author: Marie Csete
date: 2010
references:
title: Q&A: What can microfluidics do for stem-cell research?

# What can microfluidics do for stem-cell research?

Stem-cell biology and microfluidics have both been hotbeds of research activity for the past few years, yet neither field has been able to successfully commercialize a clinical 'killer application'. Stem-cell behavior is exquisitely sensitive to environmental cues, and the important cues are difficult to establish, manipulate and quantify in traditional cell culture. Because the microenvironment can be controlled in microfluidics platforms, microfluidics has a lot to offer stem-cell biology and there are many good reasons for the fields to join forces.

# What exactly is microfluidics?

Microfluidics is the characterization and manipulation of fluids on the nanoliter or picoliter scale. The behavior and properties of fluids change as amounts decrease from the macroscale (volumes used for everyday applications) to the microscale. This means that microfluidic devices cannot be built by simply scaling down macroscale devices. For instance, at low microliter volumes, fluids act more like solids, and two fluids flowing alongside each other in a microchannel will not mix well (except by diffusion); therefore, a variety of techniques (pumps, valves, electrokinetics) are used in microfluidics platforms to actuate mixing and fluid flows. Most microfluidics applications in research labs concentrate on the 10 to 100 μm scale, basically the diameter of a single cell.

Microfluidics lab-on-a-chip devices allow standard laboratory analyses, such as sample purification, labeling, detection and separation, to be carried out automatically as the sample is moved, via microchannels, to different regions of a chip. Various methods have been used to produce microfluidic devices, but inkjet printers offer an easily accessible way of printing channels and other features directly onto the device. This technique has been used to print precise patterns of proteins or protein gradients onto a surface on which cells can subsequently be cultured to investigate or control their behavior. A technically more advanced use of microfluidics is the integration of microchannels with nanoelectrospray emitters for preparing material for mass spectrometry in high-throughput proteomics analyses of biologic samples \[1\].

# What background do you need for microfluidics?

Physics (in particular fluid dynamics), mechanical engineering, or bioengineering backgrounds, the common feature of these being a strong mathematical foundation.

# Why should stem-cell biologists care about miniaturization of cell culture and analysis tools?

On the one hand, scientists working on the development of pluripotent stem cells for clinical use are encountering a major challenge in scaling up cell cultures for master banks to be used as sources of cell therapies for large numbers of patients. Microfluidics is clearly not the answer to this problem. But on the front end of developing therapies from stem cells, rigorous identification of the starting stem cell and its progeny is a major technical challenge and a regulatory requirement, analogous to the precise chemical identity of a drug. Classically, identification of stem cells is done clonally (at the single-cell level), and it is generally difficult to follow or analyze single cells in mass cell culture. Microfluidics techniques can be used for sensitive discrimination of gene expression (and protein) levels at the single-cell level and they are therefore increasingly useful in stem-cell biology to understand the heterogeneity of stem-cell populations.

Separation of rare stem cells (or rare cancer cell types) from a mixed population is also not easy using flow cytometers developed for clinical use; harsh conditions imposed on the cells during standard flow cytometry mean that cell recovery is low. Microfluidics-based, benchtop flow cytometry allows separation of small numbers of stem cells under direct visualization, and is less damaging to cells than traditional cell sorters. For both analysis and separation, microfluidics offers the means of controlling the cells' environment rigorously. Several groups have also reported that stem cells (and stem cells committed to a particular lineage) can be separated from mixed cell populations using their dielectric properties (electric and magnetic energy).

# In what ways are microfluidics culture conditions superior to those of traditional mass cell culture?

Stem-cell fate (growth, death, differentiation, migration) is highly dependent on environmental cues, but the usual cell culture environment does not mimic the *in vivo* microenvironment in several fundamental ways (20% oxygen is unphysiologically high; physiologic fluid flow and shear stresses are not present; three-dimensional environments cannot be standardized), and overall the environment in conventional cell culture is not controllable. For example, pH inevitably drifts in conventional tissue culture, but in well-designed microfluidics devices, the pH can be held constant by controlling medium inflow and outflow. In other words, engineers can provide steady-state conditions for cells, as well as fast and predictable changes in the environment surrounding the cells. Of particular importance, the best microfluidics devices are supported by mathematical descriptions of the microenvironment, and information from experiments can be fed back into mathematical models to determine optimal design features to promote specific stem-cell behaviors.

Gradient cues, so important in embryonic development, can be constructed quite precisely on microfluidics devices, as noted above. For example, migration of stem cells in response to chemotactic gradients is often studied in mass cultures using repeated studies in Boyden chambers (two chambers separated by a filter through which cells migrate), but molecular gradients established with microfluidics tools yield inherently more detailed and precise information because gradient characteristics such as slope and concentration can be quantified and correlated to migration behavior. Overall, flexibility in the configuration of microchips is a major advantage of microfluidics-based cell-culture systems, and the ease with which fluid flows can be controlled over time and space.

Human embryonic stem cells (hESCs) are particularly sensitive to handling in culture, and automation of hESC growth and differentiation *in vitro* on microfluidics platforms produces more standardized outcomes. Many investigators believe that the stress of manual handling of hESCs is an important factor in their instability over time, and therefore automated techniques for passaging and expansion may be a method for overcoming the problem of karyotypic instability.

Three-dimensional mass culture systems are especially 'noisy' and difficult to control using conventional tissue-culture methods. Embryoid bodies - floating aggregations of undifferentiated cells - are often used as an intermediate stage in differentiation protocols, and are generated from hESCs by passaging the cells onto non-adherent plates. The resulting embryoid bodies are widely heterogeneous in size unless special engineering protocols are used. This size heterogeneity means that diffusion patterns for signaling through the embryoid bodies and cell-cell interactions are also heterogeneous, resulting in lack of control over the differentiation patterns. Printed topographic features of various shapes on microchips or microchannels are a proven method for gaining control over how cells aggregate. The size and development of embryoid bodies can be controlled with microfluidics techniques, providing a more predictable differentiation pattern and organization of the cells into phenotypically distinct layers. In fact, engineers have successfully manipulated parts of embryoid bodies in different ways using microfluidics tools to alter distinct fates for different parts of the cell aggregates.

The 'micro' in microfluidics plus the configurability of channels can be used to look at simultaneous signals to two parts of a single cell, for example the apical versus basal signals that will be encountered by a polarized cell. In traditional mass culture, cells align in random fashion, and although matrix coatings on tissue-culture plastic can be used to line cells up relative to the matrix, it is impossible to present signals to separate subcellular domains. Epithelial cells are the classical polarized cell in which specific receptors are largely confined to either the apical or basal surface, and signals received at these subcellular domains determine cell function. At the very small scale of microfluidics devices, the apical and basal faces of a cell can be exposed to separate chambers whose composition can be defined and manipulated independently, making it possible to determine the hierarchy of stimuli that determine cell behavior.

An obvious advantage of microfluidics is that it provides economy in terms of reagent use, especially for high-throughput assays. Of course, this economy will only be realized if device fabrication is also inexpensive.

# What are some of the major limitations of microfluidics-based cell culture systems?

Not surprisingly, from a biologist's perspective, the materials-cell interface is still a problem. Polydimethylsiloxane (PMDS) is commonly used to make microchips because it is cheap, optically transparent, gas permeable, and can be manipulated outside a clean room. Although many groups have reported using PDMS chips for hESC studies, my experience is that PDMS has to be considerably modified (and coated), because it is very toxic to the cells. Other, more biocompatible surfaces are available, but the ideal material for exquisitely sensitive cells such as hESCs has not been developed. Again from the biologist's perspective, cellular debris can occlude small channels, so that optimal washing methods in some applications need improvement.

Engineers have pointed out that the best mathematical framework for handling models, such as differentiation, that start at small scales but result in large-scale processes is still evolving \[2\]. So along with the constant improvement in hardware and software needed to make inexpensive devices work optimally, the mathematical tools that make microfluidics approaches so valuable also need continuous refinement. Ultimately, the feedback between biologists using the devices and engineers designing them is the essential key for moving microfluidics-based cell culture forward.

A major issue limiting wide application of microfluidics is that the devices still require experts to operate them, and are not yet biology user-friendly.

# What major problems in translational stem-cell biology can be addressed using microfluidics tools?

Here again, microfluidics techniques afford the ability to define the microenvironment surrounding stem cells. The disease environment into which stem cells will be transplanted is certain to alter their behavior, and is not adequately mimicked in most animal models of disease. Microfluidics-controlled environments can be used to test the tolerance of cells to mechanical and shear forces, gases, oxidants and other extracellular cues that characterize the disease environment. Physical, mechanical and biochemical factors can be tested quantitatively at relatively high throughput on the benchtop using microfluidics to help predict behavior of stem cells *in vivo*.

Overall, microfluidics tools can be used for spatiotemporal control over the stem-cell microenvironment, so that the ideal *ex vivo* niche for cell survival and differentiation can be defined quantitatively and in high throughput. Control over the culture environment also allows investigators to perturb cell fate to generate desired outcomes, and to define the limits of physical, mechanical and biochemical factors that are tolerated by stem cells at different stages of differentiation.

# What have been the important contributions of microfluidics in biology in general?

George Whitesides points out that one of the best developed applications of microfluidics is in protein crystallographic studies, to screen the conditions that encourage growth and protection of crystals \[3\]. For cell biologists, the major impact has been in cell separation, single-cell resolution of the dynamics of gene expression, and insights into how mechanical forces applied to individual cells determine their behavior.

# Where can I find out more?

Cai L, Friedman N, Xie XS: **Stochastic protein expression in individual cells at the single molecule level**. *Nature* 2006, **440**:358-362.

Cimetta E, Figallo E, Cannizzaro C, Elvassore N, Vunjak-Novakovic G: **Micro-bioreactor arrays for controlling cellular environments: Design principles for human embryonic stem cell applications**. *Methods* 2009, **47**:81-89.

Melin J, Quake SR: **Microfluidic large-scale integration: the evolution of design rules for biological automation**. *Annu Rev Biophys Biomol Struct* 2007, **36**:213-231.

Stroock AD, Dertlinger SK, Ajdari A, Mezic I, Stone HA, Whitesides GM: **Chaotic mixer for microchannels**. *Science* 2002, **295**:647-651.

Tung Y-C, Torisawa Y, Futai N, Takayama S: **Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics**. *Lab Chip* 2007, **7**:1497-1503.


abstract: # Background
  .
  Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes.
  .
  # Methods
  .
  Surgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, \[tumor DNA\]/\[NB DNA\]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers.
  .
  # Results
  .
  Purification of DNA from ultrasonic surgical aspirations was rapid (\<3 days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level (≥ 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level (≥3.0 & \<6.0 CN ratio) amplifications detected in FFPE were also detected as being increased (≥ 2.0 CN ratio) in the aspirations.
  .
  # Conclusions
  .
  Buffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE.
  .
  # Virtual slides
  .
  <http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466>
author: Long N Truong; Shashikant Patil; Sherry S Martin; Jay F LeBlanc; Anil Nanda; Mary L Nordberg; Marie E Beckner
date: 2012
institute: 1Department of Biological Sciences, Louisiana State University - Shreveport, One University Place, Shreveport, LA 71115, USA; 2Department of Neurosurgery, Baylor College of Medicine, 1327 Lake Point Pkwy, Suite 400, Sugar Land, TX 77478, USA; 3Delta Pathology Group, One Saint Mary Place, Shreveport, LA 71101, USA; 4Department of Emergency Medicine, Long Medical Center, Louisiana State University Health Sciences Center – New Orleans, 5825 Airline Hwy, Baton Rouge, LA 70805, USA; 5Department of Neurosurgery, Louisiana State University Health Sciences Center – Shreveport, Rm. 3-215, 1501 Kings Highway, Shreveport, LA 71130, USA; 6Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center – Shreveport, Rm. B-215, 1501 Kings Highway, Shreveport, LA 71130, USA; 7Departments of Pediatrics & Medicine, Louisiana State University Health Sciences Center-Shreveport, Rm. 2-303, 1501 Kings Highway, Shreveport, LA 71130, USA; 8Delta Pathology Molecular Diagnostics, One Saint Mary Place, Shreveport, LA 71101, USA; 9Department of Neurology, Louisiana State University Health Sciences Center – Shreveport, Rm. 3-438, 1501 Kings Highway, Shreveport, LA 71130, USA
references:
title: Rapid Detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors

# Background

Oncogenes encode proteins that promote tumor growth, survival under adverse conditions, and invasion. Many studies have detected amplified oncogenes in high grade brain tumors, especially glioblastoma or glioblastoma multiforme (GBM). Therefore, assays to identify amplified genes are proposed to become critical in patient care as monoclonal antibodies and small molecules that inhibit proteins encoded by oncogenes become available. Identification of amplified genes in a rapid, multiplex manner is relevant for stratifying patients to clinical trials and treatments. Brain tumor specimens for molecular studies include ultrasonic surgical aspirations available at the time of surgery and small, diagnostic biopsies that are processed as formalin-fixed, paraffin-embedded (FFPE) samples. Sometimes tumor fragments are resected and are also processed as FFPE samples. Although tissue frozen for storage at the time of surgery can also be released for DNA studies, it is not usually available outside of research settings. Ultrasonic surgical aspirations are not routinely used for diagnostic purposes and represent an untapped source of abundant, fresh tumor DNA. With laboratory support for processing cellular fluids and the expertise for confirming tumor cell content morphologically, pathology departments are well suited for providing cellular or nuclear pellets from ultrasonic surgical aspirations to provide tumor DNA needed in molecular testing. Surgical aspirations can quickly provide DNA to speed up turnaround times and produce higher yields than FFPE sections of small biopsies.

Brain tumors debulked by ultrasonic surgical aspiration yield suspensions of single cells, minute tissue fragments, and blood in saline. Following introduction of this technique in the 1970's, ultrasonic aspiration became routine in microneurosurgery \[1\]. Unlike the characteristically small diagnostic biopsies of brain tumors, ultrasonic surgical aspirations contain ample tumor cells in large volumes of fluid. The ultrasonic apparatus consists of a computerized control unit with settings for different amplitudes of ultrasonic waves and speeds of irrigation and aspiration. Tumor tissues are targeted and selectively aspirated. An advantage of the ultrasonic aspiration technique over tissue dissection is the reduced need for retraction of normal brain in the patient. Accordingly, the use of ultrasonic surgical aspiration to debulk intracranial tumors is common and explains the unfortunate paradox of having only small diagnostic tissue biopsies from large brain tumors available for molecular studies outside of research settings. Although ultrasonic aspiration specimens have yielded viable tumor cells for experimental studies \[2-4\], their lack of tissue architecture greatly diminishes their usefulness in diagnostic pathology. For example, regions of tumor necrosis that are a useful diagnostic feature in high grade tumors are prone to disintegrate during the aspiration procedure. Although some institutions preserve small portions of ultrasonic aspirations in FFPE blocks, the intact tissue fragments from biopsies are preferred and routinely relied upon for diagnostic evaluation of primary and metastatic brain tumors. Ultrasonic surgical aspirations, that contain the bulk of brain tumor tissue in a dispersed and disaggregated form, seldom provide the complete architectural features of the tumors that aid in determining morphologic diagnoses.

In this study, ultrasonic aspirations of brain tumors were tested to see if they would yield tumor DNA that is appropriate for studies to detect amplified oncogenes. Testing methods that identify amplified genes as potential treatment targets in individual tumors would ideally be performed as soon after surgery as possible. The genes tested with commercial MLPA kits are known to be amplified in association with malignancy. The amounts of DNA purified from ultrasonic aspirations of GBMs were found to be especially ample. These DNA samples were analyzed for amplifications in seventy-nine oncogenes, with results compared to those from FFPE-derived DNA and with fluorescence *in situ* hybridizaiton (FISH) results for *EGFR* gene amplification in FFPE tissue sections. The MLPA assays on ultrasonic aspirations identified high-level amplified genes within a few days at low cost and served as a preview of the more sensitive FFPE assay results that became available later.

# Methods

## Patient Population, Specimen Retrieval and DNA Purification

Permission from our Institutional Review Board was obtained for retrieval and study of the excess amounts of ultrasonic aspiration and FFPE specimens taken from brain tumors resected in 2008–2009 in a pilot study of using these specimens for molecular tests. Ultrasonic aspirations from seven brain tumors, including four glioblastomas (GBM1-4), two lung carcinomas metastases (LCM1-2) and an ependymoma (EP1), were tested. The patients (all male except for one female with a GBM) were 44 to 81 yrs of age. Samples were obtained in saline with a Cavitron Ultrasonic Aspirator Excel (Valleylab-Tyco International, Boulder, CO) set at 23 to 36 MHz. The volume (cc) of each tumor present preoperatively and postoperatively was estimated by the neurosurgeon. The ultrasonic surgical aspirations were not designated to be used for any diagnostic purpose. The specimens in this study were delivered to the laboratory within 24 hr of surgical resection, either fresh or after overnight refrigeration. Portions of ultrasonic aspiration specimens, 15 to 30 ml, were centrifuged at 500 g for 10 min. Tumor cells sedimented as large buffy coats above the erythrocytes and beneath floating necrotic debris. According to the manufacturer's recommendations (Qiagen Midi Kits, Valencia, CA), 500 μl of each buffy coat were pipetted and combined with purification kit components to obtain a nuclear pellet that was processed immediately or frozen for later purification. All specimens yielded measureable amounts of DNA. For comparisons in each tumor, DNA from routine FFPE sections (10 μm thick, 10 per tumor) was purified with a Qiagen FFPE DNA purification kit. In each case tumor cells represented at least 90% of nucleated cells in the tissue sections. Proteinase K digestions at 55°C were extended as needed to achieve digestion of the tissues. Purified DNA was evaluated and quantified with spectrometry using absorbance measurements at 260 and 280 nm (JENWAY Genova, Jenway Limited, Essex, England).

## Histological Review of Tumor Specimens

Diagnostic FFPE tissue sections, available from all tumors, were reviewed microscopically to characterize the pathological features. Portions of ultrasonic aspiration specimens that had also been processed as FFPE specimens were available. Hematoxylin and eosin stained FFPE sections were evaluated. The number of mitotic figures in ten high power fields (HPFs) (400X magnification) was determined for each tumor in duplicate counts. Also, stains for Ki67 reactive cells were performed at the time of diagnosis for some tumors using a pre-diluted antiKi67 antibody (Ventana Medical Systems, Oro Valley, AZ) for direct staining on an automated immunohistochemical stainer (Benchmark XT, Ventana Medical Systems). Pre-diluted, non-reactive mouse monoclonal negative control (Ventana) solutions were applied to separate tissue sections. The percentages of tumor cells positive for Ki67 specific staining were determined with an automated cellular imaging system (Chromavision ACIS, San Juan Capistrano, CA) with verification by manual counts at the time of review. If interference from background staining was present, manual counts were substituted.

## *EGFR* FISH

Inclusion of probe sets for *EGFR* in the MLPA kits permitted comparison of MLPA and FISH copy number (CN) data for this gene in each tumor. FISH probes for a control locus, 7p11.1-q11.1, D7Z1, and the *EGFR* band region, 7p11.2-7p12, (Vysis Locus Specific Identifier (LSI) *EGFR* SpectrumOrange/CEP 7 SpectrumGreen, Abbott Molecular Inc., Des Plaines, IL) were hybridized to interphase nuclei. Paraffin sections of all tumors and cytologic smears prepared from some of the ultrasonic aspiration specimens were examined. Smears from aspirations were fixed, denatured, and hybridized with probes overnight. Un-hybridized probes were washed away. Diamino-phenylindole (DAP1) fluorescent blue (Abbott Molecular) stained the nuclei. Slides were scanned on a fluorescent microscope (Leica DMR, Wetzlar, GM) for analysis with images captured using a digital camera (Applied Imaging, San Jose, CA) and CytoVision v4.02 imaging software (Applied Imaging). Sections from FFPE tissue were cut at a thickness of five microns, deparaffinized (Paraffin Pretreatment Reagent Kit II, Abbott Molecular), processed on a VP2000 Processor (Abbott Molecular) and then FISH probes for *EGFR* and CEP 7 were applied and analyzed as described above. Amplifications of *EGFR* were observed as average *EGFR* to CEP 7 signal ratios greater than two for at least 20 cells (usually many more). The results were determined by averages of all cells that could be evaluated. Proteinaceous debris hindered FISH interpretations in some of the ultrasonic surgical aspiration specimens. Ratios of total *EGFR* and CEP 7 signals per tumor, individual *EGFR*/CEP 7 signals per cell and medians of the signal ratios per cell were described.

## MLPA

The PCR-based technique, MLPA, involved multiple steps, including exposure of DNA to gene-specific probe sets, enzymatic ligation, PCR, and capillary electrophoresis (CE) to separate PCR products. Briefly, as explained earlier \[5\], 200 μg of DNA from each tumor sample and normal brain in buffer were denatured at 98°C for 30 min in a thermocycler (MasterCycler personal Eppendorf, Hamburg, GM) and were then hybridized to MLPA probes (SALSA P171, P172 and P173 kits, MRC-Holland, The Netherlands) according to kit instructions. The manufacturer selected genes according to literature documenting amplification in tumors. In addition to gene specific sequences, the probes also contained universal PCR primers, X and Y, and stuffer sequences for subsequent identification of specific gene amplicons by size using CE. Ligase65 (MRC-Holland) generated ligations specific for perfectly hybridized probe pairs (sets) at ligation junctions in the DNA and the enzyme was then heat inactivated. Ligated probes were PCR amplified with polymerase (MRC-Holland) according to instructions. PCR products were separated on a CEQ 8000 (Beckman-Coulter, Fullerton, CA) and were then identified according to the length of amplicons.

Fragment analysis of PCR products produced a series of linear peaks (signals of fluorescence) corresponding to the relative quantities of PCR products that were proportional to initial amounts of DNA (or CN) of the targeted genes. Slightly less efficient amplifications of longer amplicons accounted for minor reductions in their peak heights. All of the genes tested in the three MLPA kits, P171, P172, and P173, are listed as follows: *AKT1, AURKA, BCAR2, BCAS1-2, BCL2, BCL2A1, BCL2L1*, *11* and *13, BCL6, BCLG, BIRC1-5, BRAF, BRMS1, CCNA1, CCND1-2, CCNE1, CDK4* and *6, CENPF, CYP27B1, EGFR, EMS1, ERBB2* and *4, ESR1, EVI1, FGF3* and *4, FGFR1, FLJ20517, GNAS, GSTP1, HMGA1, IGF1R, IGFBP2,4* and *5, IRS2, JAK2, MDM2* and *4, MET, MMP7, MOS, MYCL1, MYBL1* and *2, MYC, MYCN, NFKBIE, NRAS, NTRK1-3, PDGFRA* and *B, PIK3C2B, PIK3CA, PPM1D, PSMB4, PTK2, PTP4A3, PTPN1, RELA, RNF139, RUNX1, SERBINB2, 7* and *9, TERT* and *TOM1L2*.

Occasional failures to see peaks in one of the eight capillary tubes during a CE run were attributed to low current whenever the peak was obtained after repeating CE. Successful concurrent results for NB were required for a CE run of tumor samples to be further analyzed.

## Normalization of CN

For detection of somatic changes found in highly malignant tissues, there were no known reference probes that could be relied on to maintain their normal CN at the time of this study. Large numbers of genes are lost or amplified in malignant tumors leading to considerable variability in the CN of individual tumors. Also, genetic variations and mutations in tumors, such as nucleotide deletions, insertions, or substitutions near the ligation junctions could potentially impact ligation efficiency. In this study the result of each probe set in each tumor specimen was normalized with CN for the same probe set in DNA from non-neoplastic brain (NB) assayed concurrently. Comparisons of all tumors with the same source of NB controlled for assay to assay variation. Aliquots of NB were from an 82 year old woman's normal occipital lobe (Biochain, Hayward, CA).

The CN ratios, or fold-differences from normal for each gene, are represented by the ratio, \[tumor DNA\] / \[NB DNA\], derived from measurements of the CE peaks. The CN ratio was calculated after peaks of non-amplified genes on CE graphs, representing relative amounts of DNA, were matched with the average of two NB samples analyzed in the same assay run. Peak heights of non-amplified genes in tumor samples and NB were adjusted to approximately the same scale as graphical printouts from CE were produced. Final, finer adjustments were made in spreadsheets by maximizing alignments of trendlines for non-amplified genes. Scatterplots were evaluated to check the reactions of NB DNA with the MLPA probes compared to expected values provided by the manufacturer. Overlays of NB results obtained in GBM1's assays of FFPE, using P171, P172, and P173 kits, with graphs of normal values provided in the MLPA kits' literature, demonstrated comparable overall detection of the two populations of normal DNA data points (Figure 1).

Regression analysis of NB's scatterplots demonstrated that results obtained using one of the two probe sets for *ERBB4* was an outlier. In P171 data points, *ERBB4*'s probe set result was at the 95% confidence limit for the overall NB sample population. All other probe set results fell above the lower 95% confidence limit for the NB sample. Also, three probe sets were slightly out of range in the opposite direction so that missing a low level amplification was a concern but other probe sets for these genes were within the 95% confidence limits so that missing a significant CN gain after their results were averaged would be unlikely. The probe sets with results above the confidence limits included 1 of 2 for *IGFBP2*, 1 of 2 for *NTRK1*, and 1 of 4 for *IGF1R*.

The normal CN range was set at ≥ 0.75 to ≤ 1.50, with CN ratios ≥ 2.0 considered to be amplified. High-level gene amplifications in glioblastomas have been previously set at 6-fold greater than diploid or 12 or more copies per nucleus \[6\]. In this study CN ratios ≥ 6.0 were also designated as high-level amplifications and lesser amplifications were set at CN ratios of ≥ 3.0 and \< 6.0 for mid-level and ≥ 2.0 and \< 3.0 for low-level amplifications. Averaged results for two replicates of amplified and non-amplified genes in ultrasonic aspiration tumor DNA were compared to results for two replicates of genes in NB for each gene's probe set (or ligated pair). Results for multiple probe sets for a gene were averaged. MLPA analysis using the kits, P171, P172, and P173, was performed on FFPE DNA in two replicates from each of 7 tumors in 15 of the 21 assays and in single replicates in 6 due to DNA depletion from the small specimens.

## Statistical methods and correction factors

Excel was used to prepare graphs for images, determine Spearman rank correlation coefficients, regression analysis, etc. Outlier identification was performed with regression analysis to detect data points at or beyond 95% confidence intervals for residuals. A Bland-Altman plot to compare MLPA results of cavitronic ultrasonic surgical aspiration and FFPE assays was generated with R, version 2.10 (The R Foundation for Statistical Computing, http//<http://www.R-project.org>), statistical software.

# Results

## DNA purification yields

Successful DNA purification was achieved for all of the tumors. The greatest yields of DNA (43.0 to 77 μg) were obtained from buffy coats of glioblastoma ultrasonic aspiration specimens. The FFPE sections from all of the tumors yielded less DNA (6.4 to 20.5 μg) as seen in Table 1.

DNA data according to sample type, cavitronic ultrasonic surgical aspiration (CUSA) or formalin-fixed, paraffin-embedded (FFPE) sections

| **Brain tumors** | **Specimen type** | **260/280 Absorbance (nm)** | **DNA yield per specimen (μg)** |
|:---|:---|:---|:---|
| GBM1 | CUSA | 1.38 | 49.1 |
|   | FFPE | 1.98 | 6.7 |
| GBM2 | CUSA | 1.84 | 77.0 |
|   | FFPE | 1.98 | 7.9 |
| GBM3 | CUSA | 1.84 | 43.3 |
|   | FFPE | 1.86 | 6.4 |
| GBM4 | CUSA | 1.67 | 44.6 |
|   | FFPE | 1.90 | 7.0 |
| LCM1 | CUSA | 1.85 | 17.9 |
|   | FFPE | 1.81 | 11.3 |
| LCM2 | CUSA | 1.16 | 14.6 |
|   | FFPE | 1.63 | 20.5 |
| EP1 | CUSA | 1.88 | 12.1 |
|   | FFPE | 1.55 | 10.1 |

GBM = glioblastoma, LCM = Lung carcinoma metastasis, EP1 = Ependymoma.

## Clinicopathologic features of the tumors

Features of the tumors were typical for their diagnostic classification as glioblastomas, metastatic tumors, and an ependymoma. Photomicrographs of the ultrasonic surgical aspirations (100X and 400X magnifications) and FFPE sections (400X magnification) illustrates the similarity of tumor cells in the two types of specimens (Figure 2). There were too few cases to evaluate whether characteristic morphologic profiles for amplified gene(s) in these tumors were present. Mitotic figures among the glioblastomas were most numerous in GBM1 and mitoses among all tumors were most numerous in LCM2 (Table 2).

Selected clinicopathologic features of the brain tumors in this study

| **Tumor** | **Age & sex** | **Tumor (cc)** |  | **Mitoses per 10 HPF** | **Ki67 positive tumor cells** |
|:---|:---|:---|:---|:---|:---|
|   |   | Pre Op | Post Op |   |   |
| GBM1 | 50 yr M | 80.8 | 19.1 | 46.5 ± 6.4 | 61 % |
| GBM2 | 81 yr M | 51.4 | 0.0 | 19.5 ± 2.1 | 55 % |
| GBM3 | 52 yr F | 124.0 | 96.1 | 10 ± 2.8 | 38 % |
| GBM4 | 44 yr M | 35.6 | 0.0 | 18.5 ± 2.1 | Not tested |
| LCM1 | 55 yr M | 19.0 | 0.0 | 51.5 ± 9.2 | Not tested |
| LCM2 | 61 yr M | 6.5 | 0.0 | 64.5 ± 5.0 | Not tested |
| EP1 | 73 yr M | 20.6 | 1.38 | 0 | 10 % |

The volumes of tumor were provided as estimates by the neurosurgeon. HPF = high power field (400X).

## *EGFR* FISH

Analysis of *EGFR* with FISH was used to correlate with MLPA assay results. Signals were counted in the FFPE sections. The CN for *EGFR* was increased in the four glioblastomas with ratios of *EGFR* to CEP 7 increased to values of more than 20 in at least some cells of each glioblastoma. In the metastatic tumors averages of CNs for *EGFR* were 2.5 to 2.8 per cell with *EGFR* to CEP 7 ratios remaining below 2.0. Distributions of the *EGFR*/CEP 7 ratios are shown in Figure 3A and illustrate "tailing off" in the data towards high-level amplifications in individual cells. Representative cells with amplified *EGFR* from the glioblastomas and from one of the metastases lacking amplification are shown in Figure 3B. The EGFR/CEP 7 ratios are listed for all tumors in Table 3. The CN of CEP 7 varied and was increased (averages of 2.7 to 3.8) in glioblastomas. The CN of CEP 7 varied in LCM1 and LCM2 with averages of 2.5 and 2.6, respectively.

**FISH versus MLPA results for *EGFR* **

| **Tumor** | ***EGFR*/CEP 7 ratios in FISH assays on FFPE** |  | ***EGFR*'s tumor/NB DNA ratios (normalized CN) Average of 2 MLPA probe sets ± SD** |  |
|:---|:---|:---|:---|:---|
|   | Total *EGFR/*Total CEP 7 signals | Median ratio | CUSA | FFPE |
| GBM1 | 7.3 | 7.2 | 8.8 ± 0.2 | 21.1 ± 2.0 |
| GBM2 | 8.6 | 9.3 | 22.5 ± 7.9 | 19.3 ± 7.2 |
| GBM3 | 10.3 | 9.0 | 10.5 ± 3.8 | 33.1 ± 17.2 |
| GBM4 | 11.1 | 11.6 | 19.5 ± 9.3 | 26.7 ± 6.5 |
| LCM1 | 1.0 | 1.0 | 1.2 ± 0.5 | 1.2 ± 0.4 |
| LCM2 | 1.1 | 1.0 | 2.3 ± 0.9 | 2.3 ± 1.4 |
| EP1 | 1.0 | 1.0 | 1.7 ± 0.7 | 2.9 ± 2.6 |

Although amplification of *EGFR* seen in individual cells of GBMs varied greatly and MLPA provided collective results of numerous cells, both techniques demonstrated corresponding large elevations in copy number for this oncogene in GBMs. Polysomy may have accounted for low-level amplifications found with MLPA in LCM2 and EP1. Median ratios were derived from the distributions of signal ratios in individual cells. *EGFR* = *Epidermal Growth Factor Receptor*. CEP 7 = Centromere of chromosome 7, FISH = Fluorescence *in situ* hybridization. SD = Standard deviation. CUSA = Cavitronic ultrasonic surgical aspiration. FFPE = Formalin-fixed, paraffin embedded tissue.

## MLPA molecular results

Productive CE runs were obtained for all tumors. Prior to normalization of tumor CN, the output of CE data from all four glioblastomas and one of two metastases revealed obvious amplifications in at least one gene for each tumor. Measurements of the peaks for each gene were connected to create line graphs. The obvious peaks representing high-level and some mid-level gene amplifications in the glioblastomas are shown in Figure 4. Genes are listed along the *x-*axes according to amplicon size within each kit (P171, 172 and 173). Easily recognized amplifications occurred for *EGFR*, *CDK4*, *MDM2*, *CYP27B1*, and *PDGFRA* in one or more GBMs*,* and also for *CCNE1* in one of the brain metastases (not shown) using DNA from ultrasonic surgical aspirations. Comparable amplifications for the same genes were also detected in corresponding FFPE specimens (not shown). Results for non-amplified genes in the tumors and in normal brain (assayed concurrently) constitute the baselines of graphs in Figure 4. The *EGFR* data obtained with MLPA is included in Table 3 along with FISH data.

Following NB normalization to generate CN ratios, Bland-Altman analysis (*n* = 889, derived from 127 data points/tumor x 7) found that data in ultrasonic surgical aspiration assays corresponded to data from FFPE assays for the same tumor within 1.96 SD limits, except for some of the amplifications that exceeded CN ratios of 15 (log value = 1.176). A normal CN ratio of 1 corresponds to a log value of 0 (Figure 5A). Correlation coefficients for comparisons ranged from 0.71 to 0.99 in studies of each tumor according to the kits used. Consistency was high with the exception of LCM2 that showed slightly less correspondence, as seen in Figure 5. The *n*'s were 42, 42, and 43 for P171, P172, and P173 kits, respectively, in each tumor.

Normalized CN ratios for specific genes in glioblastomas, including those with high-level amplifications (CN ratio ≥ 6.0) and those with no alterations (all CN ratios ≥0.75 and ≤1.5), are shown in Figure 6 (A & B, respectively). Both the ultrasonic surgical aspiration and corresponding FFPE results from the same tumors are shown. The only high-level gene amplification in the brain metastases was *EVI1* (7.02 CN ratio) in LCM2 FFPE but it was not amplified in the corresponding aspiration specimen. The metastases, LCM1 and LCM2, had 2 and 6 mid-level gene amplifications, respectively. The ependymoma (EP1) only had 1 mid-level gene amplification. Among the 10 genes with no alterations in any of the GBMs, including replicates of all probe sets for a specific gene, there were 9 genes that were also unaltered in EP1, and 9 and 6 genes that were unaltered in LCM1 and LCM2, respectively. Totals of all deviations (\<0.75 or ≥2.0 in CN ratios) from normal values in ultrasonic aspiration and FFPE specimens are shown in Table 4. Alterations in CN were more frequent in FFPE than in ultrasonic aspiration specimens, 85% and 49% of total changes, respectively. However, FFPE did not completely encompass all of the changes. Restriction of some genomic gains and losses to only one type of specimen from the same tumor is consistent with tumor heterogeneity.

Copy number changes in formalin-fixed, paraffin-embedded (FFPE) and cavitronic ultrasonic surgical aspiration (CUSA) specimen DNA

| **Tumors** | **Genes with normalized CN ratios ≥ 2.0 (gains in copy number)** |  |  |  |  | **Genes with normalized CN ratios \< 0.75 (losses in copy number)** |  |  |  |  |
|:---|:---|:---|:---|:---|:---|:---|:---|:---|:---|:---|
|   | Total | FFPE | CUSA | Only FFPE | Only CUSA | Total | FFPE | CUSA | Only FFPE | Only CUSA |
| GBM1 | 18 | 17 | 7 | 11 | 1 | 16 | 12 | 6 | 10 | 4 |
| GBM2 | 10 | 10 | 5 | 5 | 0 | 21 | 17 | 9 | 12 | 4 |
| GBM3 | 13 | 13 | 7 | 6 | 0 | 21 | 18 | 8 | 13 | 3 |
| GBM4 | 10 | 8 | 8 | 2 | 2 | 15 | 13 | 7 | 8 | 2 |
| LCM1 | 7 | 7 | 5 | 2 | 0 | 15 | 12 | 11 | 4 | 3 |
| LCM2 | 15 | 13 | 5 | 10 | 2 | 32 | 22 | 18 | 14 | 10 |
| EP1 | 7 | 7 | 3 | 4 | 0 | 16 | 15 | 6 | 10 | 1 |
| Totals | 80 | 75 | 40 | 40 | 5 | 136 | 109 | 65 | 71 | 27 |

GBM1-4 had increased CN ratios for 22 genes (*AURKA, BCL2A1, BCL6, BIRC1,2,4, BRAF, CDK4,6, CYP27B1, EGFR, ERBB4, ESR1, EVI1, GNAS, GSTP1, MDM2, NRAS, PDGFRA, PIK3CA, RNF139,* &*SERPINB9*) and decreased CN ratios for 35 (*AKT1, BCAR3, BCAS1, BCL2, BCL2L13, BCLG, BIRC5, BRMS1, CCND1, CCNE1, CDK4, CENPF, EMS, FGF4, FGFR1, FLJ20517, GSTP1, IGF1R, IGFBP2, IRS2, JAK2, MDM4, MYBL2, MYC, MYCL1, MYCN, NFKBIE, NTRK1, PDGFRB, PIK3C2B, PTK2, PTP4A3, PTPN1, TERT,* &*TOM1L2*). LCM1-2 had increased CN ratios for 16 genes that overlapped with those for GBM1-4 plus *CCNE1, MET, PPM1D,* and *PSMB4*, and decreased CN ratios were found for thirty six genes that overlapped with those listed for GBM1-4 plus *BCL2L1, BIRC3, CYP27B1, MMP7, NTRK2, NTRK3, PDGFRA, RELA,* &*SERPINB2*. EP1 had increased CN ratios for genes among those for GBM1-4 and decreased CN ratios for those for GBM1-4 or LCM1-2 plus *CCND3, HMGA1*, &*MOS.*

# Discussion

## Overview of tumor genomics

Brain tumors are known for harbouring genetic abnormalities. Tumor genomes are being evaluated to varying extents by FISH, array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) arrays, specific mutation detection with various PCR methods, methylation studies, targeted sequencing of selected genes, and whole genome sequencing. Therapies are planned accordingly in some institutions. Ideally, molecular testing for potential treatment targets occurs at the time of diagnosis and again in tumor recurrences to indicate appropriate treatment alterations.

## Amplified oncogenes in tumor fluids

This study demonstrates that high-level amplified oncogenes can be quickly detected by inexpensive multiplex, PCR-based studies of cellular fluids using standard molecular laboratory equipment and techniques. Fluids with significant tumor cellularity offer the opportunity to retrieve fresh tumor DNA in abundant amounts. Advantages in testing the DNA of tumor cells in fluids rather than FFPE tissue sections, include avoidance of exposure to formalin, heat, and organic solvents, and enrichment for tumor via pelleting, filters, and various other methods, such as those developed for isolating tumor cells from blood. Tumor cells in fluids not needed diagnostically constitute as a potential source of DNA for molecular assays. This study demonstrated detection of amplified oncogenes despite concerns regarding ultrasonic, mechanical, and osmotic distortions and mixing of the tumor cells with saline and blood. Endothelial cells were the predominant type of non-tumor cell present but these do not exceed what was originally present in the tumor parenchyma. A tendency for short sections blood vessels to remain intact and sediment differently from tumor cells was noted but was not investigated.

## Adaptive and Co-amplification of Oncogenes

It is proposed that oncogenes can be adaptively amplified in tumor cells to increase key gene products while circumventing promoters and other traditional methods of gene regulation \[7,8\]. Tumor cells are already well known for developing resistance to chemotherapy by being permissive to amplification of a gene that encodes a key protein that aids drug metabolism \[7\]. Although genetic instability of brain tumors may account for some genes being amplified by chance, amplification of multiple oncogenes involved in a similar function, such as cellular proliferation or resistance to apoptosis, suggests an adaptive response. Co-amplification of multiple oncogenes that provoke redundant malignant behavior or adaptation to cancer treatments strengthens the rationale for planning multi-agent therapies based on identification of amplified oncogenes with multiplex techniques.

Although co-amplifications of several oncogenes have been reported in glioblastomas, the lack of predictable patterns in individual tumors indicates that each tumor needs to be tested. Despite a tendency for genes in close proximity to be amplified together in tumor genomes, the span of amplifications in these regions is variable and frequently interrupted. Amplified expanses of chromosomes include both "driver" and "passenger" or "bystander" genes in regard to their effects on tumor behaviour. The benefits of amplified "driver" genes could underlie retention of amplified chromosomal regions in malignant tumor clones.

## Specific oncogenes amplified in glioblastomas

Several oncogenes are commonly amplified in brain tumors. Amplification of *EGFR* or one of its variants, *EGFRvIII,* whose encoded protein is constitutively active, is well-known to occur in primary glioblastomas. Amplification of *EGFR* has also been found in anaplastic astrocytomas \[9\]. Gains of *EGFR* occurred in 70% of 40 glioblastomas in one study with high levels of gene amplification occurring as double minutes in 42% of the cases. Lower levels of *EGFR* amplification occurred as insertions of extra gene copies distributed along chromosome 7 \[10\]. In a previous MLPA study of 104 glioblastomas, 74 (71%) had additional copies of *EGFR*\[11\]. Although high levels of *EGFR* most likely underlie some malignant adaptations in glioblastomas, only small subsets of patients have responded to therapies targeted to EGFR in clinical trials \[12-15\]. Five moderate to high-level amplified genes in glioblastomas identified in this study, *EGFR, PDGFRA, CDK4, MDM2* and *CYP27B1*, have also been previously reported, sometimes with co-amplification \[16-20\]. Although proximity of the locations for *CDK4CYP27B1* and *MDM2* at 12q14, 12q13.1-q13.3 and 12q14.3-q15, respectively, contributes to co-amplification, this cannot be assumed to occur for all three genes in individual tumors. In 20 glioblastomas that harbored at least one of these amplifications, only 7 had amplification of all 3 genes \[20\]. In another study when 5 glioblastomas contained amplification of either *MDM2* or *CDK4*, both genes were amplified in only 3 \[18\]. In another study, among the 5 glioblastomas that contained amplification of either *CDK4* or *MDM2*, only one tumor had both amplified \[17\]. In a survey of 456 glioblastomas, 13.4% had amplifications of *CDK4* but only 9.2% had amplification of *MDM2*\[16\]. In our study, 2 of 4 glioblastomas had co-amplifications of *CDK4* and *CYP27B1* and only one also had amplification of *MDM2.* Amplification of *EGFR* was present in all four glioblastomas.

## Amplifications of oncogenes in other brain tumors

Brain metastases from lung primaries in this study also contained amplified oncogenes suggesting that genomic analysis of metastases will detect amplified genes to serve as treatment targets, such as *CCNE1* that encodes cyclin E1 \[21,22\]. Additional analyses of brain metastases are indicated to identify the full range of oncogenes that can be amplified. Interestingly, in a previous MLPA study of non-typical meningiomas, 19 oncogenes were found to have amplifications in two or more invasive/atypical/anaplastic (mostly Grade II) meningiomas (total of 15) and the sums of copy numbers were inversely correlated with patient age \[5\]. Some of those genes were among the high-level amplified genes detected here but none of the amplifications in the non-typical meningiomas were in the high-level range. In this study, sums of CN ratios normalized with NB for the same 19 genes were higher in all 4 glioblastomas and in 1 of the 2 metastases than in the non-typical meningiomas studied previously (not shown).

## Implications of amplified oncogenes

As products of amplified genes become treatment targets, combinations of specific inhibitors and monoclonal antibodies will need to be tailored for individual patients according to amplifications found in the tumor DNA. Additionally, there is a tendency for amplified oncogenes to undergo mutations. Therefore, testing each brain tumor for oncogene amplifications would be useful for detecting key molecular treatment targets.

## Methods for assessments of oncogenes

With multiplex PCR methodology, rapid assessment of a relatively large group of oncogenes can be obtained using routine molecular laboratory techniques and equipment. In comparison to FISH assays, numerous genes can be tested simultaneously with comparisons to multiple non-amplified genes, whereas the number of FISH targets is limited to fewer genes. However, in this study, FISH did offer the advantage of detecting centromere duplication as a surrogate of chromosomal duplication so that low levels of *EGFR* amplification attributed to polysomy could be predicted. In comparison to the specificity of other PCR-based assays, amplification occurred with MLPA only if the probes hybridized to the gene targets in pairs (probe sets) and then underwent enzymatic ligation based on perfect sequence correspondence at their junctions to the tumor DNA. Also, the use of only one-fourth of the reaction volume for PCR in MLPA further reduced the chances of non-specific carryover of PCR amplicons from previous reactions. However, our study did not compare MLPA with other multiplex PCR techniques. Quantitative real-time PCR using multiwell plates is among other rapid molecular techniques to consider for testing multiple oncogenes simultaneously at low cost.

The validity of MLPA has been supported by other techniques. Detection of gene amplification (*ERBB2* or *Her- 2/neu*) with fluorescent and chromogenic *in situ* hybridization has closely correlated with MLPA results \[23,24\]. Results of *EGFR* amplification with MLPA have been comparable to detection of increased protein and RNA expression using immunohistochemistry and non-MLPA PCR, respectively \[10\]. In this study, FISH confirmed MLPA results for the presence or absence of mid and high-level *EGFR* amplification. Correlations were not quantified due to the potential for FISH signals to merge in tandem repeats or as aggregates of double minutes and also the heterogeneity of results in individual tumor cells.

The cost of kit reagents in a single MLPA assay is less than \$15. Although testing a tumor and normal brain DNA with two replicates each in three reactions to encompass probes for 79 genes increases the cost, testing multiple tumors with the same control DNA could be performed. Also, the number of genes to be screened could be reduced so that only 1 kit would be needed. Although high-level gene amplification results were obtained from the ultrasonic aspirations within a few days and the content of tumor DNA was plentiful, it should be noted that the FFPE assays demonstrated higher sensitivity in detection of low to mid-level gene amplifications. Therefore, ideally both types of specimens would be tested if detection of all levels of oncogene amplifications is desired. Multiplex detection of high-level amplifications using both types of samples strengthens confidence in the results at a relatively low cost and helps to negate concerns about tumor heterogeneity.

Although DNA from patients' blood cells has been used as normal controls when searching for somatic genetic alterations in tumors, quantifying high-level amplified oncogenes pose a problem when using blood samples. Very low numbers of circulating tumor cells with high-level amplifications or their free DNA in the blood could bias analyses. Thus this study used normal brain DNA that was comparable to normal values expected with MLPA that were provided by the manufacturer. Pooled DNA from multiple normal donors should be considered for future studies. Based on this pilot study, automation of steps in the MLPA procedure and subsequent data analysis are future goals so that this type of assay can be streamlined, validated, and used clinically.

# Conclusions

In summary, multiplex detection of high-level amplifications among oncogenes was successful in ultrasonic surgical aspiration DNA obtained from malignant brain tumors when compared with FFPE from the same tumors. The results indicate that morphologic evaluation of ultrasonic surgical aspirations to confirm tumor cell content and retrieval of DNA would aid molecular testing of brain tumors for oncogene amplifications. Many of the oncogenes with copy number gains encode proteins that are potential therapeutic targets. Therefore rapid identification of high-level gene amplifications could stratify patients to clinical trials and treatment plans shortly after surgical resections of brain tumors. Although ultrasonic surgical aspiration specimens are less sensitive than FFPE in detecting low to mid-level amplifications, the bulk of the tumor is present in the aspirations, they are fresh and homogenously mixed, and high-level amplifications can be detected in them. Abundant tumor DNA harvested from cellular fluids could also be used for targeted sequencing of amplified oncogenes to detect activating mutations.

# Abbreviations

CE: Capillary electrophoresis; CGH: Comparative genomic hybridization; CN: Copy number; CUSA: Cavitronic ultrasonic surgical aspiration; DAP1: Diamino-phenylindole; EP1: Ependymoma; FFPE: Formalin-fixed: paraffin-embedded; FISH: Fluorescence in-situ hybridization; GBM: Glioblastoma multiforme; HPF: High power field; MLPA: Multiplex ligation-dependent probe amplification; LCM: Lung carcinoma metastasis; MHz: Mega Hertz; NB: Non-neoplastic (normal) brain; PCR: Polymerase chain reaction; SNP: Single nucleotide polymorphism.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

MEB, LNT, SSM, and JFL carried out the molecular genetic studies. MEB, LNT, and SP drafted the manuscript. SSM and MLN performed the FISH assays. MEB, SP, MLN and AN participated in the design of the study. SP, JFL, and AN participated in and coordinated specimen and clinical data retrieval and characterization. MEB conceived and coordinated the study. All authors read and approved the final manuscript. This study was conducted with approval from Institutional Review Board (IRB) of LSUHSC-S.

## Acknowledgements

We acknowledge Lee Ellen Brunson, Dwain D'Souza, JoAnn Dismuke and Neelam Joshi, Department of Pathology, Louisiana State University Health Sciences Center-Shreveport (LSUHSC-S), for advice and technical assistance and also Ashley B. Flowers, Kristopher M. Katira, and Raj B. Patel, while medical students at LSUHSC-S School of Medicine, for technical assistance. We thank the Feist-Weiller Cancer Center and the Department of Pathology, LSUHSC-S, for providing equipment. This study was funded by a LSUHSC-S Grant-in-Aid Award (2008–2010) to Marie E. Beckner, MD with the purpose of encouraging high-value intramural research.


abstract: Peer review is the foundation quality in science, but now, in the US, the system is broken. What's the best way to fix things.
author: Gregory A Petsko
date: 2006
institute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
title: Instructions for repair

Last month I wrote about the sharp decline in the success rate for scientific research proposals submitted to the US National Institutes of Health (NIH) and other agencies. That column provoked numerous responses from both administrators of the funding organizations and life scientists. The administrators, while not denying some of the problems I discussed, argued that things aren't quite as bad as they seem, and that a large part of the difficulty stems from sizeable increases in the number of grant applications and the amounts requested, rather than from poor choices in managing the doubling of the NIH budget that took place not long ago. The scientists, on the other hand, all said that things were even worse than I had claimed.

Care has to be taken in drawing conclusions from either of these sources. I'm sure that people who have experienced difficulty in obtaining funding are more likely to respond to that column than those who've had success. And administrators probably feel the need to defend themselves, and their agencies, from what they might, with some justification, see as an attack by someone who doesn't know the whole story the way they do.

Nevertheless, although I think both sets of comments are useful, I also think both largely missed the point. People who wrote to me were all concerned, in one way or another, with the amount of money available for research and how it is being allocated. That's what seems to be on everybody's minds, and it's certainly worth talking about. Whether or not we're allocating the available funding sensibly is something that ought to be engaging officials as well as researchers in an ongoing dialog about priorities in science. (But that dialog isn't taking place. Somehow it just seems easier to keep asking for more money.) Yet, that wasn't the main point of the column. What concerns me is that, whether there really is a crisis in scientific funding or not, the perception that there is - and believe me, that is the perception on the part of just about every researcher I have talked to - has crippled the peer-review system.

Peer review is the foundation of quality in science. It prevents widespread cronyism and slowly weeds out unproductive lines of inquiry. But it requires that reviewers be both fair and wise. When the perception is that there's not nearly enough money to fund even all of the highest-quality proposals, a defensive turf-protection replaces a spirit of curiosity and egalitarianism. When it seems as if the primary job of a reviewer is to eliminate most proposals rather than to fight for the good ones, nit-picking replaces generosity. When the feeling is that every dollar counts so much that no risks dare be taken, conservatism and incremental advances get rewarded at the expense of bold new ideas. And when all of these things happen - and I believe they are happening, now, in the US - then the system is broken.

Societies based on scarcity tend to become hierarchical, with a well-fed elite and starving masses. As can be seen from publicly available data <http://grants1.nih.gov/grants/financial/QA_Doubling_Period.doc>, during the recent doubling of the NIH budget over a seven-year period, the number of investigators getting funded changed very little. Where did the money go? Besides a very large increase in the funding for NIH's own intramural research program, it seems to have gone to large increases in funding for established investigators who renewed their grants successfully during this period, or wrote additional ones. Instead of bringing lots of new people into the system, we ended up with more money for roughly the same set of grant holders. Now that funding is tight, those bloated operations are under tremendous pressure to at least maintain their size, which makes it even more difficult for new investigators - or new ideas - to enter the system. The average age at which a scientist receives his or her first NIH grant in the US is currently 42 for PhDs (even older for MDs), and in this time of perceived scarcity a broken peer-review system is not likely to change that.

What's the best way to fix things? It could be argued that the problem is temporary, and that when funding loosens up again, as it always has in past boom-bust cycles, peer review will recover along with everything else. After all, that's what happened in the 1970s. No need to tamper with the system. Time will take care of the problem.

I have my doubts. There's one big difference between peer review in 1975 and peer review today: the number of senior investigators participating in the process. Back then most review panels had a preponderance of such scientists, who provided the system with institutional memory of the way things were supposed to work. Nowadays, most established investigators feel they are too busy to put in the considerable time required to deal with the glut of proposals that every panel faces. The result is that less experienced scientists, with no history of a different gestalt, are being fed into a system where fault-finding and conservatism are the norm, so when the funding situation improves, there's no guarantee that the peer-review system will improve with it. (If you doubt this, consider the former Soviet Union. When it collapsed in 1989, newer Soviet-block countries like Poland and Hungary and Czechoslovakia, where there was a generation of people who still had a memory of how a market-based economy should work, did much better than Russia, where no one alive had experienced any system but communism.) In addition, the insistence that the composition of the panels must satisfy a requirement for geographic and institutional balance means that it's hard to have a large number of top scientists on any panel, even if they wanted to serve.

So my first repair instruction is simple: Do away with the misguided concept of balance, and require that all holders of research grants serve at least one year on a reviewing panel for every five years of funding they receive, regardless of seniority. Renewal of funding would be contingent on fulfillment of this service. If there is a surplus of available talent, then grants administrators could forgive the obligation for any given five-year cycle, but the requirement would kick in again when a grant was renewed. There would need to be a mechanism to deal with people who hold multiple grants - perhaps they would only incur a single one year debt for every five years of total funding, or the length of service could scale with the total budget; these details can be worked out. The important point is to create a pool of the best researchers, and to make sure that they represent the majority on all peer-review panels. As a dyed-in-the-wool advocate of personal freedom, the coercive aspects of this suggestion do trouble me somewhat, but it isn't really all that different from the way things work in the other main form of peer review - the jury system.

My second idea for how fix things is meant to address the problem of reviewer morale. When someone is given twelve grants to review, and knows that there is only a small probability that even the best one is actually going to be funded, he or she rapidly becomes discouraged. It's even more depressing when some less knowledgeable reviewer nit-picks one's best proposals, and depression is not the best mindset from which to make judgments. I suspect the program officers at the funding agencies must feel equally demoralized: it's no fun having to say "no" all the time, and to watch conservative study sections pass over the most exciting new ideas in favor of more of the usual. The solution, I think, is to give the program officers more autonomy in funding decisions. Some NIH institutes and centers claim that they do this, but in practice I have found that program officers rarely go against the recommendations of the reviewing panels. I suggest taking at least 10% of the budget of each institute or center and allowing the program officers to use it to fund grants that they believe to be exciting but that would otherwise miss the payline cut-off. They would need to justify each decision to the council, of course, but this suggestion would empower them to rectify some of the worst mistakes of the panels. In my experience, funding officials tend to be bright, committed individuals with a good broad knowledge of their field; I have no hesitation in giving them more autonomy. This is the way things actually work at the National Science Foundation, a funding agency that many believe has a better long-term history of supporting innovative research than does the NIH.

There also needs to be a way to improve the judgments coming out of the panels. Having more experienced reviewers would help, but it's hard to deal thoroughly and fairly with each proposal when the number being reviewed has increased so greatly. The way to solve the problem of proposal overload is to reduce proposal size. NIH proposals now are limited to 25 pages for the scientific description (that includes background and significance, progress during the past budget period, and the plan for future research). I think that should be shortened to 15 pages. If you can't describe clearly in 15 pages what you've already done, what you intend to do, and why it's important, you probably can't do it in 50.

But I think the proposals should be structured differently for different investigators. Scientists submitting their first proposal need to spend more space detailing how they are planning to carry out the work than established investigators should. In fact, I would argue that established investigators shouldn't have to describe their proposed methods in any detail at all, except if these are novel. To ask someone who has demonstrated for years that they can deliver the goods to prove that they know what they're doing is silly and borders on insulting. It also provides the nit-pickers with extra ammunition. People who tout stocks are constantly warning investors that past performance is no guarantee of future returns. But there is one area where it is: scientific research. The best predictor I know of as to whether a project will work is the track record of the principal investigator. Someone who has been consistently successful is not likely to fail, even when doing something risky. We need to stop pretending that isn't true. Most organizations that award pre- and post-doctoral fellowships spend very little time picking over the details of the applicant's research proposal, because they know that these young people haven't had any experience writing proposals and anyway usually end up doing something different, or in a very different way, from what they propose. Instead, fellowship reviewers tend to consider the qualities of the individual to be the most important factor on which to base their judgments. I think that makes sense at all levels of science. We need to be much less concerned with the details of projects, and put our bets on people and ideas.

While we're waiting for funding levels to improve, we need additional mechanisms to get young people started. The observation that, while investigator funding went way up during the NIH budget doubling, the number of investigators changed very little, suggests that we should consider putting a cap on the size of each award so as to make more money available for funding new projects and people. This is a serious matter, because it potentially has an impact on current employees, so if we implement a cap we will need to phase it in gradually. I am not proposing that we limit the total amount of funding that an individual can have - I think if someone can justify the need for millions of dollars to do first-rate science they should be able to obtain it. But I do think that we should exercise more scrutiny in such cases, and one way to do that is to force someone who claims to need, say, a million dollars to support a project to submit two or three proposals instead of one. I also think we will do better science, as a community, if we have more individual investigator-initiated projects and fewer mega-sized 'me-too' programs. Most innovation comes from small projects by relatively new people.

Two final ideas pertain to the machinery of the reviewing process itself. Turf protection is one of the biggest problems in peer review: as fields try to survive in a time of scarce resources, they often fight to fund their own mediocre science at the expense of quality in other areas. This largely stems from the personal and professional relationships that develop among members of a particular discipline. It's less of a problem when there's more money to go around, but right now we need to fight it. Here's a heretical and possibly crazy idea: I think we should consider not allowing people to review grants in their own field. Instead, they should only be allowed to comment on any questions of technical feasibility that come up during the review. This may seem absurd, but I'm not sure it is. If we follow my suggestion to bet on people rather than projects, detailed technical expertise isn't so important. And if we have the best, most experienced people back on our review panels, they usually will have a pretty broad knowledge of genomics, or biology, or whatever the main subject is. That will allow them to assess the importance of the proposed research and the impact of the applicant's previous contributions, which I maintain are the only two criteria that really should matter. Reviewing outside one's primary area of technical expertise happens all the time on fellowship panels, and they usually make pretty good decisions. After all, you don't have to be able to lay an egg in order to tell a good one - or to smell a bad one.

The second procedural change we should consider is aimed at addressing the issue of possible bias in the system. In times of scarce dollars, reviewers worried about their own chances of obtaining funding have an incentive to prevent others from being funded. Even if we assume such a thing rarely happens, we should want to ensure that proposals are reviewed wisely as well as fairly. I think the best way to guarantee the quality of the peer-review process is to review the reviewers. The data to do so exist, because there is a record of how every member of a panel has voted on every grant. Since most panelists only read their assigned proposals in detail, we need only be concerned with how a reviewer's scoring of such applications compares with the average score awarded to those same applications by the other assigned reviewers. Abnormally high or low scores would not be damning in and of themselves (there's plenty of room for legitimate differences of opinion in science) but a consistent pattern of low or high scores could indicate either poor judgment or bias. You may wonder how to be sure about such an evaluation, but it's actually easy, because we can compare each reviewer with him or herself. Bias or territoriality should be relatively easy to detect by examining how a suspect reviewer treats the same grants when they are resubmitted after revision. Since unsuccessful applicants try hard to answer the criticisms raised by the previous review, the scores of resubmitted grant proposals should improve, on average. If a reviewer's scoring on such resubmissions remains abnormally low compared with other reviewers who are also seeing the proposal for a second time, then there is reason to question the impartiality, or the judgment, of that reviewer, and they can be eased off the panel. There might even be no need to evaluate every reviewer all the time: random checking might be all that is needed to discourage trying to rig the game.

When fear and discouragement drive peer-review decisions, then the system is broken. But it's not broken beyond repair. The suggestions I've offered here can help mend it; at the very least, I hope they will start a dialog about what should be done. The worst thing we can do as a community is to throw up our hands in despair or pretend that everything will right itself magically when more money becomes available. Peer review is too important to give up on, or be left to chance. If we're serious about funding the best science and making our profession attractive to the brightest, most creative young minds, then we need to fix the system so that it once again serves those ends. Let's get to work.

## Figures and Tables


abstract: Lessons in personal genome analysis, social networking or health information?
author: Gregory A Petsko
date: 2009
institute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
title: What my genome told me - and what it didn't

Well, it turns out I'm not descended from Genghis Khan. I'm sure that's as surprising to you as it is to me. I mean, according to what we hear from people who use genomics to track human migrations, a huge percentage of the human race is actually descended from Genghis Khan. But not me.

That's one of the things I learned when I submitted a sample of my DNA for genome-wide single nucleotide polymorphism (SNP) analysis by one of the companies that have sprung up to perform such tests for ordinary individuals for a fee. I was curious to see what sort of information they provide, and wanted to know something about my own genomic makeup, to be honest, so I followed the directions of the company I had selected and spat into a plastic container until I produced the required volume of saliva, mailed it in, and awaited the results. Would I have an allele that doomed me to a rare genetic disorder as I got older? Was I at much higher than normal risk for heart disease, diabetes, or any of the other thousand natural shocks that flesh is heir to? Was I descended from Genghis Khan?

The company I sent my saliva sample to doesn't actually do any DNA sequencing or hybridization itself; it contracts this out to a specialist laboratory. Once the lab received my sample, DNA was extracted from cheek cells in the saliva and amplified by PCR to produce enough material for the genotyping step. Next, the DNA was cut by restriction digestion into smaller, more manageable pieces. These DNA pieces were then applied to a DNA chip, which in this specific case is a small glass slide with millions of microscopic beads on its surface. Attached to each bead are the probes - bits of DNA complementary to those specific sites in the human genome where important SNPs are located. There is a pair of probes for each SNP, corresponding to the 'normal' and 'non-normal' version of each SNP. Hybridization to the particular probe, detected by fluorescence just as in the case of any other DNA chip experiment, serves to identify the allele.

The DNA chip that this particular company uses reads 550,000 SNPs that are spread across the entire genome. Although this is still only a fraction of the 10 million SNPs that are estimated to be in the human genome, these 550,000 SNPs are specially selected 'tag SNPs' - because many SNPs are linked to one another, the genotype at many SNPs can often be determined by looking at one SNP that 'tags' its group. This tagging procedure maximizes the information from every SNP analyzed, while keeping the cost of analysis low.

In addition, all the DNA analysis companies have hand-picked tens of thousands of additional SNPs of particular interest from the scientific literature and added their corresponding probes to the DNA chip. These SNPs include risk factors for common and rare human diseases, genetic traits such as color blindness, and so on.

Access to the resulting data is through the company's website, which includes the ability to download the entire set of SNP information. Once I was notified that my results were in, I did that, and being a scientist I performed my own bioinformatics on the data, but the website actually does a pretty good job of providing the customer with specific information about alleles for various illnesses, physical traits, and so on.

Here are a few of the things I leaned about myself, physically speaking:

According to my genome, my eye color is likely to be brown (good guess). I should be lactose tolerant (I am). My cytochrome P450 data show that I would be quite sensitive to the anti-clotting drug warfarin if I ever had to take it (which I hope I never do - it's a nasty drug). The SNPs in my androgen receptor gene say that I am considerably decreased in risk for male pattern baldness (I have news for them; I'm getting rather thin on top). I have a SNP in a dopamine receptor gene that, in one German study, was found to be associated with reduced efficiency in learning to avoid errors (unless I got the facts wrong). According to a single SNP in one gene associated with insulin metabolism, I have increased odds of living to be 100 (that is, if all the mistakes I don't learn to avoid don't get me first). There are a number of SNPs that have, in some studies, been associated with increased athletic performance (faster running, quicker reaction times, and so on). I don't have any of them, which will come as no surprise to any of my gym teachers.

I am at slightly increased risk, relative to the norm, for rheumatoid arthritis and psoriasis (that last one is interesting, because my father suffered from it). I am at slightly decreased risk for Celiac disease, Crohn's disease, type 1 diabetes, and prostate cancer. In all cases, the change is small - less than two-fold difference, and not enough to cause me to consider any lifestyle changes.

But one thing did jump out at me when I looked at my data. I have a guanine at rs1799945, which is located in the gene coding for a protein called HFE. HFE is the protein mutated in hereditary hemochromatosis. Hereditary hemochromatosis, the most common form of iron overload disease, is an autosomal recessive genetic disorder that causes the body to absorb and store too much iron. Excess iron is stored throughout the body in organs and tissues, including the pancreas, liver, and skin. Without treatment, the iron deposits can damage these organs and tissues. There are two primary variants that give rise to this disease.

Genetic variant 1 (C282Y/rs1800562) is in the *HFE* gene. The *HFE* gene makes a membrane protein that is similar structurally to MHC class I-type proteins and associates with β2-microglobulin. It is thought that HFE helps cells in the intestines, liver, and immune system control iron absorption by regulating the interaction of the transferrin receptor with transferrin. The C282Y substitution disrupts the interaction between HFE and its β2-microglobulin light chain and prevents cell-surface expression. Pamela Bjorkman's 2.6 Å resolution crystal structure of HFE confirms that, as predicted from its sequence, Cys282 (residue 260 in the mature form of the protein) is involved in a disulfide bridge analogous to those found in class I MHC α3 domains. Loss of the disulfide destabilizes the native fold of the protein. The second most common variant is also in the HFE gene. It is a change of histidine 63 to aspartic acid. In the crystal structure of HFE, His63 (41 in the sequence of the mature form) is involved in a crucial salt bridge, which would be destroyed by mutation to a negatively charged residue, thereby also destabilizing the protein. Thus, like so many other hereditary disorders, hemochromatosis is a protein conformational disease.

In the US, variant 1 is the more frequent. The 'normal' Cys282 allele has guanine in both strands and is found in about 876 out of 1,000 people of European ancestry. The most common form of hereditary hemochromatosis is typically associated with people homozygous for an adenine in both positions; this occurs in about 4 out of 1,000 people of European ancestry (0.4%). However, penetrance is incomplete: only about a third to a half of the homozygotes will show elevated iron levels and perhaps fewer than 10% of the males (and 1 to 2% of the females) will develop the full clinical symptoms of the disease, which include joint pain, fatigue, abdominal pain, liver dysfunction, and heart problems. As Ernest Beutler has pointed out, the hemochromatosis mutation is relatively common; the hemochromatosis disease is rare. Mutation of the HFE gene is a necessary, but not sufficient, condition. The challenge, as in the case of so many diseases in the age of genomics, is to understand what other genetic, epigenetic, and environmental factors determine why a few homozygotes for the C282Y (or H63D) mutations develop severe iron-storage disease, while the majority go through life pretty much unscathed by this genotype.

Heterozygotes for C282Y have an adenine in only one strand and represent about 120 in 1,000 people of European ancestry; they almost never develop clinical symptoms. Heterozygotes for H63D are less common, but also are unlikely to develop clinical symptoms. Like one person in ten in the United States, I am a hemochromatosis carrier. I am heterozygous for H63D.

Now that I know that, what does it do for me? Not much, I guess, but I will remember it, and should I ever develop any of the symptoms of iron overload, I will probably tell my physician to check my iron levels. Maybe that's worth knowing.

But if you go to the website of the company that did my analysis, you will find that the sort of information I've talked about here is actually not that prominently displayed. What is displayed are all manner of data connecting to ancestry. I spoke to the CEO of the company, and she confirmed that, much to their surprise, people who use their service are much more interested in tracing their roots, genetically speaking, than they are in things related to their health or physical condition. The site offers several tools for connecting yourself with others who share your ancestry, genetically speaking. In other words, for the present, the primary use of personal genome-wide SNP analysis is social networking.

My maternal haplogroup is T2b2. Haplogroup T originated about 33,000 years ago in the Middle East, as modern humans first expanded out of eastern Africa. Its present-day geographic distribution is strongly influenced by multiple migrations out of the Middle East into Europe, India, and eastern Africa after about 15,000 years ago. T2 is currently widespread in northern Africa and Europe. My mother's family most recently came from Italy, so I guess this makes sense. You can find famous people with your haplotype on the site: for example, if your maternal haplotype is H4a, you have the same type as Warren Buffet, one of the richest men in the world. You'll be delighted - and perhaps not surprised - to know that the only famous person the site lists as sharing my maternal haplotype is the notorious old-west outlaw Jesse James.

My paternal haplotype is I2. Haplogroup I2 is most abundant in eastern Europe and on the Mediterranean island of Sardinia, where it is found in 40% of the male population. Like its brother haplogroup, I1, I2 expanded northward at the end of the Ice Age about 12,000 to 14,000 years ago. But unlike I1, which expanded from the Iberian peninsula into northwestern Europe, I2 radiated outward from the Balkans and southwestern Russia into the eastern half of the continent. That again makes sense, as my father's family were Cossacks. If my paternal haplotype were the extremely common C3, I would be descended from Genghis Khan. I'm not. If it were type T, I would share paternal lineage with the great American president and founding father Thomas Jefferson. I don't. In fact, the company website doesn't list a single famous person with paternal haplotype I2 (unless you count me, of course).

So now, thanks to my own personal genome SNP analysis, I know that I'm not likely to be exceptionally athletic and that I'm not a blue-eyed balding blonde, neither of which comes as any surprise whatsoever. But I have also learned that I'm not descended from Genghis Khan. So I've got that going for me. Which is something, I suppose.


abstract: Aortic valve replacement (AVR) is the gold standard for the treatment of severe symptomatic aortic stenosis. Complications directly related to surgical procedure are relatively infrequent. Coronary ostial stenosis is, generally, referred as late complication. Anecdotal reports concern coronary ostial stenosis as acute complication. A unique fatal case of intraoperative, bilateral coronary ostial obstruction by prosthetic valve leading to an extensive myocardial infarction is reported. Surgeons must have a high level of vigilance regarding the occurrence of acute myocardial ischemia and sudden death soon after AVR.
author: Emanuela Turillazzi; Gabriele Di Giammarco; Margherita Neri; Stefania Bello; Irene Riezzo; Vittorio Fineschi
date: 2011
institute: 1Department of Forensic Pathology, University of Foggia, Ospedale Colonnello D'Avanzo, Via degli Aviatori 1, 71100 Foggia, Italy; 2Institute of Cardiac Surgery, University of Chieti, Via dei Vestini, 29, 66100 Chieti, Italy
references:
title: Coronary ostia obstruction after replacement of aortic valve prostesis

# Background

Aortic valve replacement (AVR) remains the gold standard for the treatment of severe symptomatic aortic stenosis. Late coronary ostial stenosis is described as late complication of the surgical procedure \[1\]. Anecdotal reports concern coronary ostial stenosis as acute complication: right ostial occlusion from aortotomy suture, ostial thrombosis as traumatic consequence from an aortic retractor, coronary artery spasm, calcium debris embolization and partial direct occlusion by the device or edematous reaction have been described \[2-4\].

A unique fatal case of intraoperative, bilateral coronary ostial obstruction by prosthetic valve leading to myocardial infarction is reported.

# Case presentation

## Clinical findings

A 50 -year old woman, previously submitted to surgical aortic valve replacement in 2003 for aortic valve stenosis was admitted to a cardiac surgery unit for replacement of dysfunctioning mechanical valve prosthesis. Echocardiographic evaluation documented a prosthetic dysfunction with the evidence of an increased peak gradient up to 105 mmHg and a prosthetic valve area by 0.58 cm^2^ in a normal left ventricular function. Prosthetic valve replacement was then performed in August 2010. Cardiopulmonary bypass (CPB) was instituted between right atrium and ascending aorta and a moderate hypothermia was reached. Myocardial protection was achieved by retrograde injection of blood cardioplegia through coronary sinus as induction; it was completed by antegrade injection directly in both coronary ostia. After ascending aorta had been transversely opened, prosthetic dysfunction was evident as one of the hemidisks appeared to be locked by pannus ingrowth and fresh pivotal thrombi. No perivalvular leaks were described. The prosthesis was then removed; both coronary ostia were described quite close to the aortic annulus. Bovine pericardium bioprosthesis (Edwards Magna Ease 21 mm) was then implanted in supraannular position. Aortotomy was sutured using Teflon felt strips as reinforcement due to an extremely thin aortic wall and patient was uneventfully weaned from CPB. During skin suture patient experienced sudden severe hypotension; an ECG demonstrated signs of transmural myocardial ischemia. CPB was promptly reinstituted. Severe dilatation and hypokinesia of right ventricle and severe hypokinesia of interventricular septum were registered. Intraortic ballon pumping (IABP) was inserted through a femoral artery and a saphenous vein graft to right coronary artery (RCA) immediately added on a beating heart in the hypothesis of RCA ostial obstruction. At the end of the operation cardiopulmonary support by means of Extracorporeal Membrane Oxygenator (ECMO) through femoro-femoral access was initiated and IABP removed. Patient was then transferred to Intensive Care Unit (ICU) for postoperative course. Graft verification was not performed, either intraoperatively and postoperatively. Two days after, following progressive decrease of ECMO support along with a satisfying hemodinamical recovery, she was transferred to the operating theatre to remove mechanical assistance. One hour after the new admission to the ICU, cardiac arrest occurred pushing surgical team to reposition an emergent ECMO at the ICU bed. The day after patient was transferred again to the theatre to remove thrombi from left atrium evident at ecographic inspection. At that moment cardiac activity was silent in a very compromised systemic condition. Two days after the new admission to the ICU under a new femoro-femoral support she was declared dead.

## Pathological findings

A complete post mortem examination was performed. Cerebral and pulmonary edema was detected. Cardiac size was mildly increased (11.5 × 10 × 7.7 cm), with conical shape; heart weight was increased (547g). At the inspection, a horizontal aortotomy was evident with prosthetic valve in situ after aorta was opened. Meticulous attention was devoted to observe the coronary ostia: the right one was not found at the inspection of inner surface of sinus of Valsalva. Using a metal probe inserted into right coronary artery distally cut, the entrapment of the ostium in a stitch at the level of prosthetic annulus became evident. Left coronary ostium was identified behind one of the bioprosthesis post that was responsible of its complete obstruction (Figure 1). Coronary vessels were fully patent during their course. Equally patent appeared to be saphenous graft to RCA. The inspection of both ventricles showed many foci of pale myocardium diffused to either free wall and interventricular septum.

Histological investigations showed haemostasis of all organs and mild cerebral and pulmonary edema. Specimens taken from the heart showed diffuse stretching of the myocardium with elongation of the sarcomeres and nuclei. Polymorphonuclear leucocyte infiltration was well evident at the periphery of the necrosis zones (Figure 2). Numerous foci of hypercontracted myocardial cells with markedly short sarcomeres and anomalous, extreme thickening of Z lines and rexis of the myofibrillar apparatus into cross-fibre, anomalous and irregular, up to a total granular disruption were also observed. Myocells pathological aspects were also studied on confocal laser scanning microscope: stretching of the myocardium in flaccid paralysis, resulting in a very early elongation of sarcomeres and nuclei; polymorphonuclear leucocyte infiltration was well evident.

Extramural coronary arteries showed nodular hyperplasia of smooth muscle cells and elastic tissue with fibrous replacement. Proteoglycan accumulation in the deep intima between tunica media and the fibrous cap was observed too. No critical stenosis was evident.

Final diagnosis of extensive myocardial infarction due to the intraoperative occlusion of both coronary ostia by bioprosthesis malpositioning was established as the cause of death.

# Conclusions

In the present case, extensive myocardial infarction due to the intraoperative occlusion of both coronary ostia by prosthetic valve was recorded as the cause of death. Since there are, to date, no reports of similar deaths in patients undergone to AVR, our report provides useful information on this complication of AVR.

Coronary ostial stenosis following AVR is believed to occur in 1% to 5% of AVR procedures. It is a life threatening complication which, generally, becomes evident from 1 to 6 months after the operation \[5\]. Several different mechanisms which include the possibility of microinjuries and local hyperplastic reaction related to the infusion pressure and/or low temperature of cardioplegic solution and overdilation of the vessel by the tip of the cardioplegic catheters are thought to be involved \[1,5\]. Other mechanisms were hypothesized such as widespread intimal thickening and fibrous proliferation in proximity of the aortic root, presumably as a reaction to turbulence around aortic ball valve prostheses. An immunological reaction to the heterograft after AVR has been considered in cases of bilateral ostial coronary arteries stenoses revealing several months following the surgical procedure \[1,2\]. On the basis of these evidences the exact mechanism underlying late coronary ostial stenosis following AVR is unclear.

Anecdotal reports describe the rare occurrence of acute coronary ostial stenosis; right ostial occlusion from aortotomy sutures and ostial post-traumatic thrombosis due to aortic retractor have been described \[2\]. In exceptional cases, embolism from debris, more often calcium related to aortic valve decalcification, or left atrial thrombectomy can be involved \[1,2\]. Coronary artery spasm has been recognized as a possible cause of hemodynamic and arrhythmic instability after aortic valve replacement \[6\]. Occasionally, secondary fibrosis in the area of suture placement may occur causing ostial stenosis \[7\]. Finally, use of surgical glue in aortic surgery, or compression from outside due to the glue used to protect the anastomosis may cause stenosis of one or both coronary ostia \[8\].

Conclusively, there is a reasonable body of evidence that acute coronary ostial stenoses may occur, even if rarely, after AVR and this complication may be life threatening if not promptly recognized, leading to myocardial ischemia, infarction, or fatal arrhythmia. Consequently, it is important to have an high index of diagnostic suspicion if circulatory collapse and/or signs of myocardial ischemia occur soon after surgery. Transesophageal echocardiography can be useful in diagnosis of acute complications of cardiac surgery; however urgent coronary angiography remains the gold diagnostic tool \[8\].

In our case, both coronary ostia were iatrogenically occluded by the prosthesis: the right one appeared to be entrapped by a stitch and the left one was occluded by prosthetic post. Neither intraoperative transesophageal echocardiography or postoperative coronary angiography were performed, so impeding to reach a prompt diagnosis.

From a clinical point of view, surgeons must have an high level of vigilance regarding the occurrence of acute myocardial ischemia soon after AVR and must be ready to perform either an intraoperative verification of patency or an early coronary angiography during ICU stay since these diagnostic tools may reveal mechanisms underlying ischemia which could make necessary a surgical approach (coronary stenting, device removal and/or re-replacement with a smaller valve size with or without annular enlargement) \[3,8\].

# Consent statement

Written informed consent was obtained from the Medical Examiner Department, Court of Justice, for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

# List of abbreviations

AVR: aortic valve replacement; CPB: cardiopulmonary bypass; IABP: intraortic ballon pumping; RCA: right coronary artery; ECMO: Extracorporeal Membrane Oxygenator; ICU: intensive care unit.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

SB drafted the manuscript. MN carried out the istological analysis. IR performed the microscopic analysis. ET, GDG and VF conceived of the study, and participated in its design and coordination and helped to draft the manuscript.

All authors read and approved the final manuscript.