Homologous recombination suppresses transgenerational DNA end resection and chromosomal instability in fission yeast.

Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.

Homologous recombination repair intermediates promote efficient de novo telomere addition at DNA double-strand breaks.

The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast.

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.

Arthroscopic Superior Capsular Reconstruction With Acellular Dermal Allograft for the Treatment of Massive Irreparable Rotator Cuff Tears: Short-Term Clinical Outcomes and the Radiographic Parameter of Superior Capsular Distance.

PURPOSE: This outcome analysis presents 88 consecutive shoulders presenting with irreparable rotator cuff tears that we treated with arthroscopic superior capsular reconstruction (SCR) using an acellular dermal allograft. We also present the concept of superior capsular distance to quantitatively measure the decreased distance present upon restoration of superior capsular integrity. METHODS: A retrospective review was conducted of patients treated with arthroscopic SCR with a minimum 12-month follow-up. Outcome analysis was performed via an internet-based outcome-tracking system to evaluate visual analog scale (VAS) and American Shoulder and Elbow Surgeons (ASES) scores. Radiographic analysis of anteroposterior radiographs analyzed acromiohumeral interval and superior capsular distance. Digital dynamometric strength and functional range of motion assessments were also obtained. The main inclusion criteria for patients in this analysis was all patients who underwent superior capsular reconstruction during the time period of this report. RESULTS: Eighty-six patients with an average age of 59.4 years presented with massive rotator cuff tears (Cofield >5 cm). Outcome data revealed improvement in VAS (4.0-1.5), and ASES (52-82) scores at 1 year (P = .005). Radiographic analysis showed increase in acromiohumeral interval (mean 7.1 mm preoperatively to mean 9.7 mm at 1 year) (P = .049) and superior capsular distance (mean 52.9 mm preoperatively to mean 46.2 mm at 1 year) (P = .011). Strength improved significantly (forward flexion/abduction/external rotation of 4.8/4.1/7.7 lb preoperatively to 9.8/9.2/12.3 lb at 1 year) as well as range of motion (forward flexion/abduction of 120°/103° preoperatively to 160°/159° at 1 year) (P = .044/P = .007/P = .02). At follow-up, 90% of patients were satisfied. CONCLUSIONS: This analysis reveals that arthroscopic SCR with acellular dermal allograft has been successful in decreasing pain and improving function in this patient subset. Radiographic analysis has also shown a consistent and lasting decrease in superior capsular distance and increase in acromiohumeral interval, indicating maintenance of superior capsular stability. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast.

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.

Brain tumour differentiation: rapid stratified serum diagnostics via attenuated total reflection Fourier-transform infrared spectroscopy.

The ability to diagnose cancer rapidly with high sensitivity and specificity is essential to exploit advances in new treatments to lead significant reductions in mortality and morbidity. Current cancer diagnostic tests observing tissue architecture and specific protein expression for specific cancers suffer from inter-observer variability, poor detection rates and occur when the patient is symptomatic. A new method for the detection of cancer using 1 µl of human serum, attenuated total reflection-Fourier transform infrared spectroscopy and pattern recognition algorithms is reported using a 433 patient dataset (3897 spectra). To the best of our knowledge, we present the largest study on serum mid-infrared spectroscopy for cancer research. We achieve optimum sensitivities and specificities using a Radial Basis Function Support Vector Machine of between 80.0 and 100 % for all strata and identify the major spectral features, hence biochemical components, responsible for the discrimination within each stratum. We assess feature fed-SVM analysis for our cancer versus non-cancer model and achieve 91.5 and 83.0 % sensitivity and specificity respectively. We demonstrate the use of infrared light to provide a spectral signature from human serum to detect, for the first time, cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and the organ of origin of metastatic disease from the same sample enabling stratified diagnostics depending upon the clinical question asked.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.

Diagnostic challenges, management and outcomes of midline low-grade gliomas.

INTRODUCTION: Low-grade gliomas (LGGs) are slow-growing and diffusely infiltrating tumours constituting 25-30 % of adult gliomas. Rarely, these tumours may arise in the cerebral midline, including the thalamus, hypothalamus, tectum and brainstem. Here we present a contemporary experience with midline LGGs. METHODS: Midline LGGs were identified from a retrospective database of adult patients who received a histological diagnosis of WHO grade II glioma between 2006 and 2012 at a single institution. Location, radiological data and clinical outcomes were collected. IDH1 status was assessed by immunohistochemistry. RESULTS: Eighteen patients with midline LGGs were identified, with a median age of 45. Most received biopsy upon diagnosis, though asymptomatic patients with tectal tumours underwent active surveillance. Oligodendroglial tumours were much less common than in a comparable group of lobar tumours (6 vs. 38 %, Fisher's exact test, p = 0.007). Only one tumour was immunopositive for IDH1 (1/17). Radiological diagnosis correlated with histology in only 71 % of patients. Median survival of midline LGGs was 48 months (3-90 months) and radiological features such as contrast enhancement, size and radiological diagnosis did not predict survival in this cohort. Median overall survival of midline LGGs was less than lobar LGGs (log-rank, p = 0.006), though differences became insignificant when considering only biopsied astrocytomas in both locations (log-rank, p = 0.491). CONCLUSIONS: Diagnosis of midline LGGs is complicated by both limitations of biopsy and imaging. Midline tumours have a poorer prognosis compared to lobar equivalents and survival differences are probably due to the absence of significant surgical intervention in midline locations.

Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast.

Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double-strand break (DSB)-induced extensive LOH, and its suppression, using a non-essential minichromosome, Ch(16), in fission yeast. We find extensive LOH to arise from a new break-induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1-dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break-induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genome-wide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair.

Cellularity and apparent diffusion coefficient in oligodendroglial tumours characterized by genotype.

PURPOSE: Apparent diffusion coefficient (ADC) describes water diffusion within tissues. Previous studies report a negative linear correlation between minimum ADC and tumour cellularity in different types of gliomas, but there are no studies in oligodendroglial tumours. This study evaluated the relationship between ADC and tumour cellularity in oligodendroglial tumours characterized by genotype. METHODS: ADC was assessed in 17 patients with known 1p/19q status: 3 grade II oligodendrogliomas (OII), 9 grade II oligoastrocytomas (OAII), 5 grade III oligoastrocytomas (OAIII). Regions of interest were placed on ADC maps around tumour margins to generate mean tumour ADC, and over minimum and maximum tumour ADC. Histopathology assessment of tumour cellularity determined minimum, maximum and mean cell density in serial stereotactic biopsies. RESULTS: 1p/19q loss was present in 2/3 OII, 5/9 OAII, 2/5 OAIII. Grade III tumours had higher maximum cell density than grade II tumours (17.2 vs. 10.57%: Mann Whitney U; P = 0.20). Oligoastrocytoma were more likely to have a lower minimum cell density than oligodendrogliomas (Mann Whitney U; P = 0.032). There was no relationship between cell density and genotype. There was no linear correlation between mean ADC and mean cell density (Spearman's rho; r = 0.486: P = 0.438), minimum ADC and maximum cell density (Spearman's rho; r = 0.158: P = 0.660), and maximum ADC and minimum cell density (Spearman's rho; r = 0.039: P = 0.985). CONCLUSIONS: In oligodendroglial tumours there is no relationship between quantitative assessment of cellularity and ADC. This may reflect differences in oligodendroglial tumour biology compared to other gliomas, although the composition of the extracellular matrix may influence ADC more than cellularity.

Phase II trial of intratumoral BCNU injection and radiotherapy on untreated adult malignant glioma.

DTI-015 (BCNU dissolved in ethanol) utilizes solvent facilitated perfusion (SFP) for intratumoral drug delivery. A phase II clinical trial of DTI-015 and fractionated external beam radiotherapy on newly diagnosed, malignant gliomas investigated early changes in tumour physiology and metabolism, clinical outcome and safety. Pre- and post DTI-015 injection neuro-imaging included computed tomography (CT) cerebral blood flow and volume, glucose and thallium single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Clinical status was determined before and after DTI-015, prior to radiotherapy and 3 monthly thereafter until progression (defined by Macdonald criteria). Primary endpoint was radiographic response. Secondary endpoints were progression free (PFS) and overall survival (OS). Twelve patients were enrolled; eight glioblastoma multiforme (GBM), four anaplastic astrocytoma (AA). Three days after DTI-015 injection, mean tumour blood flow (Paired t-test; P < 0.001) and blood volume (Paired t-test; P = 0.001) were significantly reduced. There was a significant decrease in glucose utilization (Paired t-test; P < 0.001) and thallium uptake (Paired t-test; P < 0.001) at 6 days. Tumour blood volume had a sustained reduction (Paired t-test; P = 0.001) at 26 days after DTI-015. There were two serious adverse events. Two patients with AA achieved a partial response. Median PFS was 39 weeks for AA and 27 weeks for GBM; median OS for GBM was 47 weeks and 132 weeks for AA. The imaging data forms a biological basis for understanding the effects of high dose BCNU delivered intratumorally by SFP, and suggests early effects on tumour vasculature and metabolism.

Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition.

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.

Molecular genetics, imaging and treatment of oligodendroglial tumours.

The discovery of a genetic signature of chemosensitivity and prognosis in oligodendroglial tumours prompted a new optimism in glioma management. After more than a decade since the initial reports, where do we stand in the current management of oligodendroglial tumours? This review focuses on the latest molecular genetics, imaging characteristics, and recent trials of treatment paradigms for these tumours.

Quality Outcomes in Appendicitis Care: Identifying Opportunities to Improve Care.

INTRODUCTION: Appendicitis is one of the most common causes of acute abdominal pain requiring surgical intervention, but the variability of diagnosis and management continue to challenge the surgeons. Aim: This study assessed patients undergoing appendectomy to identify opportunities to improve diagnostic accuracy and outcomes. METHODS: An ethically approved retrospective cohort study was undertaken between March 2016 and March 2017 at a single university hospital of all consecutive adult and paediatric patients undergoing appendectomy. Demographic data including age, gender, co-morbidities, presentation and triage timings along with investigation, imaging and operative data were analysed. Appendicitis was defined as acute based on histology coupled with intraoperative grading with the American Association for the Surgery of Trauma (AAST) grades. Complications using the Clavien-Dindo classification along with 30-day re-admission rates and the negative appendectomy rates (NAR) were recorded and categorised greater and less than 25%. The use of scoring systems was assessed, and retrospective scoring performed to compare the Alvarado, Adult Appendicitis Score (AAS) and the Appendicitis Inflammatory Response (AIR) score. Results: A total of 201 patients were studied, 115 male and 86 females, of which 136/201 (67.6%) were adults and 65/201 (32.3%) paediatric. Of the adult group, 83 were male and 53 were female, and of the paediatric group, 32 were male and 33 were female. Median age was 20 years (range: 5 years to 81 years) and no patient below the age of 5 years had an appendectomy during our study period. All patients were admitted via the emergency department and median time from triage to surgical review was 2 h and 38 min, (range: 10 min to 26 h and 10 min). Median time from emergency department review to surgical review, 55 min (range: 5 min to 6 h and 43 min). Median time to operating theatre was 21 h from admission (range: 45 min to 140 h and 30 min). Out of the total patients, 173 (86.1%) underwent laparoscopic approach, 28 (13.9%) had an open approach and 12 (6.9%) of the 173 were converted to open. Acute appendicitis occurred in 166/201 (82.6%). There was no significant association between grade of appendicitis and surgeons' categorical NAR rate (p = 0.07). Imaging was performed in 118/201 (58.7%); abdominal ultrasound (US) in 53 (26.4%), abdominal computed tomography (CT) in 59 (29.2%) and both US and CT in 6 (3%). The best cut-off point was 4 (sensitivity 84.3% and specificity of 65.7%) for AIR score, 9 (sensitivity of 74.7% and specificity of 68.6%) for AAS, and 7 (sensitivity of 77.7% and specificity of 71.4%) for the Alvarado score. Twenty-four (11.9%) were re-admitted, due to pain in 16 (58.3%), collections in 3 (25%), 1 (4.2%) wound abscess, 1 (4.2%) stump appendicitis, 1 (4.2%) small bowel obstruction and 1 (4.2%) fresh rectal bleeding. CT guided drainage was performed in 2 (8.3%). One patient had release of wound collection under general anaesthetic whereas another patient had laparoscopic drain placement. A laparotomy was undertaken in 3 (12.5%) patients with division of adhesions in 1, the appendicular stump removed in 1 and 1 had multiple collections drained. CONCLUSION: The negative appendectomy and re-admission rates were unacceptably high and need to be reduced. Minimising surgical variance with use of scoring systems and introduction of pathways may be a strategy to reduce NAR. New systems of feedback need to be introduced to improve outcomes.

Using Pulsed-Field Gel Electrophoresis to Analyze Schizosaccharomyces pombe Chromosomes and Chromosomal Elements.

Pulsed field gel electrophoresis (PFGE) uses alternatively oriented pulsed electrical fields to separate large DNA molecules. Here, we describe PFGE protocols and conditions for separating and visualizing chromosomes between 0.5 and 6 Mb (optimal for analyzing the endogenous fission yeast chromosomes of 5.7, 4.6, and 3.5 Mb), and for shorter chromosomal elements of between 50 and 600 kb, such as the 530 kb Ch16 minichromosome. In addition to determining chromosome size, this technique has a wide range of applications, including determining whether DNA replication or repair is complete, defining the molecular karyotype of cells, analyzing chromosomal rearrangements, assigning genes or constructs to particular chromosomes, and isolating DNA from specific chromosomes.

Histological growth patterns and genotype in oligodendroglial tumours: correlation with MRI features.

Oligodendroglial neoplasms with the -1p/-19q genotype are more indolent with longer survival and increased therapeutic responsiveness than those with intact 1p/19q, but the biological basis for these clinical differences is unclear. Recent research suggests that oligodendrogliomas with and without the -1p/-19q genotype may be distinguished by their magnetic resonance imaging (MRI) appearance, suggesting possible differences in growth characteristics. This study examined the relationship between genotype and histological growth patterns of oligodendroglial neoplasms in association with MR imaging characteristics. Tumour imaging features assessed on MRI included sharp-versus-indistinct border, smooth-versus-irregular contour, homogeneous-versus-heterogeneous signal, contrast enhancement and paramagnetic susceptibility effect. Growth patterns (solid : mixed : infiltrative), tumour-margin transitions in cellularity and calcification were determined histopathologically. Allelic imbalance in chromosomes 1p36 and 19q13 was determined. Thirty-three oligodendrogliomas (25 with 1p/19q loss) and 53 oligoastrocytomas (18 with 1p/19q loss) were investigated. Solid, mixed or infiltrative growth patterns were seen in grade II and grade III tumours with or without 1p/19q loss, but infiltrative growth was more common in tumours with intact 1p/19q (chi2: P = 0.029). Grade III tumours were more likely to have a solid growth pattern (chi2: P = 0.046) associated with contrast enhancement (chi2: P = 0.011). Transition in cellularity at the radiological margin did not differ according to genotype. All cases with T1 or T2 signal homogeneity had intact 1p/19q. Tumours with sharp/smooth borders were more likely to have intact 1p/19q than those with indistinct/irregular borders (chi2: P < 0.001), but this was not related to histological growth characteristics. This study identified a group of oligodendroglial tumours with intact 1p/19q displaying distinctive MR imaging features that were unrelated to the histopathology characteristics.

Sum1, a component of the fission yeast eIF3 translation initiation complex, is rapidly relocalized during environmental stress and interacts with components of the 26S proteasome.

Eukaryotic translation initiation factor 3 (eIF3) is a multisubunit complex that plays a central role in translation initiation. We show that fission yeast Sum1, which is structurally related to known eIF3 subunits in other species, is essential for translation initiation, whereas its overexpression results in reduced global translation. Sum1 is associated with the 40S ribosome and interacts stably with Int6, an eIF3 component, in vivo, suggesting that Sum1 is a component of the eIF3 complex. Sum1 is cytoplasmic under normal growth conditions. Surprisingly, Sum1 is rapidly relocalized to cytoplasmic foci after osmotic and thermal stress. Int6 and p116, another putative eIF3 subunit, behave similarly, suggesting that eIF3 is a dynamic complex. These cytoplasmic foci, which additionally comprise eIF4E and RNA components, may function as translation centers during environmental stress. After heat shock, Sum1 additionally colocalizes stably with the 26S proteasome at the nuclear periphery. The relationship between Sum1 and the 26S proteasome was further investigated, and we find cytoplasmic Sum1 localization to be dependent on the 26S proteasome. Furthermore, Sum1 interacts with the Mts2 and Mts4 components of the 26S proteasome. These data indicate a functional link between components of the structurally related eIF3 translation initiation and 26S proteasome complexes.

Phenotype versus genotype in gliomas displaying inter- or intratumoral histological heterogeneity.

PURPOSE: Molecular classification of gliomas is becoming increasingly important clinically as an adjunct to histopathological diagnosis. Whereas histological heterogeneity of gliomas is well recognized, less is known of the relationship between histological heterogeneity and genetic alterations. Our objective was to investigate the relationship between genotype and phenotype for markers of potential clinical utility in histologically heterogeneous gliomas. EXPERIMENTAL DESIGN: We have used laser capture microdissection to sample the various histological phenotypes present in 42 tumors from 25 glioma cases with either inter- or intratumoral histological heterogeneity, and multiple simultaneous PCR amplification of microsatellite markers and capillary electrophoresis to determine allelic imbalance in chromosomes 1p, 19q, 17p, 10p, and 10q. RESULTS: Loss of 1p36 and 19q13 was seen only in oligodendroglial histology in 7 of 13 oligodendrogliomas. 17p13 loss was found in 14 of 41 tumors in astrocytic, oligoastrocytic, oligodendroglial, and glioblastomatous histologies. Chromosome 10 loss was seen in all of the high-grade histologies in 7 of 7 glioblastomas with an oligodendroglial component and in 1 of 5 low-grade oligodendroglial regions present within high-grade tumors. Seven tumors from 5 cases had no detectable losses of any markers investigated. In 13 tumors with intratumoral heterogeneity, identical genetic losses were present in all areas of histological differentiation. Additional losses were seen in some but not all of the histologies within 2 tumors and were associated with progression in 3 cases. CONCLUSIONS: The gliomas in this study were more homogeneous in their genotype than their histological phenotype with regions of differing histological subtype indistinguishable by the genetic markers investigated, supporting a monoclonal origin of these tumors.

Correlation of molecular genetics with molecular and morphological imaging in gliomas with an oligodendroglial component.

PURPOSE: Since the recognition that oligodendrogliomas may be chemosensitive, their diagnosis and clinical management has become highly controversial. Histopathology diagnosis remains challenging and new tools such as molecular genetics or molecular imaging require evaluation. EXPERIMENTAL DESIGN: In a single-center, population-based prospective study, allelic imbalance in chromosomes 1p36, 19q13, 17p13, 10p12-15, and 10q22-26 has been investigated in 19 oligodendroglioma WHO grade 2 (OII), 20 oligoastrocytoma WHO grade 2 (OAII), 8 oligodendroglioma WHO grade 3 (OIII), and 12 oligoastrocytoma WHO grade 3 (OAIII), and compared with pretherapy histopathology, computed tomography and/or magnetic resonance (CT and/or MR), [fluorine-18]fluoro-2-deoxyglucose (18F-FDG), and thallium-201 single-photon emission computed tomography (201Tl SPECT). RESULTS: In 50 cases, 18F-FDG uptake correlated with 201Tl uptake; however, 8 cases had increased 201Tl uptake but were hypometabolic for 18F-FDG, and 1 case was hypermetabolic with normal 201Tl uptake. Sixteen cases enhanced on CT/MR but failed to show 201Tl uptake; and 2 low-grade non-enhancing oligodendrogliomas had increased 201Tl uptake. Increased metabolism was more likely in high-grade cases, with 201Tl uptake more strongly correlated with grade than was 18F-FDG uptake. Tumors with 1p/19q loss were more likely to show increased 201Tl uptake and, to a lesser degree, increased 18F-FDG uptake than those without these losses. Elevated metabolism in 28% of low-grade tumors was significantly more common in tumors with 1p/19q loss, and increased uptake of both 18F-FDG and 201Tl in low-grade cases was found only in those with 1p/19q loss. CONCLUSIONS: In this study, dissociation of uptake of contrast agents and radiotracers suggests independent deregulation of the blood-brain barrier breakdown and metabolism during disease progression of oligodendroglial neoplasms, and the association of elevated metabolism with 1p/19q loss, particularly in low-grade tumors, may have implications for clinical management.

Implementing root cause analysis in an area health service: views of the participants.

PURPOSE: This study identifies the attitudes of participants in the root cause analysis (RCA) process and barriers to it's implementation within one New South Wales area health service. METHOD: Employees and consumer representatives of the former South Western Sydney Area Health Service who participated in an RCA as either a team member or a team leader between December 2002 and October 2003 completed a self-administered survey. RESULTS: Thirty seven of 39 eligible participants completed the survey (response rate 95%). The respondents identified formulation of causal statements, ensuring the causal statements met the "rules of causality" outlined by New South Wales Health, and arranging times for interviews as most difficult. Team leader respondents (n = 7) ranked keeping the team focused, organising the first meeting within 7 days of the incident, and completing the RCA in three 2-hour meetings as barriers to the process. CONCLUSIONS: Training was valued by participants, however greater emphasis on the development of causal statements could be beneficial. Team leaders expressed difficulty in keeping the team focused and meeting the stipulated RCA timeframes, suggesting that additional support for RCA participants may be warranted.