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Evaluation of Thermal Mechanisms to Predict the Transient Electroplastic Effect in Aluminum and an Investigation of Electrically Assisted Drilling

The objective of this research is twofold: first, to evaluate if the microscale Joule heating theory can predict the transient electroplastic effect in 7075-T6 aluminum. Second, to determine if electrical application can have a significant impact on drilling of 1500MPa steel, and if the operation is predictable using a modified Merchant’s machining model. Both 7075-T6 and 1500 MPa steel are of interest to the automotive industry due to their high strength-to-weight ratios. These metals are important to aid in lightweighting to meet increasingly strict governmental fuel economy standards. However, the strength of the steel makes it difficult to machine in post-forming operations. The ductility of the aluminum makes it impossible to form using conventional methods, especially for deep parts such as a body side outer. A potential fix to these problems is electrical augmentation to locally or globally soften the metal. It has been shown that electricity can increase ductility/formability in metals while also decreasing the forming loads and stresses required (this group of phenomena is termed the electroplastic effect). While the effects of electricity are well known, the underlying mechanisms are not, resulting in four key theories, two of which have already been disproven. This research examines one of the remaining two theories to predict the transient electroplastic effect. The microscale Joule heating theory suggests that microscale hot spots develop inside of the metal in areas of high electrical resistivity, such as grain boundaries where dislocations pile up during deformation. A coupled mechanical-thermal-electrical model was partitioned with grains, grain boundaries, and precipitates. Temperature and dislocation density-dependent electrical resistivity was used in order to evaluate the microscale Joule heating theory. It was found that this theory cannot fully explain the resultant stress drop caused during the transient phase of electrically-assisted pulsed tension. During model testing it was discovered that electricity changes the strain hardening behavior of aluminum. To further investigate, the effect of electricity on precipitates was explored through measurement of precipitate size and distribution in specimens treated with different electrical treatments. An electrically-assisted drilling experiment was designed, fabricated, and tested to determine the effect of electricity on a drilling process. A design of experiments study was conducted on 1008 steel to determine if electric current had a significant effect on process temperature, axial force, and tool wear compared to inputs of feedrate and spindle RPM. It was found that current was dominant and that tool wear and cutting forces could be decreased with electric current. The first electrically-assisted drilling model was created by modifying Merchant’s machining model. This model was found to have shortcomings due to knowledge limitations on friction and equipment limitations on temperature measurement. The knowledge generated from the 1008 experiments was used to further the constraining limits of the drilling process, leading to 1000% tool life improvement on drilling of 1500 MPa steel while increasing the achievable feedrate for cutting by 200%

Clinicopathological predictors of benefit from adjuvant chemotherapy for stage C colorectal cancer: microsatellite unstable cases benefit

Aim: In colorectal cancer (CRC), adjuvant therapy is offered on the basis of stage and attempts to identify factors to better target treatment have not been successful. Recent work suggested that mismatch repair deficient CRCs may not benefit from 5FU adjuvant chemotherapy but studies remain conflicting. We aimed to determine if gender, tumor site, tumor pathological characteristics and microsatellite instability (MSI) predict survival benefit from adjuvant chemotherapy in stage C CRC. Methods: Data were collated on ACPS (Australian Clinico-pathological Staging System) stage C CRC cases that underwent curative resection over a 23-year period. Pathology was reevaluated, DNA was extracted from the formalin-fixed paraffin specimen, and MSI status was established by BAT26 instability. Multivariate analysis was performed using Cox proportional hazard model and effects modification interaction testing. Results: In total 814 unselected cases were included, of whom 37% received chemotherapy. Seventy-seven cases exhibited MSI. Overall, adjuvant chemotherapy produced a cancer-specific survival benefit (HR 0.52, 95% CI 0.39-0.70; P <0.0001). On interaction testing, none of the examined parameters significantly influenced the magnitude of that survival benefit. Chemotherapy was beneficial in both the MSI (HR 0.08, 95% CI 0.02-0.27; P = <0.0001) and the microsatellite stable cohort (HR 0.62, 95% CI 0.47-0.81; P = 0.001). Conclusion: These results suggest that survival benefit from 5FU adjuvant chemotherapy for stage C CRC does not vary according to gender, site of tumor, pathological characteristics or MSI status. This study suggests that it would be unwise to exclude patients from being offered adjuvant chemotherapy on the basis of MSI.Michelle L. Thomas, Peter J. Hewett, Andrew R. Ruszkiewicz, and James W.E. Moor

Can we accurately report PTEN status in advanced colorectal cancer?

BACKGROUND: Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results. METHODS: We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman® copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman® CNV was assessed. RESULTS: Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%). CONCLUSION: Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer

Metallothionien 3 expression is frequently down-regulated in oesophageal squamous cell carcinoma by DNA methylation

BACKGROUND: Metallothionein 3 (MT3) inhibits growth in a variety of cell types. We measured MT3 gene expression by RT-PCR, and DNA methylation in the MT3 promoter by combined bisulphite restriction analysis, in four oesophageal cancer cell lines and the resected oesophagus from 64 patients with oesophageal squamous cell carcinoma (SCC). RESULTS: MT3 expression was not detected in one of the four oesophageal cell lines. The MT3 promoter was methylated in all of the oesophageal cell lines, but the degree of methylation was greater in the non-expressing cell line. After treatment with 5-aza-2'-deoxycytidine there was a reduction in the degree of methylation, and an increase in MT3 expression, in each of the cell lines (p < 0.01). Methylation was detected in 52% (33 of 64) of primary SCC and 3% (2 of 62) of histologically normal resection margins. MT3 expression was measured in 29 tumours, 17 of which had methylation of MT3. The expression of MT3 was significantly less in the methylated tumours compared to either the unmethylated tumours (p = 0.03), or the matched margin (p = 0.0005). There was not a significant difference in MT3 expression between the tumour and the margin from patients with unmethylated tumour. No correlations were observed between methylation of MT3 and survival time, patient age, gender, smoking or drinking history, tumour stage, volume, or lymph node involvement. CONCLUSION: We conclude that MT3 expression is frequently down-regulated in oesophageal SCC, by DNA methylation, but that this is not a prognostic indicator

Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa

Division of Translational and Clinical Oncolog

The La antigen is over-expressed in lung cancer and is a selective dead cancer cell target for radioimmunotherapy using the la-specific antibody APOMAB(R)

Background: The lupus-associated (La)-specific murine monoclonal antibody DAB4 (APOMAB®) specifically binds dead cancer cells. Using DAB4, we examined La expression in human lung cancer samples to assess its suitability as a cancer-selective therapeutic target. We evaluated the safety and effectiveness of radioimmunotherapy (RIT) using DAB4 radiolabeled with Lutetium-177 (177Lu) in the murine Lewis Lung (LL2) carcinoma model, and determined whether combining RIT with DNA-damaging cisplatin-based chemotherapy, a PARP inhibitor (PARPi), or both alters treatment responses. Methods: The expression of La mRNA in human lung cancer samples was analysed using the online database Oncomine, and the protein expression of La was examined using a TissueFocus Cancer Survey Tissue Microarray. The binding of DAB4 to cisplatin-treated LL2 cells was assessed in vitro. LL2 tumour-bearing mice were administered escalating doses of 177Lu-DAB4 alone or in combination with chemotherapy, and tumour growth and survival measured. Biodistribution analysis was used to determine tissue uptake of 177Lu-DAB4 or its isotype control (177Lu-Sal5), when delivered alone or after chemotherapy. PARPi (rucaparib; AG-014699) was combined with chemotherapy and the effects of combined treatment on tumour growth, tumour cell DNA damage and death, and intratumoural DAB4 binding were also analysed. The effect of the triple combination of PARPi, chemotherapy and 177Lu-DAB4 on tumour growth and survival of LL2 tumour-bearing mice was tested. Results: La was over-expressed at both mRNA and protein levels in surgical specimens of human lung cancer and the over-expression of La mRNA conferred a poorer prognosis. DAB4 bound specifically to cisplatin-induced dead LL2 cells in vitro. An anti-tumour dose response was observed when escalating doses of 177Lu-DAB4 were delivered in vivo, with supra-additive responses observed when chemotherapy was combined with 177Lu-DAB4. Combining PARPi with chemotherapy was more effective than chemotherapy alone with increased tumour cell DNA damage and death, and intratumoural DAB4 binding. The combination of PARPi, chemotherapy and 177Lu-DAB4 was well-tolerated and maximised tumour growth delay. Conclusions: The La antigen represents a dead cancer cell-specific target in lung cancer, and DAB4 specifically targeted tumour tissue in vivo, particularly after chemotherapy. Tumour uptake of DAB4 increased further after the combination of PARPi and chemotherapy, which generated new dead tumour cell-binding targets. Consequently, combining 177Lu-DAB4 with PARPi and chemotherapy produced the greatest anti-tumour response. Therefore, the triple combination of PARPi, chemotherapy and RIT may have broad clinical utility.Alexander H Staudacher, Fares Al-Ejeh, Cara K Fraser, Jocelyn M Darby, David M Roder, Andrew Ruszkiewicz, Jim Manavis and Michael P Brow

Similarity of aberrant DNA methylation in Barrett's esophagus and esophageal adenocarcinoma

Background Barrett's esophagus (BE) is the metaplastic replacement of squamous with columnar epithelium in the esophagus, as a result of reflux. It is the major risk factor for the development of esophageal adenocarcinoma (EAC). Methylation of CpG dinucleotides of normally unmethylated genes is associated with silencing of their expression, and is common in EAC. This study was designed to determine at what stage, in the progression from BE to EAC, methylation of key genes occurs. Results We examined nine genes (APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3, and TMEFF2), frequently methylated in multiple cancer types, in a panel of squamous (19 biopsies from patients without BE or EAC, 16 from patients with BE, 21 from patients with EAC), BE (40 metaplastic, seven high grade dysplastic) and 37 EAC tissues. The methylation frequency, the percentage of samples that had any extent of methylation, for each of the nine genes in the EAC (95%, 59%, 76%, 57%, 70%, 73%, 95%, 74% and 83% respectively) was significantly higher than in any of the squamous groups. The methylation frequency for each of the nine genes in the metaplastic BE (95%, 28%, 78%, 48%, 58%, 48%, 93%, 88% and 75% respectively) was significantly higher than in the squamous samples except for CDKN2A and RBP1. The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC. The methylation extent was an estimate of both the number of methylated alleles and the density of methylation on these alleles. This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3. There was no significant difference in methylation extent for any gene between high grade dysplastic BE and EAC. Conclusion We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC. This is also the first report of gene silencing by methylation of ID4 in BE or EAC. This study suggests that metaplastic BE is a highly abnormal tissue, more similar to cancer tissue than to normal epithelium.Eric Smith, Neville J De Young, Sandra J Pavey, Nicholas K Hayward, Derek J Nancarrow, David C Whiteman, B Mark Smithers, Andrew R Ruszkiewicz, Andrew D Clouston, David C Gotley, Peter G Devitt, Glyn G Jamieson and Paul A Dre

Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis

Background: Hyperplastic polyposis of the colorectum is a precancerous condition that has been linked with DNA methylation. The polyps in this condition have been distinguished from typical small hyperplastic polyps and renamed sessile serrated adenomas. Sessile serrated adenomas also occur sporadically and appear to be indistinguishable from their counterparts in hyperplastic polyposis. Aims and methods: The existence of distinguishing molecular features was explored in a series of serrated polyps and matched normal mucosa from patients with and without hyperplastic polyposis by assessing mutation of BRAF, DNA methylation in 14 markers (MINTs 1, 2 and 31, p16, MGMT, MLH1, RASSF1, RASSF2, NORE1 (RASSF5), RKIP, MST1, DAPK, FAS, and CHFR), and immunoexpression of MLH1. Results: There was more extensive methylation in sessile serrated adenomas from subjects with hyperplastic polyposis (p<0.0001). A more clearcut difference in patients with hyperplastic polyposis was the finding of extensive DNA methylation in normal mucosa from the proximal colon. Conclusions: A genetic predisposition may underlie at least some forms of hyperplastic polyposis in which the earliest manifestation may be hypermethylation of multiple gene promoters in normal colorectal mucosa. Additionally, some of the heterogeneity within hyperplastic polyposis may be explained by different propensities for MLH1 inactivation within polyps.P Minoo, K Baker, R Goswami, G Chong, W D Foulkes, A R Ruszkiewicz, M Barker, D Buchanan, J Young, J R Jas

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype

Division of Translational and Clinical Oncolog