Integral fixation titanium/polyetheretherketone cages for cervical arthrodesis: Two-year clinical outcomes and fusion rates using ß-tricalcium phosphate or supercritical carbon dioxide treated allograft.

CONTEXT: Despite increasing promising reports regarding composite titanium (Ti)/PolyEtherEtherKetone (PEEK) cages, further longer-term, quality research is required. Synthetic bone graft substitutes are another rapidly developing area of spinal surgical research. AIMS: The purpose of this study is to evaluate the outcomes of an integral fixation composite Ti/PEEK cage for anterior cervical discectomy and fusion (ACDF) and compare a synthetic bone graft substitute (ß-tricalcium phosphate; [ßTCP]) with allograft processed using supercritical fluid technology. METHODS AND DESIGN: Data from 195 consecutive patients were prospectively collected from a single centre. Indications were largely degenerative. Allograft and ßTCP were used in a 3:1 randomization protocol. Patients were followed up for a minimum of 6 months and up to 48 months. Clinical outcomes included visual analogue scale and neck oswestry disability index. Radiographic outcomes included fusion rates, subsidence rates and implant complications. RESULTS: Graft sub-cohorts were largely comparable and included 133 and 52 patients in the allograft and ßTCP sub-cohorts, respectively. Clinical outcomes overall significantly improved (P < 0.001), with no significant inter-cohort differences. There were no implant-related complications. Overall fusion rate was 94.1% (175/186). The allograft cohort produced a significantly greater fusion rate of 97.7% (126/129) compared to 77.6% (38/49) for the ßTCP cohort (P = 0.001). CONCLUSIONS: This study demonstrates the viability of an integral fixation composite Ti/PEEK ACDF device in effectively and safely improving patient outcomes and achieving fusion. Allograft is more effective in achieving fusion compared to ßTCP, though both were similarly efficacious in improving clinical outcomes.

C3-C5 Chordoma Resection and Reconstruction with a Three-Dimensional Printed Titanium Patient-Specific Implant.

BACKGROUND: With this case report, we aim to add to the clinical literature on the use of three-dimensional printed patient-specific implants in spinal surgery, show the current state of the art in patient-specific implant device design, present thorough clinical and radiographic outcomes, and discuss the suitability of titanium alloy as an implant material for patients with cancer. CASE DESCRIPTION: A 45-year-old man presented with neck and left arm pain combined with shoulder weakness. Imaging revealed significant destruction of the C3-C5 vertebrae, and chordoma diagnosis was confirmed by biopsy. Gross total tumor resection including multilevel corpectomy was performed in combination with reconstruction using a three-dimensional printed titanium custom implant. Custom-designed features aimed to reduce reconstruction time and result in good clinical and radiographic outcomes. Clinical scores improved postoperatively and remained improved at 17-month postoperative follow-up: visual analog scale score 10/10 preoperatively improved to 2-6/10 at 17 months; Neck Disability Index 46% preoperatively improved to 32% at 17 months. Neither dysphagia nor dysphonia remained after surgical soft tissue swelling subsided. The patient was successfully treated with proton beam therapy after surgery, with no tumor recurrence at 17-month follow-up. Radiographic assessment showed incomplete fusion at 3 months, with clinically insignificant implant subsidence (2.7 mm) and no implant migration or failure at 14 months. CONCLUSIONS: Computer-aided preoperative planning with three-dimensional printed biomodels and custom implant resulted in relatively quick and simple reconstruction after tumor resection, with good clinical and radiographic outcomes at 17 and 14 months, respectively. For patients with primary tumors who may require follow-up radiotherapy or postoperative magnetic resonance imaging, metals used in the devices cause significant imaging artifact.

Anterior Lumbar Interbody Fusion Using a Personalized Approach: Is Custom the Future of Implants for Anterior Lumbar Interbody Fusion Surgery?

BACKGROUND: Spine surgery has the potential to benefit from the use of three-dimensional (3D) printing technology (additive manufacturing), particularly in cases of complex anatomic diseases. Custom devices have the potential to reduce operative times, reduce blood loss, provide immediate stability, and improve fusion rates. CASE DESCRIPTION: A 34-year-old man presented with 3-year history of bilateral L5 radiculopathy caused by bilateral L5 pars defect, L5/S1 degenerative disc disease, and severe foraminal stenosis. Anterior lumbar interbody fusion surgery was determined to be the most efficacious method for distraction of the disc space to increase the foraminal volume and stabilization of the motion segment. Surgical decompression and reconstruction was performed in combination with a 3D printed custom interbody implant. Custom design features included corrective angulation to restore lumbar lordosis, preplanned screw holes in the 3D implant, and device end plate interface geometry designed to shape-match with the patient's end plate anatomy. CONCLUSIONS: The use of patient-specific implants has reduced operative time significantly, which may offset costs of increased time spent preplanning the procedure. Surgical procedures can be preplanned using 3D models reconstructed from patient computed tomography and/or magnetic resonance imaging scans. Planning can be aided by 3D printed models of patient anatomy, which surgeons can use in training before performing complex procedures. When considering implants and prostheses, the use of 3D printing allows a superior anatomic fit for the patient compared with generic devices, with the potential to improve restoration of nonpathologic anatomy.

L5 En-Bloc Vertebrectomy with Customized Reconstructive Implant: Comparison of Patient-Specific Versus Off-the-Shelf Implant.

BACKGROUND: Spine surgery has the potential to benefit from additive manufacturing/3-dimensional printing (3DP) technology with complex anatomical pathologies requiring reconstruction, with the potential to customize surgery to reduce operative times, reduce blood loss, provide immediate stability, and potentially improve fusion rates. We report a unique case of intraoperative trial placement of a custom patient-specific implant (PSI) versus the final implantation of a customizable off-the-shelf (OTS) implant. Data collected for comparison included time to implant, ease of implantation, firmness of press-fit, and fixation options after implantation. CASE DESCRIPTION: A 64-year-old man presented with low back pain. Computed tomography and magnetic resonance imaging revealed a solitary lesion in the L5 vertebral body, confirmed by positron emission tomography scan. Removal of the L5 vertebral body was performed, and reconstruction was achieved with an expandable cage. The time of implant insertion was minimal with the PSI (90 seconds) versus the OTS (>40 minutes). Immediate press-fit and "firmness" of implantation was clearly superior with the PSI, although this was an intraoperative subjective assessment. Other benefits include integral fixation that is predetermined with the PSI, reduced time and blood loss, and ease of bone grafting with a PSI. CONCLUSIONS: Use of 3DP has been able to reduce operative time significantly. Surgeons can train before performing complex procedures, which enhances their presurgical planning, with the goal to maximize patient outcomes. When considering implants and prostheses, the use of 3DP allows a superior anatomical fit for the patient, with the potential to improve restoration of anatomy.

Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer.

Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

Evaluation of an assay for methylated BCAT1 and IKZF1 in plasma for detection of colorectal neoplasia.

BACKGROUND: Specific genes, such as BCAT1 and IKZF1, are methylated with high frequency in colorectal cancer (CRC) tissue compared to normal colon tissue specimens. Such DNA may leak into blood and be present as cell-free circulating DNA. We have evaluated the accuracy of a novel blood test for these two markers across the spectrum of benign and neoplastic conditions encountered in the colon and rectum. METHODS: Circulating DNA was extracted from plasma obtained from volunteers scheduled for colonoscopy for any reason, or for colonic surgery, at Australian and Dutch hospitals. The extracted DNA was bisulphite converted and analysed by methylation specific real-time quantitative PCR (qPCR). A specimen was deemed positive if one or more qPCR replicates were positive for either methylated BCAT1 or IKZF1 DNA. Sensitivity and specificity for CRC were estimated as the primary outcome measures. RESULTS: Plasma samples were collected from 2105 enrolled volunteers (mean age 62 years, 54 % male), including 26 additional samples taken after surgical removal of cancers. The two-marker blood test was run successfully on 2127 samples. The test identified 85 of 129 CRC cases (sensitivity of 66 %, 95 % CI: 57-74). For CRC stages I-IV, respective positivity rates were 38 % (95 % CI: 21-58), 69 % (95 % CI: 53-82), 73 % (95 % CI: 56-85) and 94 % (95 % CI: 70-100). A positive trend was observed between positivity rate and degree of invasiveness. The colonic location of cancer did not influence assay positivity rates. Gender, age, smoking and family history were not significant predictors of marker positivity. Twelve methylation-positive cancer cases with paired pre- and post-surgery plasma showed reduction in methylation signal after surgery, with complete disappearance of signal in 10 subjects. Sensitivity for advanced adenoma (n = 338) was 6 % (95 % CI: 4-9). Specificity was 94 % (95 % CI: 92-95) in all 838 non-neoplastic pathology cases and 95 % (95 % CI: 92-97) in those with no colonic pathology detected (n = 450). CONCLUSIONS: The sensitivity for cancer of this two-marker blood test justifies prospective evaluation in a true screening population relative to a proven screening test. Given the high rate of marker disappearance after cancer resection, this blood test might also be useful to monitor tumour recurrence. TRIAL REGISTRATION: ACTRN12611000318987 .

A two-gene blood test for methylated DNA sensitive for colorectal cancer.

BACKGROUND: Specific genes are methylated with high frequency in colorectal neoplasia, and may leak into blood. Detection of multiple methylated DNA biomarkers in blood may improve assay sensitivity for colorectal cancer (CRC) relative to a single marker. We undertook a case-control study evaluating the presence of two methylation DNA markers, BCAT1 and IKZF1, in circulation to determine if they were complementary for detection of CRC. METHODS: Methylation-specific PCR assays were developed to measure the level of methylated BCAT1 and IKZF1 in DNA extracted from plasma obtained from colonoscopy-confirmed 144 healthy controls and 74 CRC cases. RESULTS: DNA yields ranged from 2 to 730 ng/mL plasma (mean 18.6ng/mL; 95% CI 11-26 ng/mL) and did not correlate with gender, age or CRC status. Methylated BCAT1 and IKZF1 DNA were detected in respectively 48 (65%) and 50 (68%) of the 74 cancers. In contrast, only 5 (4%) and 7 (5%) controls were positive for BCAT1 and IKZF1 DNA methylation, respectively. A two-gene classifier model ("either or" rule) improved segregation of CRC from controls, with 57 of 74 cancers (77%) compared to only 11 of 144 (7.6%) controls being positive for BCAT1 and/or IKZF1 DNA methylation. Increasing levels of methylated DNA were observed as CRC stage progressed. CONCLUSIONS: Detection of methylated BCAT1 and/or IKZF1 DNA in plasma may have clinical application as a novel blood test for CRC. Combining the results from the two methylation-specific PCR assays improved CRC detection with minimal change in specificity. Further validation of this two-gene blood test with a view to application in screening is now indicated.

CAHM, a long non-coding RNA gene hypermethylated in colorectal neoplasia.

The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097-163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the gene QKI, which encodes an RNA binding protein. Deep bisulphite sequencing of ten colorectal cancer (CRC) and matched normal tissues demonstrated frequent hypermethylation within the CAHM gene in cancer. A quantitative methylation-specific PCR (qMSP) was used to characterize additional tissue samples. With a threshold of 5% methylation, the CAHM assay was positive in 2/26 normal colorectal tissues (8%), 17/21 adenomas (81%), and 56/79 CRC samples (71%). A reverse transcriptase-qPCR assay showed that CAHM RNA levels correlated negatively with CAHM % methylation, and therefore CAHM gene expression is typically decreased in CRC. The CAHM qMSP assay was applied to DNA isolated from plasma specimens from 220 colonoscopy-examined patients. Using a threshold of 3 pg methylated genomic DNA per mL plasma, methylated CAHM sequences were detected in the plasma DNA of 40/73 (55%) of CRC patients compared with 3/73 (4%) from subjects with adenomas and 5/74 (7%) from subjects without neoplasia. Both the frequency of detection and the amount of methylated CAHM DNA released into plasma increased with increasing cancer stage. Methylated CAHM DNA shows promise as a plasma biomarker for use in screening for CRC.

A panel of genes methylated with high frequency in colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Discovery and validation of molecular biomarkers for colorectal adenomas and cancer with application to blood testing.

BACKGROUND & AIMS: Colorectal cancer incidence and deaths are reduced by the detection and removal of early-stage, treatable neoplasia but we lack proven biomarkers sensitive for both cancer and pre-invasive adenomas. The aims of this study were to determine if adenomas and cancers exhibit characteristic patterns of biomarker expression and to explore whether a tissue-discovered (and validated) biomarker is differentially expressed in the plasma of patients with colorectal adenomas or cancer. METHODS: Candidate RNA biomarkers were identified by oligonucleotide microarray analysis of colorectal specimens (222 normal, 29 adenoma, 161 adenocarcinoma and 50 colitis) and validated in a previously untested cohort of 68 colorectal specimens using a custom-designed oligonucleotide microarray. One validated biomarker, KIAA1199, was assayed using qRT-PCR on plasma extracted RNA from 20 colonoscopy-confirmed healthy controls, 20 patients with adenoma, and 20 with cancer. RESULTS: Genome-wide analysis uncovered reproducible gene expression signatures for both adenomas and cancers compared to controls. 386/489 (79%) of the adenoma and 439/529 (83%) of the adenocarcinoma biomarkers were validated in independent tissues. We also identified genes differentially expressed in adenomas compared to cancer. KIAA1199 was selected for further analysis based on consistent up-regulation in neoplasia, previous studies and its interest as an uncharacterized gene. Plasma KIAA1199 RNA levels were significantly higher in patients with either cancer or adenoma (31/40) compared to neoplasia-free controls (6/20). CONCLUSIONS: Colorectal neoplasia exhibits characteristic patterns of gene expression. KIAA1199 is differentially expressed in neoplastic tissues and KIAA1199 transcripts are more abundant in the plasma of patients with either cancer or adenoma compared to controls.