimester and adverse perinatal outcomes in an Australian antenatal population. METHODS: A retrospective analysis of ultrasound scans was performed between March 1, 2004 and March 1, 2009 at The Royal Women's Hospital, Melbourne, Vic., Australia. Cases reported as having FEB on second trimester ultrasound were included. Medical records of each case were reviewed and information concerning additional investigations and perinatal outcomes were extracted. RESULTS: A total of 66 cases were identified in our database. Three patients (5%) were excluded from further analysis as they were lost to follow-up, leaving 63 (95%) cases in this series. Thirty-two fetuses (52%) underwent karyotyping via amniocentesis, 5 (16%) of which were found to have chromosomal defects. Maternal serology for cytomegalovirus (CMV) was performed in 49 (78%) cases. Investigations indicated a total of 5 women who had CMV infection during their pregnancy. Thirty-three pregnancies (53%) were tested for cystic fibrosis (CF) and 1 baby was confirmed to have CF postnatally. Among the 50 liveborn infants, 3 cases of fetal growth restriction were apparent. Overall, 42 of the 50 liveborn infants (84%) and 67% of the entire cohort of 63 patients with a midtrimester diagnosis of FEB had a normal short-term neonatal outcome. CONCLUSION: This study reiterates the increased prevalence of aneuploidy, CMV, CF and fetal growth restriction in pregnancies complicated by the midtrimester sonographic finding of FEB. However, reassuringly, 67% of cases with ultrasound-detected echogenic bowel in the second trimester had a normal short-term neonatal outcome in this multiethnic Australian population.

OS046. Genome-wide association scans identify novel maternalsusceptibility loci for preeclampsia.

INTRODUCTION: We have successfully utilized a family-based study design to localize several positional candidate preeclampsia susceptibility genes to chromosomes 2q22(ACVR2A,LCT,LRP1B,RND3,GCA),5q (ERAP2) and 13q(TNFSF13B). We now report on our continued positional cloning efforts using an alternative genome-wide association (GWA) mapping strategy in large Caucasian case-control cohorts from Australia and Norway. OBJECTIVES: To identify maternal genetic risk loci for preeclampsia. METHODS: The unrelated Australian samples (545 cases,547 controls) were genotyped using Illumina BeadChip technology (700K loci) and have been analyzed using PLINK. All unrelated Norwegian samples were genotyped across several Illumina BeadChip substrates and consist of 847 cases (700K loci) and 638 controls. The Norwegian control samples originate from other HUNT studies pertaining to migraine (n=95,700K loci), lung cancer (n=89,370K loci) and normal brain pathology (n=454,2.5M loci). To analyze a concordant set of 2.5-3 million genotypes across all Norwegian samples we are currently using MaCH to impute those loci not directly genotyped. The Norwegian GWA data will be analyzed in SOLAR utilizing empirical kinship estimates to account for any distant relatedness. RESULTS: 1078 Australian samples (538 cases,540 controls) and 648, 175 SNPs passed our quality control metrics. Two SNP associations (rs7579169,p=3.6×10(-7); rs12711941,p=4.3×10(-7)) satisfied our genome-wide significant threshold (p<5.1×10(-7)). These SNPs reside less than 15kb downstream from the 3 terminus of the Inhibin, beta B (INHBB) gene on 2q14.2. Sequencing of the INHBB locus in our patient cohort identified a third intergenic SNP to significantly associate with preeclampsia (rs7576192,p=1.5×10(-7)). These three SNPs confer risk (OR>1.56) and are in strong linkage disequilibrium with each other (r(2)>0.9) but not with any other genotyped SNP ±200kb. The analysis of the Norwegian GWAS is underway. CONCLUSION: The Australian GWAS has identified a novel preeclampsia risk locus on chromosome 2q. The INHBB gene closest to our SNP associations is a plausible positional candidate susceptibility gene. There is a substantive body of evidence implicating inhibins, activins and other members of the TGF-ßsuperfamily to have a role in the development of preeclampsia. The biological connection between ACVR2A and INHBB leads us to speculate that our linkage-based and GWA-based study designs, respectively, have identified a key biological pathway involved in susceptibility to preeclampsia.

Tissue expansion for burn sequelae: Jeitawe Burn Center, Lebanon.

Burn sequelae used to be treated with skin grafts and local or distant flaps with a high morbidity on the donor site. The purpose of treatment today by skin expansion is to achieve aesthetic amelioration, as the advantage of this technique is that it becomes possible to obtain local flaps with the same characteristics of colour, texture, hair, and sensitivity as normal skin. This is a review of 14 cases of burn patients treated between 2006 and 2010 at our burn centre at Jeitawe Hospital, Lebanon. The patients' ages ranged from 6 to 50 yr. The regions expanded were the scalp, forehead, neck, trunk, and the upper and lower limbs. The implants were positioned on the fascial layer; antibiotics and drainage were routinely used. The inflation of the expander began two weeks after surgery and continued for an average time of three months. Complications were rare. Results were good with an improvement of scars and minimal morbidity. Fifty per cent of our patients underwent another expansion.

Induction of sodium iodide symporter gene and molecular characterisation of HNF3 beta/FoxA2, TTF-1 and C/EBP beta in thyroid carcinoma cells.

Thyroid carcinoma cells often do not express thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). Treatment of thyroid carcinoma cells (four papillary and two anaplastic cell lines) with histone deacetylase inhibitors (SAHA or VPA) modestly induced the expression of the NIS gene. The promoter regions of the thyroid-specific genes contained binding sites for hepatocyte nuclear factor 3 beta (HNF3 beta)/forkhead box A2 (FoxA2), thyroid transcription factor 1 (TTF-1), and CCAAT/enhancer binding protein (C/EBP beta). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed decreased expression of HNF3 beta/FoxA2 and TTF-1 mRNA in papillary thyroid carcinoma cell lines, when compared with normal thyroid cells. Forced expression of these genes in papillary thyroid carcinoma cells inhibited their growth. Furthermore, the CpG island in the promoter region of HNF3 beta/FoxA2 was aberrantly methylated; and treatment with 5-aza-2-deoxycytidine (5-Az) induced its expression. Immunohistochemical staining showed that C/EBP beta was localised in the nucleus in normal thyroid cells but was detected in the cytoplasm in papillary thyroid carcinoma cells. Subcellular fractionation of papillary thyroid carcinoma cell lines also demonstrated high levels of expression of C/EBP beta in the cytoplasm, suggesting that a large proportion of C/EBP beta protein is inappropriately localised in the cytoplasm. In summary, these findings reveal novel abnormalities in thyroid carcinoma cells.

Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas.

Most human lymphomas originate from transformed germinal center (GC) B lymphocytes. While activating mutations and translocations of MYC, BCL2 and BCL6 promote specific GC lymphoma subtypes, other genetic and epigenetic modifications that contribute to malignant progression in the GC remain poorly defined. Recently, aberrant expression of the TCL1 proto-oncogene was identified in major GC lymphoma subtypes. TCL1 transgenic mice offer unique models of both aggressive GC and marginal zone B-cell lymphomas, further supporting a role for TCL1 in B-cell transformation. Here, restriction landmark genomic scanning was employed to discover tumor-associated epigenetic alterations in malignant GC and marginal zone B-cells in TCL1 transgenic mice. Multiple genes were identified that underwent DNA hypermethylation and decreased expression in TCL1 transgenic tumors. Further, we identified a secreted isoform of EPHA7, a member of the Eph family of receptor tyrosine kinases that are able to influence tumor invasiveness, metastasis and neovascularization. EPHA7 was hypermethylated and repressed in both mouse and human GC B-cell non-Hodgkin lymphomas, with the potential to influence tumor progression and spread. These data provide the first set of hypermethylated genes with the potential to complement TCL1-mediated GC B-cell transformation and spread.

DLK1: increased expression in gliomas and associated with oncogenic activities.

DLK1 (delta-like) is a transmembrane and secreted protein in the epidermal growth factor-like homeotic family. Although expressed widely during embryonic development, only a few tissues retain the expression in adults. Neuroendocrine tumors often highly express this protein; therefore, we hypothesized that brain tumors might also express it. This study found that the expression of DLK1 in gliomas was higher than that in normal brain (P < 0.05). After stable transfection of a DLK1 cDNA expression vector into GBM cell lines, their proliferation was increased. Furthermore, they lost contact inhibition, had enhanced anchorage-independent growth in soft agar, and had significantly greater capacity to migrate. Western blot studies showed that expression of cyclin D1, CDK2, and E2F4 were increased, and Rb levels were decreased in these cells. DLK1 was found on the cell surface and secreted in the medium from the transfected GBM cells. DLK1-enriched condition medium stimulated the growth of glioblastoma multiforme cell lines and explants. DLK1 antibody blocked cell growth stimulated by DLK1. In summary, these results suggest that DLK1 may play a role in the formation or progression of gliomas.

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes.

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.

Interferon-alpha induces dendritic cell differentiation of CML mononuclear cells in vitro and in vivo.

The ability of interferon-alpha (IFN-alpha) to induce dendritic cell (DC) differentiation in chronic myeloid leukemia (CML) was evaluated. Peripheral blood mononuclear cells from CML patients cultured with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) developed a dendritic morphology. Fluorescence in situ hybridization demonstrated that the DCs harbored the bcr/abl translocation. The DCs prepared with IFN-alpha/GM-CSF expressed significantly higher levels of class I and II HLA than those grown in interleukin-4 (IL-4) and GM-CSF. The DCs prepared from newly diagnosed CML patients using IFN-alpha/GM-CSF expressed immunoregulatory proteins at levels comparable to normal DCs. In contrast, DCs cultured from CML patients who did not achieve a cytogenetic response to IFN-alpha expressed significantly lower levels of class I HLA, CD40, CD54, CD80 and CD86 than normal DCs. The expression of CD86 by CML DCs was enhanced when they were cultured with IFN-alpha/IL-4/GM-CSF, or when IFN-alpha/GM-CSF-treated cells were induced to mature by CD40 ligand. The DCs from IFN-alpha failures were less stimulatory than normal DCs in the allogeneic mixed leukocyte reaction. CML patients who had a cytogenetic response to IFN-alpha initially had low numbers of bone marrow DCs that increased significantly with treatment, while nonresponders had more prevalent DCs at baseline that showed no consistent change with treatment. Therefore, IFN-alpha can induce DC differentiation from CML progenitor cells both in vitro and in vivo. The therapeutic activity of IFN-alpha in CML may be due to its ability to stimulate the generation of DCs that can present CML-specific antigens. Resistance to IFN-alpha may result when DC differentiation becomes impaired.

Analysis of the CHK2 gene in lymphoid malignancies.

The CHK2 gene encodes a protein kinase that is important for the regulation of cell cycle arrest after DNA damage. CHK2 acts downstream of ataxia teleangiecstasia mutated (ATM), modulates the function of p53 and may help mediate cell cycle arrest at G2/M by phosphorylation of Cdc25C. Recently, the human homolog of the checkpoint kinase Cds1 (CHK2) has been suggested to be a tumor suppressor gene. Heterozygous germline mutations have been reported in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype, and in sporadic colon cancer. LFS is associated with the development of lymphoid malignancies, especially childhood ALL. Therefore, we analyzed the DNA from 143 lymphoid malignancies to determine whether they had mutations of the CHK2 gene. The 14 exons of CHK2 were studied by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing of aberrantly migrating bands. One missense mutation changing serine to phenylalanine (codon 428) in an evolutionarily highly conserved domain was found in a non-Hodgkin's aggressive lymphoma. Another point mutation in the non-coding region was identified in one of adult T-cell leukemias (ATL) samples. This result suggests that mutation of the CHK2 gene may rarely be involved in the development of selected lymphomas.