Kris R. Jatana

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patients blood from 4.05 × 109 to 8.04 × 103 cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patients blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534.

Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.

Significance of circulating tumor cells in patients with squamous cell carcinoma of the head and neck: initial results.

OBJECTIVES to present and discuss a high-performance negative depletion method for the isolation of circulating tumor cells (CTCs) in the blood of patients with head and neck cancer and to determine the correlation between the presence of CTCs and early clinical outcome in these patients. DESIGN prospective clinical follow-up study of patients with squamous cell carcinoma of the head and neck (SCCHN) undergoing surgical intervention, who had peripheral blood examined for the presence of CTCs. PATIENTS the study population comprised 48 patients diagnosed as having SCCHN and undergoing surgical intervention. INTERVENTION a negative depletion process to isolate and quantify CTCs from the blood of patients with SCCHN using immunomagnetic separation was developed and validated. Immunostaining for cytokeratin was performed on the enriched samples to determine the number of CTCs extracted from each patients blood sample. Correlation of the presence of CTCs, tumor stage, nodal status, clinical characteristics, and outcome was made. MAIN OUTCOME MEASURE disease-free survival. RESULTS our initial data, that have a mean follow-up of 19.0 months, suggest that patients with no detectable CTCs per milliliter of blood had a significantly higher probability of disease-free survival (P = .01). There was no correlation between the presence of CTCs with regard to age, sex, tumor site, stage, or nodal involvement. CONCLUSIONS our enrichment technology, based on the removal of normal cells, has been used on the peripheral blood of patients with head and neck cancer for which follow-up data were collected. If no CTCs were present, a statistically significant improved disease-free survival was observed in SCCHN. A blood test with such a prognostic capability could have important implications in the treatment of patients with head and neck cancer.

Pediatric button battery injuries: 2013 task force update

Over the last 10 years, there has been a dramatic rise in the incidence of severe injuries involving children who ingest button batteries. Injury can occur rapidly and children can be asymptomatic or demonstrate non-specific symptoms until catastrophic injuries develop over a period of hours or days. Smaller size ingested button batteries will often pass without clinical sequellae; however, batteries 20mm and larger can more easily lodge in the esophagus causing significant damage. In some cases, the battery can erode into the aorta resulting in massive hemorrhage and death. To mitigate against the continued rise in life-threatening injuries, a national Button Battery Task Force was assembled to pursue a multi-faceted approach to injury prevention. This task force includes representatives from medicine, public health, industry, poison control, and government. A recent expert panel discussion at the 2013 American Broncho-Esophagological Association (ABEA) Meeting provided an update on the activities of the task force and is highlighted in this paper.

Confocal Images of Circulating Tumor Cells Obtained Using a Methodology and Technology That Removes Normal Cells

A completely negative enrichment technology was used to detect circulating tumor cells, CTCs, in the peripheral blood of head and neck cancer patients. Of 32 blood samples, 63% contained CTCs and the number of CTCs identified per mL of blood collected ranged from 0 to 214. The final purity ranged from 1 CTC in 9 total cells to 1 CTC in 20,000 total cells, the final purity being both a function of the number of CTCs and the performance of the specific enrichment. Consistent with previous reports, CTC were positively identified if: (1) they contained a nucleus based on DAPI stain, (2) stained positive for cytokeratins, and (3) have a high nuclei to cytoplasmic ratio. In addition, for a blood sample to be considered positive for CTCs, the enriched sample must be positive for epithelial growth factor receptor, EGFR, as measured by RT-PCR. While most of the blood samples were obtained during surgery, a number were taken prior to and during surgery. In all of the pre- and postsurgery paired samples, significant numbers of CTCs were detected. A number of these enriched samples were observed under confocal microscope in addition to the microscopic observations under traditional wide-field fluorescent microscope. As expected, the FITC stained cytokeratins appeared in the cytoplasm and the average size of these positively stained cells, on the cytospin, was in the range of 8-12 mum. Future studies will involve the investigation if cancer stem cell and mesenchymal markers are present on these CTCs and correlations of patient outcome to the number and type of CTC present.

Mammary analogue secretory carcinoma of the parotid gland in a pediatric patient

S alivary neoplasms comprise 1% to 3% of all head and neck malignancies. Less than 5% of salivary gland malignancies are diagnosed in children. A recent study in adult patients describes a new salivary gland tumor, mammary analogue secretory carcinoma of the salivary gland (MASC). This lesion has pathological characteristics similar to secretory carcinoma of the breast, salivary acinic cell carcinoma, and low-grade cystadenocarcinoma. MASC is frequently associated with a translocation, t(12;15)(p13;q25), resulting in the fusion gene ETV6-NTRK3. This translocation has been demonstrated consistently in secretory carcinoma of the breast. The tyrosine kinase encoded by this fusion gene has been directly related to transformation of epithelial cells in mouse mammary glands. Children’s Memorial Hospital institutional review board approval was obtained, and we report the first case of MASC in the parotid gland of a child.

Effects of nasal continuous positive airway pressure and cannula use in the neonatal intensive care unit setting.

OBJECTIVE To investigate the effects of nasal continuous positive airway pressure (CPAP) and cannula use in the neonatal intensive care unit. DESIGN Cross-sectional study. SETTING Tertiary care childrens hospital. PATIENTS One hundred patients (200 nasal cavities), younger than 1 year, who received at least 7 days of nasal CPAP (n = 91) or cannula supplementation (n = 9) in the neonatal intensive care unit. INTERVENTIONS External nasal examination and anterior nasal endoscopy with photographic documentation. MAIN OUTCOME MEASURES The incidence and characteristics of internal and external nasal findings of patients with nasal CPAP or cannula use. RESULTS Nasal complications were seen in 12 of the 91 patients (13.2%) with at least 7 days of nasal CPAP exposure, while no complications were seen in the 9 patients with nasal cannula use alone. The external nasal finding of columellar necrosis, seen in 5 patients (5.5%), occurred as early as 10 days after nasal CPAP use. Incidence of intranasal findings attributed to CPAP use, in the 182 nostrils examined, included ulceration in 6 nasal cavities (3.3%), granulation in 3 nasal cavities (1.6%), and vestibular stenosis in 4 nasal cavities (2.2%). Intranasal complications were seen as early as 8 to 9 days after nasal CPAP administration. Nasal complications from CPAP were associated with lower Apgar scores at 1 (P = .02) and 5 (P = .06) minutes. CONCLUSIONS External or internal complications of nasal CPAP can be relatively frequent (13.2%) and can occur early, and patients with lower Apgar scores may be at higher risk. Close surveillance for potential complications should be considered during nasal CPAP use.

Emerging technologies for CTC detection based on depletion of normal cells.

Properly conducted, an enrichment step can improve selectivity, sensitivity, yield, and most importantly, significantly reduce the time needed to isolate rare circulating tumor cells (CTCs). The enrichment process can be broadly categorized as positive selection versus negative depletion, or in some cases, a combination of both. We have developed a negative depletion CTC enrichment strategy that relies on the removal of normal cells using immunomagnetic separation in the blood of cancer patients. This method is based on the combination of magnetic and fluid forces in an axial, laminar flow in long cylinders placed in quadrupole magnets. Using this technology, we have successfully isolated CTCs from patients with breast carcinoma and squamous cell carcinoma of the head and neck. In contrast to a positive selection methodology, this approach provides an unbiased characterization of these cells, including markers associated with epithelial mesenchymal transition.

Cuffed endotracheal tubes in infants and children: Should we routinely measure the cuff pressure?

OBJECTIVE Over the past 5 years, there has been a change in the clinical practice of pediatric anesthesiology with a transition to the use of cuffed instead of uncuffed endotracheal tubes in infants and children. However, there are few studies evaluating the current practices of inflation of these cuffs and the intracuff pressures. METHOD There was no change dictated in clinical practice for these patients. During the first 30 min of the case, the pressure in the cuff was measured using a hand held manometer. Additional data collected included the patients demographic data (age, weight, and gender), the size of the ETT, whether nitrous oxide was in use, whether the patient was breathing spontaneously or undergoing positive pressure ventilation, and the type of anesthesia provider (resident, fellow, CRNA or SRNA). RESULTS The cohort for the study included 200 patients ranging in age from 1 month to 17 years and in weight from 3.5 to 99.1 kg. The average cuff pressure was 23 ± 22 cmH(2)O in the total cohort of 200 patients. The cuff pressure was ≥ 30 cmH(2)O in 47 of the 200 patients (23.5%). The average cuff pressure was significantly higher in patients who were 8 years of age or greater compared to younger patients. Additionally, there were significantly more patients with a cuff pressure ≥ 30 cmH(2)O in the ≥ 8 year old age group. Although no difference in the mean cuff pressure was noted when comparing staff anesthesia providers (pediatric anesthesiologist or CRNA) versus trainees (SRNA, anesthesiology resident, medical student or pediatric anesthesiology fellow), the incidence of significantly excessive cuff pressures (≥ 60 cmH(2)O) was higher in the trainee group versus the faculty group (12 of 99 versus 2 of 101, p<0.0001). CONCLUSIONS Using current clinical practice to inflate the cuff, a significant percentage of pediatric patients have an intracuff pressure greater than the generally recommended upper limit of 30 cmH(2)O.

Guidelines to decrease unanticipated hospital admission following adenotonsillectomy in the pediatric population

INTRODUCTION Tonsillectomy has become one of the most commonly performed surgical procedures in the pediatric-aged patient. Many of these children are diagnosed with obstructive sleep apnea (OSA). Although polysomnography is considered the gold standard, many practioners rely on the clinical examination and parental history. Nationwide Childrens Hospital recently instituted pediatric adenotonsillectomy guidelines for hospital admission to help determine which patients should be done in main hospital OR vs. outpatient surgery facility. The main goal was to decrease unanticipated admissions. The secondary goal was to determine areas for practice improvement. METHODS Using databases for the hospital, operating room, and otolaryngology, all cases with CPT codes 42820, 42830, 42825, 42826, and 42821 were evaluated from October 2009 to August 2012 in the main operating room and 2 outpatient surgery centers. Data for each unanticipated admission were reviewed to determine whether the criteria were met according to the developed guidelines. Fishers exact test was applied to the unplanned admission rate before and after the institution of the guidelines. Non-paired t-test and a Fishers exact test were used for comparison of the demographic data between the two groups. RESULTS Following the institution of the pediatric adenotonsillectomy guidelines, the number of unanticipated admissions decreased from an absolute number of 88 to 43. This represents a decrease from 2.38% to 1.44% (p=0.008). Forty-two percent of the unanticipated admissions prior to establishing guidelines were in patients who would have met criteria for admission based on the guidelines. This decreased to 30% after establishing the guidelines. CONCLUSION We found that the institution of pediatric adenotonsillectomy guidelines for patients undergoing adenotonsillectomy significantly decreased the rate of unanticipated admission. However, there was still a significant percentage (30%) of unanticipated admissions due to non-compliance with the guidelines demonstrating the need for ongoing practice improvement.