Patient specific real-time PCR in precision medicine - Validation of IG/TR based MRD assessment in lymphoid leukemia.

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.

Iatrogenic Radial Artery Injuries: Variable Injury Patterns, Treatment Times, and Outcomes.

Background: The radial artery is commonly accessed for arterial blood sampling, invasive blood pressure monitoring, and vascular access for cardiac catheterization. Iatrogenic radial artery injury is a rare complication with potentially devastating outcomes. The purpose of our study was to identify the timing of these injuries and define a treatment algorithm. Methods: A retrospective chart review of all patients with iatrogenic radial artery injuries were identified between the years 2008 and 2018. Patient demographics, mechanism of injury, interventions, and outcomes were recorded. Results: A total of 18 patients were identified with iatrogenic radial artery injury over a 10-year period. Fifty percent of these resulted from arterial line cannulation, and 50% occurred after transradial cardiac catheterization. Thirty-three percent resulted in radial artery pseudoaneurysm (RAP), and 66% had acute radial artery thrombosis (RAT). Eleven of the 18 patients underwent operative intervention. Of the 12 patients with RAT, 4 were treated with systemic anticoagulation for 3 months. All patients with RAP who were surgically treated had resolution of symptoms on follow-up evaluation. Of the patients with RAT, 2 had persistent sensorimotor deficits after treatment, and 1 patient had multiple necrotic fingers requiring amputation. Conclusion: Radial artery injuries are an uncommon but potentially devastating complication of common invasive procedures resulting in thrombosis, pseudoaneurysm, or overt hand ischemia. The treatment options vary depending on presenting symptoms.

Does etiology of gastroparesis determine clinical outcomes in gastric electrical stimulation treatment of gastroparesis?

BACKGROUND: Gastroparesis is a condition characterized by impaired gastric motility that may result in weight loss and malnutrition. There have been promising studies demonstrating improvement in symptoms after gastric electrical stimulation (GES) implantation for medically refractory gastroparetics [1-10]. With the heterogeneous population of gastroparetics, the aim of this study was to assess if etiology correlated with response to GES. METHODS: A retrospective review and analysis was performed on patients who underwent GES over a 10-year period at a single institution. Each patient was stratified into an etiological subset (diabetes, idiopathic, post-surgical). Patients were compared by demographics, medical and surgical history, subsequent GES explantation vs continued therapy, need for supplemental nutrition postoperatively, weight gain, weight loss or weight maintenance, and readmission rates. RESULTS: 183 patients underwent GES from 2005 to 2015. 50% were diabetic (n = 91), 42% idiopathic (n = 76), and 9% post-surgical (n = 16). Diabetic patients (DM) demonstrated the highest likelihood of continued therapy compared to post-surgical (PS) and idiopathic patients (ID) (54.7% vs 9.5% vs 35.8%, respectively, p < 0.05). DM patients saw a greater incidence of weight gain > 4 kg, compared to PS and IS patients (67.6% vs 8.1% vs. 24.3%, respectively, p < 0.05). ID patients were most likely to have it removed compared to DM and PS patients (65.7% vs 28.6% vs 5.7%, respectively, p = < 0.05). PS patients were least likely to have their GES removed. They were also least likely to utilize supplemental nutrition compared to DM and ID (9.4% vs 49.1% vs 41.51%, respectively, p < 0.05). CONCLUSIONS: Patients with gastroparesis had different clinical outcomes after GES therapy based on underlying etiology. By gaining a better understanding of the effects of GES, it can be offered to the appropriate patient.

A Clinically Applicable Gene-Expression Classifier Reveals Intrinsic and Extrinsic Contributions to Consensus Molecular Subtypes in Primary and Metastatic Colon Cancer.

PURPOSE: Four consensus molecular subtypes (CMS1-4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer-related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: We developed a colorectal cancer-focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. RESULTS: Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. CONCLUSIONS: We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.

South Carolina Surgical Quality Collaborative Colon Surgery Outcomes Vary by Age and Race.

Identifying disparities in surgical outcomes among patient populations may help hospitals target patients at highest risk for complications. The South Carolina Surgical Quality Collaborative (SCSQC) is a regional collaborative made up of eight facilities whose goal is to improve the quality and value of general surgical care in South Carolina. Using SCSQC data, we reviewed colon surgery outcomes to determine whether disparities exist between specific patient populations. SCSQC colon surgery data were reviewed from August 2015 to August 2017. SSI, length of stay, return to the ED, and reoperation rates were used as outcome measures. They were evaluated in patient populations stratified by gender, race (white, black, and other), and age (<50, 50-70, and >70 years). A total of 2611 patients were included in this study. Statistically significant differences in outcomes were identified between white and black patients in length of stay (6.0 vs 7.5 days, P < 0.0001) and return to the ED (8.1% vs 14.7%, P < 0.0001), but not in SSI (6.4% vs 6.8%, P = 0.8839) or reoperation rates (6.4% vs 8.4%, P = 0.1886). Length of stay increased with increasing age (4.1 vs 7.1 vs 8.8, P < 0.0001). SSI varied by age (4.0% vs 8.2% vs 6.4%, P = 0.0005), as did return to the ED (11.2% vs 9.7% vs 769%, P = 0.0987) and reoperation rates (4.5% vs 8.1% vs 8.2%, P = 0.0034). SCSQC data indicate that race and age may place patients at risk for negative outcomes after colorectal surgery.

Development and Application of a Microfluidics-Based Panel in the Basal/Luminal Transcriptional Characterization of Archival Bladder Cancers.

In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. METHODS: We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. RESULTS: The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. CONCLUSIONS: We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Nanostructures of small-molecule organic crystals on capillary wave surfaces with controllable capillary lengths.

We report on the nanostructures of organic small-molecule pentacene crystals that have been vapor-deposited onto the capillary wave surfaces of thin liquid films. The characteristic lateral length of the capillary wave surface or the capillary length can be controlled by changing the thickness of the liquid films and, thus, the van der Waals interaction with the substrate. We find that the morphology of the organic crystals gradually varies from fractals to compact islands as the liquid film thickness increases. The square of average distance between organic crystal grains was also found to be proportional to the liquid film thickness. We discuss the possibility that these effects are driven by capillary fluctuations at the air-liquid interface.

Therapy of percutaneous infection around craniofacial implants.

This study sought to develop treatment strategies for managing percutaneous infection around craniofacial implants. The present general pathogen situation together with a bacterial resistance were determined in 57 infected peri-implant sites. Forty-four implants were randomly assigned for wound cleaning and split into three groups-two with local antibiotics of proven efficacy and one with 3% hydrogen peroxide (H2O2). The pathogen spectrum differed depending on the severity of the infection, with Staphylococcus aureus clearly correlated with the degree of inflammation (positive correlation: R = 0.72). It was observed that the use of additional local antibiotics was not superior to conventional wound cleaning with 3% H2O2. It is suggested that sulcus fluid flow rate measurements could serve as a simple and reliable objective parameter for recall examinations.

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras.

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

Impact of microenvironment on the growth of primary human epidermal cells.

Although the conditions for in vitro cultivation of adult stem cells and tissue are easily standardized, little is known about the optimal conditions for biointegration after transfer of the tissue graft, playing an important role in the treatment of defects especially soft-tissue skin injuries. To examine the influence of the microenvironment, we investigated the doubling time of primary epithelial cells in relation to the culture medium. Serum from patients of different age groups (n = 15, <20 years; n = 9, >20 years; and fetal calf serum) was pooled independently of age and added to culture medium of epithelial cells from a skin donor (10%). Number of cells was counted in vitro after 1 and 4 days of cultivation using a photometric extinction test. Results were plotted using quotient for calculating cell proliferation ([T4 -T1]:T1). Statistical significance was calculated by Wilcoxon test. Highest proliferation rate was achieved by cultivating the cells in the heterological serum admixture. Homologous serum admixtures in the cell cultures of <20 donators yielded a significantly higher proliferation rate than adult serum (P < 0.01). High regenerative capacity of skin in children has, thus far, mainly been attributed to the high plasticity of the cellular structures. Our study shows for the first time that the age-dependent regenerative capacity in vitro is also influenced by age-dependent humoral factors. In vivo cells from older patients may thus be transferred into an altogether suboptimal microenvironment. Responsible humoral factors should be more closely examined to optimize the clinical management of cellular transplants.

Cultured epithelial autografts in the treatment of facial skin defects: clinical outcome.

PURPOSE: To report the treatment of facial skin defects by cultured epithelial autografts and its clinical outcome. PATIENTS AND METHODS: Between 2002 and 2003, 18 patients with secondary facial skin defects (after tumor excision, trauma, or due to chronic wound healing dysfunction) were successfully treated with autologous cultivated keratinocytes. Overall, 12 patients were included in our study. At the time of this evaluation, the average time lapse after treatment with autologous cultivated keratinocytes was 13.1 months. From 9 of 12 patients a skin biopsy was taken, 12 of 12 patients were neurologically tested, and the results of 12 of 12 patients' esthetics were evaluated by photography and in written form with a standardized questionnaire. RESULTS: Histologically, 9 of 12 patients showed a regular epithelial layer with evidence of basal cells of the basal membrane and conspicuously arranged connective tissue. The neurologic quality of the skin was discreetly reduced in 9 of 12 patients, but this was not experienced by the patient as a limitation. The wound closure was permanent in the case of all 12 patients. Scar tissue was found frequently, when the wound size was greater than 2.5 cm2. On the basis of the standardized questionnaire, 12 of 12 patients rated the degrees of their subjective satisfaction. CONCLUSION: From the esthetic, histologic, and neurologic points of view, cultured epithelial autografts are an auspicious alternative to conventional grafting methods for facial skin replacement. Optimizing cell growth in vitro to decrease the cultivation period still remains an essential goal for the future to increase patient acceptance of the procedure as well.

Identifying children at high risk for psychological sequelae after pediatric intensive care unit hospitalization.

OBJECTIVE: To identify those patients in a pediatric intensive care unit who may be at highest risk for developing persistent psychological sequelae after hospital discharge. DESIGN: A secondary data analysis was conducted to examine data gathered in an earlier study of children's psychological responses after critical illness. The current study focused exclusively on patients who required pediatric intensive care unit hospitalization. PATIENTS: Sixty children, aged 6 to 17 yrs, hospitalized in two Canadian pediatric intensive care units. PROCEDURES: Children were categorized as either high risk or low risk for developing persistent psychological sequelae after discharge based on their level of illness severity and the number of invasive procedures to which they were exposed. Outcome data were analyzed using descriptive statistics, followed by an assessment of group differences at baseline, 6 wks, and 6 mos postdischarge. Combined effects of invasive procedures and illness severity on the outcome variables were explored. OUTCOME MEASURES: Three questionnaires were completed by all children 6 wks and 6 mos postdischarge, including the Children's Impact of Events Scale, the Children's Medical Fears Scale, and the Children's Health Locus of Control Scale. RESULTS: Children in the high risk group demonstrated more psychological sequelae 6 wks and 6 mos postdischarge. Exposure to high numbers of invasive procedures was the most important predictor of group differences 6 wks postdischarge. CONCLUSIONS: Findings suggest there is a group of children in the pediatric intensive care unit who are at higher risk for developing persistent psychological sequelae postdischarge. Exposure to high numbers of invasive procedures may be the driving force behind group differences, particularly at 6 wks postdischarge. These children warrant closer observation and follow-up.

Spontaneous scalp arteriovenous fistula in a child with hartnup disease.

PURPOSE: To report the endovascular treatment of a spontaneous scalp arteriovenous fistula (AVF) in a child with Hartnup disease. CASE REPORT: A 6-year-old girl with Hartnup disease presented with recurrent attacks of intense, migraine-like, right-sided headache; a tender, pulsatile small mass was observed in the scalp. Selective digital subtraction angiography revealed a high-flow scalp AVF fed by the frontal branch of the right superficial temporal artery draining via the scalp veins. Endovascular treatment was performed by direct puncture of the distal feeding artery and injection of 2 mL of a 50% mixture of N-butyl-cyanoacrylate and Lipiodol. Serial arteriograms performed 6 months and 2 years later documented complete resolution of the lesion. The patient has had no recurrence of clinical symptoms or local signs for recanalization. CONCLUSIONS: Scalp AVFs may progress in size, causing significantly disabling symptoms, particularly in children. We recommend endovascular treatment at the earliest possible stage.

Endovascular management of a mandibular arteriovenous malformation in a patient with severe hemophilia a.

The unusual case of a mandibular arteriovenous malformation in a patient with severe hemophilia A and hepatitis C is reported. Supplementary substitution of various coagulation factors allowed direct puncture and intralesional injection of a liquid adhesive, resulting in complete anatomic and clinical cure without peri- or postoperative bleeding. Replacement therapy providing normal levels of relevant coagulation factors enables endovascular treatment in a safe and effective manner in hemophiliac patients.