Julie Y. Tse

Local Myotoxicity from Sustained Release of Bupivacaine from Microparticles

Background:Sustained release of local anesthetics is frequently associated with myotoxicity. The authors investigated the role of particulate delivery systems and of the pattern of drug release in causing myotoxicity. Methods:Rats were given sciatic nerve blocks with bupivacaine solutions, two types of bupivacaine-containing microparticles (polymeric microspheres and lipid–protein–sugar particles), or blank particles with or without bupivacaine in the carrier fluid. Myotoxicity was scored in histologic sections of the injection sites. Bupivacaine release kinetics from the particles were measured. Myotoxicity of a range of bupivacaine concentrations from exposures up to 3 weeks was assessed in C2C12 myotubes, with or without microparticles. Results:Both types of bupivacaine-loaded microparticles, but not blank particles, were associated with myotoxicity. Whereas 0.5% bupivacaine solution caused little myotoxicity, a concentration of bupivacaine that mimicked the amount of bupivacaine released initially from particles caused myotoxicity. Local anesthetics showed both concentration and time-dependent myotoxicity in C2C12s. Importantly, even very low concentrations that were nontoxic over brief exposures became highly toxic after days or weeks of exposure. The presence of particles did not increase bupivacaine myotoxicity in vitro but did in vivo. Findings applied to both particle types. Conclusions:Whereas the release vehicles themselves were not myotoxic, both burst and extended release of bupivacaine were. A possible implication of the latter finding is that myotoxicity is an inevitable concomitant of sustained release of local anesthetics. Particles, and perhaps other vehicles, may enhance local toxicity through indirect mechanisms.

Tetrodotoxin for prolonged local anesthesia with minimal myotoxicity

Conventional local anesthetics such as bupivacaine cause considerable myotoxicity and neurotoxicity, whereas tetrodotoxin (TTX) does not. Tetrodotoxin combined with bupivacaine or vasoconstrictors produces long‐duration nerve blockade. To assess whether these prolonged blocks can be produced without increased myotoxicity, Sprague‐Dawley rats were injected with bupivacaine, TTX, and both, or TTX plus epinephrine. Median durations of thermal nociceptive blockade were, respectively, 188, 401, 882, and 972 min. On dissection 4 days later, all tissues appeared macroscopically pristine. Muscle injury was at most mild to moderate in all animals, and the muscle injury scores for the combination formulations were not higher than for bupivacaine alone. Similarly, in differentiated cells from a myoblast cell line (C2C12), TTX caused either no or minimal worsening of cell viability from bupivacaine at 2 or 7 days. Epinephrine did not worsen TTXs relatively minimal cytotoxicity. Tetrodotoxin may thus be useful in producing prolonged nerve block with minimal myotoxicity and perhaps neurotoxicity. Muscle Nerve, 2006

Site 1 sodium channel blockers prolong the duration of sciatic nerve blockade from tricyclic antidepressants.

&NA; Many recent reports in the literature address the local anesthetics efficacy of tricyclic antidepressants (TCAs). Here we investigated whether nerve block from TCAs is prolonged by site 1 sodium channel blockers such as tetrodotoxin and saxitoxin, which are known to prolong block from conventional local anesthetics. Tetrodotoxin and saxitoxin greatly prolonged block from TCAs. For example, the median duration of thermal nociceptive blocks for 10 mM amitriptyline, nortriptyline and doxepin were 0, 0, and 124 min; co‐injection with 20 &mgr;M TTX (median block duration=0), yielded blocks lasting 404, 325, and 697 min, respectively. Co‐injection of 12 &mgr;M saxitoxin (median block duration=0) with 10 mM amitriptyline resulted in a thermal nociceptive block duration of 373 min. Co‐injection of 7.7 mM bupivacaine and 7.7 mM amiptriptyline did not result in block prolongation. Systemic (subcutaneous) delivery of tetrodotoxin or amitriptyline did not result in prolongation of block from the other class of drug injected at the sciatic nerve. In TCA‐containing formulations, motor blockade was consistently longer than thermal nociceptive block; motor blockade was also prolonged by tetrodotoxin and saxitoxin. In summary site 1 sodium channel blockers prolong the duration of TCAs via a locally mediated mechanism.

Genomic landscape of colorectal cancer in Japan: Clinical implications of comprehensive genomic sequencing for precision medicine

BackgroundComprehensive genomic sequencing (CGS) has the potential to revolutionize precision medicine for cancer patients across the globe. However, to date large-scale genomic sequencing of cancer patients has been limited to Western populations. In order to understand possible ethnic and geographic differences and to explore the broader application of CGS to other populations, we sequenced a panel of 415 important cancer genes to characterize clinically actionable genomic driver events in 201 Japanese patients with colorectal cancer (CRC).MethodsUsing next-generation sequencing methods, we examined all exons of 415 known cancer genes in Japanese CRC patients (n = 201) and evaluated for concordance among independent data obtained from US patients with CRC (n = 108) and from The Cancer Genome Atlas-CRC whole exome sequencing (WES) database (n = 224). Mutation data from non-hypermutated Japanese CRC patients were extracted and clustered by gene mutation patterns. Two different sets of genes from the 415-gene panel were used for clustering: 61 genes with frequent alteration in CRC and 26 genes that are clinically actionable in CRC.ResultsThe 415-gene panel is able to identify all of the critical mutations in tumor samples as well as WES, including identifying hypermutated tumors. Although the overall mutation spectrum of the Japanese patients is similar to that of the Western population, we found significant differences in the frequencies of mutations in ERBB2 and BRAF. We show that the 415-gene panel identifies a number of clinically actionable mutations in KRAS, NRAS, and BRAF that are not detected by hot-spot testing. We also discovered that 26% of cases have mutations in genes involved in DNA double-strand break repair pathway. Unsupervised clustering revealed that a panel of 26 genes can be used to classify the patients into eight different categories, each of which can optimally be treated with a particular combination therapy.ConclusionsUse of a panel of 415 genes can reliably identify all of the critical mutations in CRC patients and this information of CGS can be used to determine the most optimal treatment for patients of all ethnicities.

Branched chain in situ hybridization for albumin as a marker of hepatocellular differentiation: evaluation of manual and automated in situ hybridization platforms.

Albumin, widely recognized as a highly sensitive and specific marker of hepatocellular carcinoma (HCC), is currently unavailable in the diagnostic laboratory because of the lack of a robust platform. In a prior study we detected albumin mRNA in the majority of intrahepatic cholangiocarcinomas using a novel branched chain RNA in situ hybridization (ISH) platform. We now explore the utility of albumin ISH as a marker of hepatocellular differentiation in HCCs, and compare its sensitivity with Hep Par 1 and Arginase-1. We evaluated 93 HCCs and its mimics including neuroendocrine tumors of the gastrointestinal tract (n=31), neuroendocrine tumors of the pancreas (n=163), melanoma (n=15), and gallbladder carcinoma (n=34). We performed ISH for albumin and immunohistochemistry for Hep Par 1 and Arginase-1. Five previously uncharacterized hepatic neoplasms from our files were also evaluated. Immunohistochemistry for Arginase-1 was performed on 59 intrahepatic cholangiocarcinomas. In addition, 43 HCCs evaluated on the manual platform were also examined on the automated instrument. Fifty-five percent of HCCs were moderately differentiated and 39% poorly differentiated. The sensitivity of ISH for albumin was 99%, with 92 of 93 HCCs staining positive for albumin. In contrast to ISH, the sensitivity of immunohistochemistry for Hep Par 1 and Arginase-1 was 84% and 83%, respectively. The sensitivity of albumin for poorly differentiated HCCs was 99%, whereas that for Arginase-1 and Hep Par 1 was 71% and 64%, respectively. Ninety-seven percent of the HCCs showed albumin positivity in >50% of tumor cells using the ISH platform, as compared with 76% and 70% for Hep Par 1 and Arginase-1 immunohistochemistry, respectively. Three of the 5 previously uncharacterized neoplasms were positive for albumin ISH. Automated albumin ISH platform performed equivalently to the manual format, with albumin reactivity in >50% of tumor cells in all 43 cases that were tested on both platforms. All non-HCCs were negative for albumin. All 59 intrahepatic cholangiocarcinomas were negative for Arginase-1. In conclusion, branched chain ISH performed on manual and automated mode is a robust assay for detecting albumin with sensitivity for poorly differentiated HCCs superior to Arginase-1 and Hep Par 1. When interpreted in conjunction with Arginase-1, albumin ISH offers a high level of sensitivity and specificity.

Microcystic adnexal carcinoma versus desmoplastic trichoepithelioma: a comparative study.

Abstract:The histologic distinction between microcystic adnexal carcinoma (MAC) and desmoplastic trichoepithelioma (dTE) can be challenging in the setting of a superficial biopsy. However, accurate diagnosis has treatment implication because the standard of care for MAC is wide local excision but more conservative care for dTE. We reviewed the histologic features of 30 MAC and 39 dTE cases and performed cytokeratin (CK) 17, CK19, and epidermal growth factor receptor (EGFR) immunostains on 20 MACs and 18 dTEs. MAC cases occurred in older patients in comparison with dTE (median, 67 years vs. 34 years). The head and neck was the most commonly involved site, 88% and 89% for MAC and dTE, respectively. In addition to features previously reported as specific for MAC, such as skeletal muscle and subcutaneous tissue invasion, perineural invasion, and ductal differentiation, we found the presence of mitotic figures to be significantly more frequent in MAC cases (P < 0.0001). In contrast, the presence of keratocyst, keratin granuloma, and calcification was significantly more frequent in dTE cases (P < 0.0001). CK19 seems to be a helpful adjunct because its expression was seen in 70% (14/20) of MAC versus 22% (4/18) of dTE cases (P = 0.0044); however, the clinical usefulness in individual cases may be limited because of the overlapping immunoprofile. CK17 and EGFR expression was seen in all the MAC and dTE cases. Low polysomy of EGFR gene was observed in only one MAC case, suggesting that molecular mechanisms other than gene amplification play a role in EGFR overexpression.

Clinical application of a cancer genomic profiling assay to guide precision medicine decisions

Aim: Develop and apply a comprehensive and accurate next-generation sequencing based assay to help clinicians to match oncology patients to therapies. Materials & methods: The performance of the CANCERPLEX® assay was assessed using DNA from well-characterized routine clinical formalin-fixed paraffin-embedded (FFPE) specimens and cell lines. Results: The maximum sensitivity of the assay is 99.5% and its accuracy is virtually 100% for detecting somatic alterations with an allele fraction of as low as 10%. Clinically actionable variants were identified in 93% of patients (930 of 1000) who underwent testing. Conclusion: The tests capacity to determine all of the critical genetic changes, tumor mutation burden, microsatellite instability status and viral associations has important ramifications on clinical decision support strategies, including identification of patients who are likely to benefit from immune checkpoint blockage therapies.

Basal cell carcinoma with osteosarcomatous component.

Abstract:Sarcomatoid carcinoma or carcinosarcomas of the skin are rare. Basal cell carcinoma (BCC) with osteosarcomatous differentiation is the second most common sarcomatoid carcinoma of the skin, following squamous cell carcinoma with heterologous mesenchymal differentiation. There are only 11 cases of BCC with osteosarcomatous component reported in the literature, with limited documented molecular analyses. The authors report the clinical and histological features of 2 cases with molecular analyses for recurrent mutations in 17 cancer genes. In both cases, the epithelial or BCC component was positive for BerEP4 and high–molecular weight cytokeratin, whereas the sarcomatous component was negative for both markers. Mutational analyses revealed TP53 mutation in 1 case with p53 expression noted in both components. The other case was negative for both p53 expression and TP53 mutation.

Implementation of an expanded point-of-care testing (POCT) site inspection checklist in a large academic medical center: Implications for the management of a POCT program

BACKGROUND Management of point-of-care testing (POCT) continues to present challenges. The use of site inspections by POCT coordinators has been a mainstay for maintaining regulatory compliance in POCT although this process is labor intensive. Improvements in device design and the use of POCT data management systems have improved the ability of hospitals to manage POCT programs. Given these developments, using regular site inspections to ensure regulatory compliance of POCT may be less relevant than in the past. METHODS We implemented an expanded POCT site inspection checklist modeled after the requirements of the Joint Commission. The checklist included categories not specifically related to POCT such as the environment of care and safety. RESULTS The average number of deficiencies per site in 2011 was 2.37 (range 0-10) out of a possible 42. Only 18% of site inspections produced no citations. In contrast in 2010 the average number of deficiencies per site was 3.17 (out of a possible 32) and only 8.7% of sites had no citations. The difference in the number of deficiencies per site was significant (t-test two tailed p=0.04). The most frequently cited deficiencies (24.2%) related to safety followed by maintenance of proper procedures and documentation of competency assessment. CONCLUSIONS Despite improvements in many POCT devices and data management systems, regular site inspections are still required to identify regulatory deficiencies and improve testing quality. The use of an expanded site inspection checklist will identify many areas for improvement that are not specifically related to POCT alone.

Melanoma arising in a nevus of Ito: novel genetic mutations and a review of the literature on cutaneous malignant transformation of dermal melanocytosis

Dermal melanocytosis refers to a spectrum of benign melanocytic proliferations that includes Mongolian spot, nevus of Ota and nevus of Ito. These lesions most commonly occur in persons of Asian or African descent and are often present at birth or develop during childhood. Very rarely, dermal melanocytoses undergo malignant transformation. There have been only 13 reports in the literature of primary cutaneous melanoma arising in dermal melanocytoses. We report a case of a Chinese woman with melanoma arising in a congenital nevus of Ito. We performed targeted next‐generation sequencing of the tumor which revealed mutations of GNAQ and BAP1, suggesting that alterations in these two genes led to malignant transformation of the nevus of Ito. We also provide a summary of reports in the literature regarding primary cutaneous melanoma arising in the context of dermal melanocytosis.