Robust envelope exchange platform for oncolytic measles virus.

The use of oncolytic viruses (OV) to precisely target and eliminate tumors ('virotherapy') is a rapidly evolving therapeutic approach to treating cancer. A major obstacle in virotherapy, especially for systemic administration, is the host's immune response towards the OV. In the case of measles virus (MeV), most individuals have been immunized against this agent leading to pre-existing neutralizing antibodies that can impair OV delivery to the tumor. These antibodies predominantly target the hemagglutinin (H) and fusion (F) envelope glycoproteins displayed at the particle's surface. Here, we introduce a novel and versatile pseudotyping platform for rapid envelope exchange of oncolytic MeV that allows for engineering of chimeric viruses invulnerable to pre-existing anti-MeV antibodies. Using this system, we have successfully exchanged the MeV F and H proteins with the glycoprotein G of vesicular stomatitis virus (VSV) and the surface proteins of Newcastle disease virus (NDV) or canine distemper virus (CDV), all of which are not endemic in the general human population. While the MeV-VSV and MeV-NDV pseudotypes were non-functional, the MeV-CDV pseudotype was successfully propagated to high-titer virus stocks. This study describes the successful generation of a robust envelope exchange platform for oncolytic MeV while also highlighting its intricate pseudotyping tolerance.

Adjuvant oncolytic virotherapy for personalized anti-cancer vaccination.

By conferring systemic protection and durable benefits, cancer immunotherapies are emerging as long-term solutions for cancer treatment. One such approach that is currently undergoing clinical testing is a therapeutic anti-cancer vaccine that uses two different viruses expressing the same tumor antigen to prime and boost anti-tumor immunity. By providing the additional advantage of directly killing cancer cells, oncolytic viruses (OVs) constitute ideal platforms for such treatment strategy. However, given that the targeted tumor antigen is encoded into the viral genomes, its production requires robust infection and therefore, the vaccination efficiency partially depends on the unpredictable and highly variable intrinsic sensitivity of each tumor to OV infection. In this study, we demonstrate that anti-cancer vaccination using OVs (Adenovirus (Ad), Maraba virus (MRB), Vesicular stomatitis virus (VSV) and Vaccinia virus (VV)) co-administered with antigenic peptides is as efficient as antigen-engineered OVs and does not depend on viral replication. Our strategy is particularly attractive for personalized anti-cancer vaccines targeting patient-specific mutations. We suggest that the use of OVs as adjuvant platforms for therapeutic anti-cancer vaccination warrants testing for cancer treatment.

The Canadian Cancer Research Conference 2019.

The 5th Canadian Cancer Research Conference (ccrc) took place 3-5 November 2019 in Ottawa, Ontario. Nearly 1000 participants-scientists, oncologists, community members, and patients-gathered to share knowledge, foster collaboration, and fuel the future of cancer research in Canada. The scientific program included 3 plenary sessions, 26 concurrent sessions, and 2 poster sessions presenting research described in more than 600 submitted abstracts, giving participants the opportunity to share health research that collectively encompassed the 4 pillars recognized by the Canadian Institutes of Health Research. In addition to the breadth of topics addressed by Canadian and international experts, the highlights of the meeting included the integration of patients and patient advocates, new rapid-fire sessions for abstract presentation, and events that enhanced learning opportunities for trainees.

School performance for children with cleft lip and palate: a population-based study.

BACKGROUND: Educational attainment is important in shaping young people's life prospects. To investigate whether being born with orofacial cleft (OFC) affects school performance, we compared school test results between children born with and without OFC. METHODS: Using record-linked datasets, we conducted a population-based cohort study of children liveborn in Western Australia 1980-2010 with a diagnosis of OFC on the Register of Developmental Anomalies, and a random sample of 6603 children born without OFC. We compared odds ratios for meeting national minimum standards in five domains (reading, numeracy, writing, spelling, grammar and punctuation), and adjusted OR (aOR) for children with cleft lip only (CLO), cleft lip and palate (CL + P) and cleft palate only (CPO) for each domain. RESULTS: Results from two testing programs (WALNA and NAPLAN) were available for 3238 (89%) children expected to participate. Most met the national minimum standards. Compared with children without OFC, children with CPO were less likely to meet minimum standards for NAPLAN reading (aOR 0.57 [95%CI 0.34, 0.96]) grammar and punctuation (aOR 0.49 [95%CI 0.32, 0.76]), WALNA writing (aOR 0.66 [95%CI 0.47, 0.92]), and WALNA and NAPLAN numeracy (aOR 0.64 [95%CI 0.43, 0.95] and aOR 0.47 [95%CI 0.28, 0.82]), respectively. Children with CL + P had significantly lower odds for reaching the spelling standard in NAPLAN tests (aOR 0.52 [95%CI 0.29, 0.94]). Children with CLO had similar odds for reaching all minimum standards. CONCLUSION: Children born with OFC, particularly children with CPO, should be monitored to identify learning difficulties early, to enable intervention to maximize school attainment.

Expression of the fusogenic p14 FAST protein from a replication-defective adenovirus vector does not provide a therapeutic benefit in an immunocompetent mouse model of cancer.

When injected directly into a tumor mass, adenovirus (Ad) vectors only transduce cells immediately along the injection tract. Expression of fusogenic proteins from the Ad vector can lead to syncytium formation, which efficiently spreads the therapeutic effect. Fusogenic proteins can also cause cancer cell death directly, and enhance the release of exosome-like particles containing tumor-associated antigens, which boosts the anti-tumor immune response. In this study, we have examined whether delivery of an early region 1 (E1)-deleted, replication-defective Ad vector encoding the reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein can provide therapeutic efficacy in an immunocompetent mouse tumor model. A high multiplicity of infection of AdFAST is required to induce cell fusion in mouse mammary carcinoma 4T1 cells in vitro, and FAST protein expression caused a modest reduction in cell membrane integrity and metabolic activity compared with cells infected with a control vector. Cells expressing FAST protein released significantly higher quantities of exosomes. In immunocompetent Balb/C mice harboring subcutaneous 4T1 tumors, AdFAST did not induce detectable cancer cell fusion, promote tumor regression or prolong mouse survival compared with untreated mice. This study suggests that in the context of the 4T1 model, Ad-mediated FAST protein expression did not elicit a therapeutic effect.

Programmable insect cell carriers for systemic delivery of integrated cancer biotherapy.

Due to cancer's genetic complexity, significant advances in the treatment of metastatic disease will require sophisticated, multi-pronged therapeutic approaches. Here we demonstrate the utility of a Drosophila melanogaster cell platform for the production and in vivo delivery of multi-gene biotherapeutic systems. We show that cultured Drosophila S2 cell carriers can stably propagate oncolytic viral therapeutics that are highly cytotoxic for mammalian cancer cells without adverse effects on insect cell viability or gene expression. Drosophila cell carriers administered systemically to immunocompetent animals trafficked to tumors to deliver multiple biotherapeutics with little apparent off-target tissue homing or toxicity, resulting in a therapeutic effect. Cells of this Dipteran invertebrate provide a genetically tractable platform supporting the integration of complex, multi-gene biotherapies while avoiding many of the barriers to systemic administration of mammalian cell carriers. These transporters have immense therapeutic potential as they can be modified to express large banks of biotherapeutics with complementary activities that enhance anti-tumor activity.

Mechanical comparison of monofilament nylon leader and orthopaedic wire for median sternotomy closure.

OBJECTIVES: To compare the mechanical properties of monofilament nylon leader and orthopaedic wire for median sternotomy closure in the dog. MATERIALS AND METHODS: Median sternotomy was performed in 14 canine cadaver sternums with the manubrium intact. The sternotomy was closed with either 80 lb monofilament nylon leader or 20G orthopaedic stainless steel wire in a peristernal figure of 8 pattern. Constructs were loaded in a servohydraulic material testing machine. Load at yield, maximum load, stiffness, displacement and mode of failure were compared between constructs subjected to a single cycle load to failure. RESULTS: No significant differences were found in load at yield, maximum load, stiffness or displacement between the monofilament nylon leader and the stainless steel wire constructs. No implant failure was evident in the stainless steel wire constructs. Four of the monofilament nylon leader constructs failed by pulling of the nylon through the crimp. CLINICAL SIGNIFICANCE: Monofilament nylon leader is mechanically comparable to stainless steel wire and potentially a suitable alternative for closure of median sternotomy in the dog.

Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model.

In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.

Non-replicating rhabdovirus-derived particles (NRRPs) eradicate acute leukemia by direct cytolysis and induction of antitumor immunity.

Rhabdoviruses (RVs) are currently being pursued as anticancer therapeutics for various tumor types, notably leukemia. However, modest virion production and limited spread between noncontiguous circulating leukemic cells requires high-dose administration of RVs, which exceeds the maximum tolerable dose of the live virus. Furthermore, in severely immunosuppressed leukemic patients, the potential for uncontrolled live virus spread may compromise the safety of a live virus approach. We hypothesized that the barriers to oncolytic virotherapy in liquid tumors may be overcome by administration of high-dose non-replicating RVs. We have developed a method to produce unique high-titer bioactive yet non-replicating rhabdovirus-derived particles (NRRPs). This novel biopharmaceutical is multimodal possessing direct cytolytic and immunomodulatory activity against acute leukemia. We demonstrate that NRRP resistance in normal cells is mediated by intact antiviral defences including interferon (IFN). This data was substantiated using murine models of blast crisis. The translational promise of NRRPs was demonstrated in clinical samples obtained from patients with high-burden multidrug-resistant acute myeloid leukemia. This is the first successful attempt to eradicate disseminated cancer using a non-replicating virus-derived agent, representing a paradigm shift in our understanding of oncolytic virus-based therapies and their application toward the treatment of acute leukemia.

Demonstration of ignition radiation temperatures in indirect-drive inertial confinement fusion hohlraums.

We demonstrate the hohlraum radiation temperature and symmetry required for ignition-scale inertial confinement fusion capsule implosions. Cryogenic gas-filled hohlraums with 2.2 mm-diameter capsules are heated with unprecedented laser energies of 1.2 MJ delivered by 192 ultraviolet laser beams on the National Ignition Facility. Laser backscatter measurements show that these hohlraums absorb 87% to 91% of the incident laser power resulting in peak radiation temperatures of T(RAD)=300 eV and a symmetric implosion to a 100 µm diameter hot core.

[Oncolytic vesicular stomatitis viruses as intravesical agents against non-muscle-invasive bladder cancer]. / Intravesikale Therapie nicht muskelinvasiver Blasentumoren mit onkolytischen Vesikular-Stomatitisviren.

Patients with high-risk bladder cancer who do not respond to bacillus Calmette-Guerin (BCG) immunotherapy represent a significant therapeutic challenge. The addition of interferon to BCG has recently evolved as a second-line treatment option; however, many high-grade tumors are nonresponsive to interferon. Thus, replication-competent oncolytic vesicular stomatitis viruses (VSV) that selectively target interferon-refractory tumors are promising intravesical agents. In vitro, wild-type VSV as well as a mutant variant (AV3) that has an impaired ability to shut down innate immunity preferentially killed undifferentiated, interferon-nonresponsive bladder cancer cells. Testing of these viruses in an orthotopic murine model of high-grade bladder cancer, which we have recently validated, revealed that both AV3 and wild-type VSV significantly inhibited orthotopic tumor growth. Despite the use of immunocompromised nude mice, there was no evidence of toxicity. In conclusion, VSV instillation therapy demonstrated strong antitumor activity and safety in an orthotopic model of high-risk disease. These findings provide preclinical proof-of-principle for the intravesical use of VSV, especially in interferon-refractory patients.

Outcomes of pregnancies in women with pre-gestational diabetes mellitus and gestational diabetes mellitus; a population-based study in New South Wales, Australia, 1998-2002.

AIM: To determine population-based rates and outcomes of pre-gestational diabetes mellitus (pre-GDM) and gestational diabetes mellitus (GDM) in pregnancy. METHODS: This was a cross-sectional study, using linked population databases, of all women, and their infants, discharged from hospital following birth in New South Wales (NSW) between 1 July 1998 and 31 December 2002. Women with, and infants exposed to pre-GDM or GDM were compared with those without diabetes mellitus for pregnancy characteristics and outcomes. RESULTS: Women with a singleton pregnancy (n = 370,703) and their infants were included: 1248 women (0.3%) had pre-GDM and 17,128 (4.5%) had GDM. Of those women with pre-GDM, 57% had Type 1 diabetes, 20% had Type 2 diabetes and for 23% the type of diabetes was unknown. Major maternal morbidity or mortality was more common in women with pre-GDM (7.9%) [odds ratio (OR) 3.2, 95% confidence interval (CI) 2.6, 3.9] and in women with GDM (3.1%) (OR 1.2, 95% CI 1.1, 1.4) when compared with women without diabetes (2.6%). Major infant morbidity or mortality occurred more frequently in infants exposed to pre-GDM compared with no diabetes (13.6% vs. 3.1%) (OR 5.0, 95% CI 4.2, 5.8) and in infants exposed to GDM compared with no diabetes (3.2% vs. 2.3%) (OR 1.4, 95% CI 1.3, 1.5). CONCLUSIONS: Pre-GDM and GDM continue to be associated with an increased risk of adverse maternal and neonatal outcomes; however, women with GDM have adverse outcomes less frequently. Rates of GDM and pre-GDM appear to be increasing over time. Clinicians should consider the potential for adverse outcomes, and arrange referral to appropriate services.

Taming the Trojan horse: optimizing dynamic carrier cell/oncolytic virus systems for cancer biotherapy.

Live cells offer unique advantages as vehicles for systemic oncolytic virus (OV) delivery. Recent studies from our laboratory and others have shown that virus-infected cells can serve as Trojan horse vehicles to evade antiviral mechanisms encountered in the bloodstream, prevent uptake by off-target tissues and act as microscale factories to produce OV upon arrival in tumor beds. However to be employed effectively, OV-infected cells are best viewed as dynamic biological systems rather than static therapeutic agents. The time-dependent processes of infection and in vivo cell trafficking will inevitably vary depending on which particular OV is being delivered, as well as the type of carrier cells (CC) employed. Understanding these parameters with respect to each unique CC/OV combination will therefore be required in order to effectively evaluate and harness their potential in preclinical study. In the following review, we discuss how early studies of OV delivery led us to investigate the use of cell carriers in our laboratory, and the approaches we are currently undertaking to compare the dynamics of different CC/OV systems. On the basis of these studies and others it is apparent that the success of any cell-based system for OV delivery rests upon the coordinated timing of three sequential phases--(1) ex vivo loading, (2) stealth delivery and (3) virus production at the tumor site. While at the current time, the timing of these processes are coupled to the natural cycle of infection and in vivo trafficking properties innate to each cell virus system, a quantitative delineation of their dynamics will lay the foundation for engineering CC/OV biotherapeutic systems that can be clinically deployed in a highly directed and controlled manner.

A mechanical evaluation of pre-tapped and self-tapped screws in small bones.

The purpose of this study was to compare the holding powers of 2.7 mm pre-tapped and self-tapped screws placed closely together and tightened in small bones. Pairs of metatarsals were collected from healthy, skeletally mature Greyhounds and part of a 2.7 mm dynamic compression plate was fixed to the dorsal surface of each bone using three 2.7 mm screws. Identical screws were used throughout but only one of each pair of bones had threads pre-cut using a tap prior to insertion. All of the screws were tightened before the constructs were mounted in a materials testing machine and the centrally placed screw was loaded incrementally until failure. Load-deformation curves were plotted and yield point, ultimate load to failure, stiffness and energy prior to yield point were measured. Mean values were recorded for each parameter and Student's T-test was used to test the null hypothesis that there is no difference in holding power between pre-tapped and self-tapped screws. Significant mechanical differences were not found between pre-tapped and self-tapped screws placed closely together and tightened into small bones. Self-tapped screws can be considered for use in small animal surgery even when multiple screws are to be placed closely together in relatively small pieces of bone.

Analysis of microsatellite variation in Pinus radiata reveals effects of genetic drift but no recent bottlenecks.

Most conifer species occur in large continuous populations, but radiata pine, Pinus radiata, occurs only in five disjunctive natural populations in California and Mexico. The Mexican island populations were presumably colonized from the mainland millions of years ago. According to Axelrod (1981), the mainland populations are relicts of an earlier much wider distribution, reduced some 8,000 years ago, whereas according to Millar (1997, 2000), the patchy metapopulation-like structure is typical of the long-term population demography of the species. We used 19 highly polymorphic microsatellite loci to describe population structure and to search for signs of the dynamics of population demography over space and time. Frequencies of null alleles at microsatellite loci were estimated using an approach based on the probability of identity by descent. Microsatellite genetic diversities were high in all populations [expected heterozygosity (H(e)) = 0.68-0.77], but the island populations had significantly lower estimates. Variation between loci in genetic differentiation (F(ST)) was high, but no locus deviated statistically significantly from the rest at an experiment wide level of 0.05. Thus, all loci were included in subsequent analysis. The average differentiation was measured as F(ST) = 0.14 (SD 0.012), comparable with earlier allozyme results. The island populations were more diverged from the other populations and from an inferred common ancestral gene pool than the mainland ones. All populations showed a deficiency of expected heterozygosity given the number of alleles, the mainland populations more so than the island ones. The results thus do not support a recent important contraction in the mainland range of radiata pine.

Oncolytic activity of vesicular stomatitis virus in primary adult T-cell leukemia.

Treatments for hematological malignancies have improved considerably over the past decade, but the growing therapeutic arsenal has not benefited adult T-cell leukemia (ATL) patients. Oncolytic viruses such as vesicular stomatitis virus (VSV) have recently emerged as a potential treatment of solid tumors and leukemias in vitro and in vivo. In the current study, we investigated the ability of VSV to lyse primary human T-lymphotropic virus type 1 (HTLV-1)-infected T-lymphocytes from patients with ATL. Ex vivo primary ATL cells were permissive for VSV and underwent rapid oncolysis in a time-dependent manner. Importantly, VSV infection showed neither viral replication nor oncolysis in HTLV-1-infected, nonleukemic cells from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and in naive CD4(+) T-lymphocytes from normal individuals or in ex vivo cell samples from patients with chronic lymphocytic leukemia (CLL). Interestingly, activation of primary CD4(+) T-lymphocytes with anti-CD3/CD28 monoclonal antibody, and specifically with anti-CD3, was sufficient to induce limited viral replication and oncolysis. However, at a similar level of T-cell activation, VSV replication was increased fourfold in ATL cells compared to activated CD4(+) T-lymphocytes, emphasizing the concept that VSV targets genetic defects unique to tumor cells to facilitate its replication. In conclusion, our findings provide the first essential information for the development of a VSV-based treatment for ATL.

Discontinuation of epidural analgesia late in labour for reducing the adverse delivery outcomes associated with epidural analgesia.

BACKGROUND: Although epidural analgesia provides the most effective labour analgesia, it is associated with some adverse obstetric consequences, including an increased risk of instrumental delivery. Many centres discontinue epidural analgesia late in labour to improve a woman's ability to push and reduce the rate of instrumental delivery. OBJECTIVES: To assess the impact of discontinuing epidural analgesia late in labour on: i) rates of instrumental deliveries and other delivery outcomes; and ii) analgesia and satisfaction with labour care. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group trials register (1 September 2003). SELECTION CRITERIA: Randomised controlled trials of epidurals discontinued late in labour compared with continuation of the same epidural protocol until birth, in women who receive an epidural for analgesia in the first stage of labour. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed study eligibility and quality and extracted the data. We analysed categorical data using relative risk (RR), and continuous data using weighted mean difference. MAIN RESULTS: We identified six studies, of which five were included (462 participants). Three of these were high quality studies whilst the other two were judged to be of lower quality because placebo was not used and the method of randomisation not described. All studies used different epidural analgesia protocols (type of drug, dosage or method of administration). Overall, the reduction in instrumental delivery rate was not statistically significant (23% versus 28%, RR 0.84, 95% confidence interval (CI) 0.61 to 1.15) nor was there any statistically significant difference in rates of other delivery outcomes. The only statistically significant result was an increase in inadequate pain relief when the epidural was stopped (22% versus 6%, RR 3.68, 95% CI 1.99 to 6.80). REVIEWERS' CONCLUSIONS: There is insufficient evidence to support the hypothesis that discontinuing epidural analgesia late in labour reduces the rate of instrumental delivery. There is evidence that it increases the rate of inadequate pain relief in the second stage of labour. The practice of discontinuing epidurals is widespread and the size of the reduction in instrumental delivery rate could be clinically important; therefore, we recommend a larger study than those included in this review be undertaken to determine whether this effect is real or has occurred by chance, and to provide stronger evidence about the safety aspects.

Antibiotics for ureaplasma in the vagina in pregnancy.

BACKGROUND: Preterm birth is a significant obstetric problem in high-income countries. Genital infection including ureaplasmas are suspected of playing a role in preterm birth and preterm rupture of the membranes. Antibiotics are used to treat women with preterm prelabour rupture of the membranes and results in prolongation of pregnancy and lowers the risks of maternal and neonatal infection. However, antibiotics may be beneficial earlier in pregnancy to eradicate potentially causative agents. OBJECTIVES: The objective of this review is to assess whether antibiotic treatment of pregnant women with ureaplasma in the vagina reduces the incidence of preterm birth and other adverse pregnancy outcomes. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group trials register (April 2003). SELECTION CRITERIA: All randomised controlled trials that compared any antibiotic regimen with placebo or no treatment in pregnant women with ureaplasma detected in the vagina. DATA COLLECTION AND ANALYSIS: Three reviewers independently assessed eligibility and trial quality and extracted data. MAIN RESULTS: One trial involving 1071 women was included. Of these, 644 randomly received antibiotic treatment (174 erythromycin estolate, 224 erythromycin sterate, and 246 clindamycin hydrochloride) and 427 received placebo. This trial did not report data on preterm birth. Incidence of low birthweight less than 2500 grams was only evaluated for erythromycin (combined) (n = 398 ) compared to placebo (n = 427) and there was no statistically significant difference between those treated and those not treated (relative risk (RR) 0.70, 95% confidence interval (CI) 0.46 to 1.07). In regards to side-effects sufficient to stop treatment, data were available for all women, and there were no statistically significant differences between any antibiotic (combined) and the placebo group (RR 1.25, 95% CI 0.85 to 1.85). REVIEWER'S CONCLUSIONS: There is insufficient evidence to show whether giving antibiotics to women with ureaplasma in the vagina will prevent preterm birth.

Detection and verification of quantitative trait loci for resistance to Dothistroma needle blight in Pinus radiata.

Six related radiata pine ( Pinus radiata) full-sib families were used to detect and independently verify quantitative trait loci (QTLs) for resistance to Dothistroma needle blight, caused by Dothistroma septospora. The detection families had from 26 to 30 individuals each, and had either a common maternal (31053) or paternal (31032) parent; one family (cross 4) consisted of progeny from both parents, 31053 x 31032. Approximately 200 additional progeny from cross 4 were clonally replicated and planted at two sites, with at least five to seven ramets of each individual per site. Marker segregation data were collected from a total of 250 RFLP and microsatellite markers, and single factor ANOVAs were conducted separately for each family and marker. A number of putative associations were observed, some across more than one family. Permutation tests were used to confirm expected probabilities of multiple associations based on chance alone. Seven markers representing at least four QTLs for resistance to Dothistroma were identified as being significant in more than one family; one of these was significant at P<0.05 in three families and highly significant at P<0.01 in a fourth. Further confirmation was obtained by testing those markers that were significant in more than one of the detection families (or highly significant in cross 4) in the clonally replicated progeny from cross 4. Four QTL positions were verified in the clonal populations, with a total percent variation accounted for of 12.5.