Darryl Lau

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering microscopy provides a rapid, label-free means of detecting tumor infiltration of brain tissue ex vivo and in vivo. Virtual Histology During brain tumor surgery, precision is key. Removing healthy tissue can cause neurologic deficits; leaving behind tumor tissue can allow cancer to spread and treatment to fail. To help the surgeon clearly see tumor versus normal tissue, Ji and colleagues developed a stimulated Raman scattering (SRS) microscopy method and demonstrated its ability to identify malignant human brain tissue. In SRS microscopy, laser beams are directed at the tissue sample to generate a series of output signals called “Raman spectra.” These spectra depend on the molecular composition of the tissue. Ji et al. implanted human brain cancer (glioblastoma) cells into mice, allowed them to infiltrate and grow into tumors, and then removed slices for SRS imaging. From the resulting spectra, the authors were able to differentiate the two major components of brain tissue—lipid-rich white matter and protein-rich cortex—as well as tumors, which are full of proteins. Intraoperatively, using an imaging window into mouse brains, the authors found that SRS microscopy could locate tumor infiltration in areas that appeared normal by eye, which suggests that this tool could be applied during surgery. Imaging fresh tissue slices ex vivo could also complement or perhaps replace standard hematoxylin and eosin (H&E) staining in the clinic because it avoids artifacts inherent in imaging frozen or fixed tissues. To this end, Ji and colleagues showed that SRS microscopy could identify hypercellular tumor regions in fresh surgical specimens from a patient with glioblastoma. Certain diagnostic features were present in these specimens and readily identified by SRS, including pseudopalisading necrosis and microvascular proliferation. The next step will be to apply SRS microscopy to a large collection of human specimens to see whether this technology may be useful in quickly distinguishing glioblastoma from healthy tissue, both outside and inside the operating room. Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Awake craniotomy to maximize glioma resection: methods and technical nuances over a 27-year period

OBJECT Awake craniotomy is currently a useful surgical approach to help identify and preserve functional areas during cortical and subcortical tumor resections. Methodologies have evolved over time to maximize patient safety and minimize morbidity using this technique. The goal of this study is to analyze a single surgeons experience and the evolving methodology of awake language and sensorimotor mapping for glioma surgery. METHODS The authors retrospectively studied patients undergoing awake brain tumor surgery between 1986 and 2014. Operations for the initial 248 patients (1986-1997) were completed at the University of Washington, and the subsequent surgeries in 611 patients (1997-2014) were completed at the University of California, San Francisco. Perioperative risk factors and complications were assessed using the latter 611 cases. RESULTS The median patient age was 42 years (range 13-84 years). Sixty percent of patients had Karnofsky Performance Status (KPS) scores of 90-100, and 40% had KPS scores less than 80. Fifty-five percent of patients underwent surgery for high-grade gliomas, 42% for low-grade gliomas, 1% for metastatic lesions, and 2% for other lesions (cortical dysplasia, encephalitis, necrosis, abscess, and hemangioma). The majority of patients were in American Society of Anesthesiologists (ASA) Class 1 or 2 (mild systemic disease); however, patients with severe systemic disease were not excluded from awake brain tumor surgery and represented 15% of study participants. Laryngeal mask airway was used in 8 patients (1%) and was most commonly used for large vascular tumors with more than 2 cm of mass effect. The most common sedation regimen was propofol plus remifentanil (54%); however, 42% of patients required an adjustment to the initial sedation regimen before skin incision due to patient intolerance. Mannitol was used in 54% of cases. Twelve percent of patients were active smokers at the time of surgery, which did not impact completion of the intraoperative mapping procedure. Stimulation-induced seizures occurred in 3% of patients and were rapidly terminated with ice-cold Ringers solution. Preoperative seizure history and tumor location were associated with an increased incidence of stimulation-induced seizures. Mapping was aborted in 3 cases (0.5%) due to intraoperative seizures (2 cases) and patient emotional intolerance (1 case). The overall perioperative complication rate was 10%. CONCLUSIONS Based on the current best practice described here and developed from multiple regimens used over a 27-year period, it is concluded that awake brain tumor surgery can be safely performed with extremely low complication and failure rates regardless of ASA classification; body mass index; smoking status; psychiatric or emotional history; seizure frequency and duration; and tumor site, size, and pathology.

Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis

Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.

Complications and perioperative factors associated with learning the technique of minimally invasive transforaminal lumbar interbody fusion (TLIF)

Before the advent of minimally invasive spine surgery (MIS), open transforaminal lumbar interbody fusion (TLIF) was performed to treat spondylosis, spondylolisthesis, and spondylolysis. Minimally invasive TLIF has recently become more popular based upon the premise that a smaller, less traumatic incision should afford better recovery and outcomes. However, the learning curve associated with this technique must be considered. To analyze the perioperative factors associated with the learning curve in patients who underwent MIS TLIF versus open TLIF, we identified 22 patients who underwent TLIF from 2005 to 2008 within levels L4-S1 by the senior author (D.C.). Patients were subdivided into two groups according to whether they underwent: (i) MIS TLIF (10 patients, the first MIS TLIF procedures performed by D.C.); or (ii) open TLIF (12 patients). Preoperative, perioperative and postoperative factors were evaluated. Patients who underwent MIS TLIF had a statistically significant lower intraoperative transfusion rate, and rate of required postoperative surgical drains; and shorter periods of required drainage, and time to ambulation. However, the MIS TLIF group tended to have a higher rate of complications, which might have been associated with the learning curve. Both groups had a minimum of 1-year follow-up.

Cannabidiol enhances the inhibitory effects of Δ9-tetrahydrocannabinol on human glioblastoma cell proliferation and survival

The cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor agonist Δ9-tetrahydrocannabinol (THC) has been shown to be a broad-range inhibitor of cancer in culture and in vivo, and is currently being used in a clinical trial for the treatment of glioblastoma. It has been suggested that other plant-derived cannabinoids, which do not interact efficiently with CB1 and CB2 receptors, can modulate the actions of Δ9-THC. There are conflicting reports, however, as to what extent other cannabinoids can modulate Δ9-THC activity, and most importantly, it is not clear whether other cannabinoid compounds can either potentiate or inhibit the actions of Δ9-THC. We therefore tested cannabidiol, the second most abundant plant-derived cannabinoid, in combination with Δ9-THC. In the U251 and SF126 glioblastoma cell lines, Δ9-THC and cannabidiol acted synergistically to inhibit cell proliferation. The treatment of glioblastoma cells with both compounds led to significant modulations of the cell cycle and induction of reactive oxygen species and apoptosis as well as specific modulations of extracellular signal-regulated kinase and caspase activities. These specific changes were not observed with either compound individually, indicating that the signal transduction pathways affected by the combination treatment were unique. Our results suggest that the addition of cannabidiol to Δ9-THC may improve the overall effectiveness of Δ9-THC in the treatment of glioblastoma in cancer patients. Mol Cancer Ther; 9(1); 180–9

Proximal junctional kyphosis and failure after spinal deformity surgery: A systematic review of the literature as a background to classification development

Study Design. Systematic review of literature. Objective. To perform a comprehensive English language systematic literature review of proximal junctional kyphosis (PJK) and proximal junctional failure (PJF), concentrating on incidence, risk factors, health related quality of life impact, prevention strategy, and classification systems. Summary of Background Data. PJK and PJF are well described clinical pathologies and are a frequent cause of revision surgery. The development of a PJK classification that correlates with clinical outcomes and guides treatment decisions and possible prevention strategies would be of significant benefit to patients and surgeons. Methods. The phrases “proximal junctional,” “proximal junctional kyphosis,” and “proximal junctional failure” were used as search terms in PubMed for all years up to 2014 to identify all articles that included at least one of these terms. Results. Fifty-three articles were identified overall. Eighteen articles assessed for risk factors. Eight studies specifically reviewed prevention strategies. There were no randomized prospective studies. There were 3 published studies that have attempted to classify PJK. The reported incidence of PJK ranged widely, from 5% to 46% in patients undergoing spinal instrumentation and fusion for adult spinal deformity. It is reported that 66% of PJK occurs within 3 months and 80% within 18 months after surgery. The reported revision rates due to PJK range from 13% to 55%. Modifiable and nonmodifiable risk factors for PJK have been characterized. Conclusion. PJK and PJF affect many patients after long segment instrumentation after the correction of adult spinal deformity. The epidemiology and risk factors for the disease are well defined. A PJK and PJF scoring system may help describe the severity of disease and guide the need for revision surgery. The development and prospective validation of a PJK classification system is important considering the prevalence of the problem and its clinical and economic impact. Level of Evidence: N/A

The transpedicular approach compared with the anterior approach: an analysis of 80 thoracolumbar corpectomies

OBJECT Whereas standard anterior approaches for thoracolumbar corpectomies have commonly been used, the transpedicular technique is increasingly used to perform corpectomies from a posterior approach. The authors conducted a study to analyze whether there was a difference in outcomes by comparing transpedicular corpectomies to standard anterior thoracolumbar corpectomies. METHODS The senior author performed thoracolumbar corpectomies in 80 patients between 2004 and 2008. The authors reviewed medical records and follow-up data, consisting of clinic visits, radiographs, or telephone interviews. Neurological outcome, complications, operative times, revision surgery rates, and estimated blood loss (EBL) were evaluated. RESULTS Thirty-four patients underwent transpedicular corpectomies, and 46 patients underwent anterior thoracolumbar approaches. Single-level transpedicular corpectomies appear to be comparable to anterior-only corpectomies in terms of EBL, operative time, and complication rates. There was a higher complication rate, increased EBL, and longer operative time with anterior-posterior corpectomies compared with transpedicular corpectomies. Patients undergoing transpedicular corpectomies had a greater recovery of neurological function than those in whom anterior-approach corpectomies were performed. CONCLUSIONS The transpedicular corpectomy appears to have a comparable morbidity rate to anterior-only corpectomies, but its morbidity rate is lower than that of anterior-posterior corpectomies.

Incidence of and risk factors for superior facet violation in minimally invasive versus open pedicle screw placement during transforaminal lumbar interbody fusion: a comparative analysis Clinical article

OBJECT A reported risk factor for adjacent-segment disease is injury to the superior facet joint from pedicle screw placement. Given that the facet joint is not typically visualized during percutaneous pedicle screw insertion, there is a concern for increased facet violation (FV) in minimally invasive fusion procedures. The purpose of this study was to analyze and compare the incidence of FV among patients undergoing minimally invasive transforaminal lumbar interbody fusion (MITLIF) and open transforaminal lumbar interbody fusion (TLIF). The impact of O-arm navigation compared with traditional fluoroscopy on FV in MITLIF is also assessed, as are risk factors for FV. METHODS The authors identified a consecutive population of patients who underwent MITLIF with percutaneous pedicle screw placement, as well as a matched cohort of patients who underwent open TLIF. Postoperative CT imaging was assessed to determine intraarticular FV due to pedicle screw placement. Patients were stratified into minimally invasive and open TLIF groups. Within the MITLIF group, the authors performed a subanalysis of image guidance methods used in cases of FV. Two-tailed Student t-test, ANOVA, chi-square testing, and logistic regression were used for statistical analysis. RESULTS A total of 282 patients were identified, with a total of 564 superior pedicle screw placements. The MITLIF group consisted of 142 patients with 284 screw insertions. The open TLIF group consisted of 140 patients with 280 screw insertions. Overall, 21 (7.4%) of 282 patients experienced FV. A total of 21 screws violated a facet joint for a screw-based FV rate of 3.7% (21 of 564 screws). There were no significant differences between the MITLIF and open TLIF groups in the percentage of patients with FV (6.3% vs 8.6%) and or the percentage of screws with FV (3.2% vs 4.3%) (p = 0.475 and p = 0.484, respectively). Further stratifying the MI group into O-arm navigation and fluoroscopic guidance subgroups, the patient-based rates of FV were 10.8% (4 of 37 patients) and 4.8% (5 of 105 patients), respectively, and the screw-based rates of FV were 5.4% (4 of 74 screws) and 2.4% (5 of 210 screws), respectively. There was no significant difference between the subgroups with respect to patient-based or screw-based FV rates (p = 0.375 and p = 0.442, respectively). The O-arm group had a significantly higher body mass index (BMI) (p = 0.021). BMI greater than 29.9 was independently associated with higher FV (OR 2.36, 95% CI 1.65-8.53, p = 0.039). CONCLUSIONS The findings suggest that minimally invasive pedicle screw placement is not associated with higher rates of FV. Overall violation rates were similar in MITLIF and open TLIF. Higher BMI, however, was a risk factor for increased FV. The use of O-arm fluoroscopy with computer-assisted guidance did not significantly decrease the rate of FV.

A prospective Phase II clinical trial of 5-aminolevulinic acid to assess the correlation of intraoperative fluorescence intensity and degree of histologic cellularity during resection of high-grade gliomas

OBJECT There is evidence that 5-aminolevulinic acid (ALA) facilitates greater extent of resection and improves 6-month progression-free survival in patients with high-grade gliomas. But there remains a paucity of studies that have examined whether the intensity of ALA fluorescence correlates with tumor cellularity. Therefore, a Phase II clinical trial was undertaken to examine the correlation of intensity of ALA fluorescence with the degree of tumor cellularity. METHODS A single-center, prospective, single-arm, open-label Phase II clinical trial of ALA fluorescence-guided resection of high-grade gliomas (Grade III and IV) was held over a 43-month period (August 2010 to February 2014). ALA was administered at a dose of 20 mg/kg body weight. Intraoperative biopsies from resection cavities were collected. The biopsies were graded on a 4-point scale (0 to 3) based on ALA fluorescence intensity by the surgeon and independently based on tumor cellularity by a neuropathologist. The primary outcome of interest was the correlation of ALA fluorescence intensity to tumor cellularity. The secondary outcome of interest was ALA adverse events. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and Spearman correlation coefficients were calculated. RESULTS A total of 211 biopsies from 59 patients were included. Mean age was 53.3 years and 59.5% were male. The majority of biopsies were glioblastoma (GBM) (79.7%). Slightly more than half (52.5%) of all tumors were recurrent. ALA intensity of 3 correlated with presence of tumor 97.4% (PPV) of the time. However, absence of ALA fluorescence (intensity 0) correlated with the absence of tumor only 37.7% (NPV) of the time. For all tumor types, GBM, Grade III gliomas, and recurrent tumors, ALA intensity 3 correlated strongly with cellularity Grade 3; Spearman correlation coefficients (r) were 0.65, 0.66, 0.65, and 0.62, respectively. The specificity and PPV of ALA intensity 3 correlating with cellularity Grade 3 ranged from 95% to 100% and 86% to 100%, respectively. In biopsies without tumor (cellularity Grade 0), 35.4% still demonstrated ALA fluorescence. Of those biopsies, 90.9% contained abnormal brain tissue, characterized by reactive astrocytes, scattered atypical cells, or inflammation, and 8.1% had normal brain. In nonfluorescent (ALA intensity 0) biopsies, 62.3% had tumor cells present. The ALA-associated complication rate among the study cohort was 3.4%. CONCLUSIONS The PPV of utilizing the most robust ALA fluorescence intensity (lava-like orange) as a predictor of tumor presence is high. However, the NPV of utilizing the absence of fluorescence as an indicator of no tumor is poor. ALA intensity is a strong predictor for degree of tumor cellularity for the most fluorescent areas but less so for lower ALA intensities. Even in the absence of tumor cells, reactive changes may lead to ALA fluorescence.

Comparison of perioperative outcomes following open versus minimally invasive transforaminal lumbar interbody fusion in obese patients.

OBJECT Minimally invasive (MI) transforaminal lumbar interbody fusion (TLIF) has proven to be effective in the treatment of spondylolisthesis and degenerative disc disease (DDD). Compared with the traditional open TLIF, the MI procedure has been associated with less blood loss, less postoperative pain, and a shorter hospital stay. However, it is uncertain whether the advantages of an MI TLIF also apply specifically to obese patients. This study was dedicated to evaluating whether obese patients reap the perioperative benefits similar to those seen in patients with normal body mass index (BMI) when undergoing MI TLIF. METHODS Obese patients-that is, those with a BMI of at least 30 kg/m(2)-who had undergone single-level TLIF were retrospectively identified and categorized according to BMI: Class I obesity, BMI 30.0-34.9 kg/m(2); Class II obesity, BMI 35.0-39.9 kg/m(2); or Class III obesity, BMI ≥ 40.0 kg/m(2). In each obesity class, patients were stratified by TLIF approach, that is, open versus MI. Perioperative outcomes, including intraoperative estimated blood loss (EBL), complications (overall, intraoperative, and 30-day postoperative), and hospital length of stay (LOS), were compared. The chi-square test, Fisher exact test, or 2-tailed Student t-test were used when appropriate. RESULTS One hundred twenty-seven patients were included in the final analysis; 49 underwent open TLIF and 78 underwent MI TLIF. Sixty-one patients had Class I obesity (23 open and 38 MI TLIF); 45 patients, Class II (19 open and 26 MI); and 21 patients, Class III (7 open and 14 MI). Overall, mean EBL was 397.2 ml and mean hospital LOS was 3.7 days. Minimally invasive TLIF was associated with significantly less EBL and a shorter hospital stay than open TLIF when all patients were evaluated as a single cohort and within individual obesity classes. Overall, the complication rate was 18.1%. Minimally invasive TLIF was associated with a significantly lower total complication rate (11.5% MI vs 28.6% open) and intraoperative complication rate (3.8% MI vs 16.3% open) as compared with open TLIF. When stratified by obesity class, MI TLIF was still associated with lower rates of total and intraoperative complications. This effect was most profound and statistically significant in patients with Class III obesity (42.9% open vs 7.1% MI). CONCLUSIONS Minimally invasive TLIF offers obese patients perioperative benefits similar to those seen in patients with normal BMI who undergo the same procedure. These benefits include less EBL, a shorter hospital stay, and potentially fewer complications compared with open TLIF. Additional large retrospective studies and randomized prospective studies are needed to verify these findings.