Evaluation of immunologic parameters in canine glioma patients treated with an oncolytic herpes virus.

Aim: To molecularly characterize the tumor microenvironment and evaluate immunologic parameters in canine glioma patients before and after treatment with oncolytic human IL-12-expressing herpes simplex virus (M032) and in treatment naïve canine gliomas. Methods: We assessed pet dogs with sporadically occurring gliomas enrolled in Stage 1 of a veterinary clinical trial that was designed to establish the safety of intratumoral oncoviral therapy with M032, a genetically modified oncolytic herpes simplex virus. Specimens from dogs in the trial and dogs not enrolled in the trial were evaluated with immunohistochemistry, NanoString, Luminex cytokine profiling, and multi-parameter flow cytometry. Results: Treatment-naive canine glioma microenvironment had enrichment of Iba1 positive macrophages and minimal numbers of T and B cells, consistent with previous studies identifying these tumors as immunologically "cold". NanoString mRNA profiling revealed enrichment for tumor intrinsic pathways consistent with suppression of tumor-specific immunity and support of tumor progression. Oncolytic viral treatment induced an intratumoral mRNA transcription signature of tumor-specific immune responses in 83% (5/6) of canine glioma patients. Changes included mRNA signatures corresponding with interferon signaling, lymphoid and myeloid cell activation, recruitment, and T and B cell immunity. Multiplexed protein analysis identified a subset of oligodendroglioma subjects with increased concentrations of IL-2, IL-7, IL-6, IL-10, IL-15, TNFα, GM-CSF between 14 and 28 days after treatment, with evidence of CD4+ T cell activation and modulation of IL-4 and IFNγ production in CD4+ and CD8+ T cells isolated from peripheral blood. Conclusion: These findings indicate that M032 modulates the tumor-immune microenvironment in the canine glioma model.

Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche.

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds.

Efficacy and safety of midostaurin in patients with advanced systemic mastocytosis: 10-year median follow-up of a phase II trial.

Patients with advanced systemic mastocytosis (SM) (e.g. aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL)) have limited treatment options and exhibit reduced survival. Midostaurin is an oral multikinase inhibitor that inhibits D816V-mutated KIT, a primary driver of SM pathogenesis. We conducted a phase II trial of midostaurin 100 mg twice daily, administered as 28-day cycles, in 26 patients (ASM, n=3; SM-AHN, n= 17; MCL, n=6) with at least one sign of organ damage. During the first 12 cycles, the overall response rate was 69% (major/partial response: 50/19%) with clinical benefit in all advanced SM variants. With ongoing therapy, 2 patients achieved a complete remission of their SM. Midostaurin produced a ⩾50% reduction in bone marrow mast cell burden and serum tryptase level in 68% and 46% of patients, respectively. Median overall survival for the entire cohort was 40 months, and 18.5 months for MCL patients. Low-grade gastrointestinal side effects were common and manageable with antiemetics. The most frequent grade 3/4 nonhematologic and hematologic toxicities were asymptomatic hyperlipasemia (15%) and anemia (12%). With median follow-up of 10 years, no unexpected toxicities emerged. These data establish the durable activity and tolerability of midostaurin in advanced SM.

Immunoglobulin (Ig)M antibodies to proteinase 3 in granulomatosis with polyangiitis and microscopic polyangiitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) appear to play an important role in the pathogenesis of ANCA-associated vasculitis (AAV). However, ANCA alone are not sufficient to generate disease, and some evidence suggests that infectious triggers may serve as inciting events for AAV disease activity. Antibodies of the immunoglobulin (Ig)M isotype often serve as markers of recent infection, and IgM ANCA have been identified previously in patients with AAV, although the frequency and clinical relevance of IgM ANCA is not well established. We sought to characterize IgM ANCA more clearly by creating a novel enzyme-linked immunosorbent assay (ELISA) for IgM antibodies to proteinase 3 [IgM proteinase 3 (PR3)-ANCA], which we applied to two large, clinically well-characterized trial cohorts of patients with granulomatosis with polyangiitis and microscopic polyangiitis. In the first cohort, IgM PR3-ANCA occurred with a frequency of 15·0%, and were associated with a higher degree of disease severity and a trend towards a higher rate of alveolar haemorrhage (29·6 versus 15·7%, P = 0·10). Analysis of follow-up samples in this cohort showed that the presence of IgM PR3-ANCA was transient, but could recur. In the second cohort, IgM PR3-ANCA occurred with a frequency of 41·1%, and were also associated with a higher degree of disease severity. A higher rate of alveolar haemorrhage was observed among those with IgM PR3-ANCA (45·3 versus 15·8%; P < 0·001). The association of transient IgM PR3-ANCA with an acute respiratory manifestation of AAV suggests a possible link between an infectious trigger and AAV disease activity.

Comparability of patients with ANCA-associated vasculitis enrolled in clinical trials or in observational cohorts.

OBJECTIVES: To analyse the differences between patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) entered into randomised clinical trials (RCTs) and those followed in large observational cohorts. METHODS: The main characteristics and outcomes of patients with generalised and/or severe GPA or MPA with a five-factor score ≥ 1 enrolled in the French Vasculitis Study Group (FVSG) or the US-Canadian-based Vasculitis Clinical Research Consortium cohorts were compared to those enrolled in one of 2 FVSG clinical RCTs (WEG91, WEGENT) or 3 European Vasculitis Society clinical trials (CYCLOPS, CYCAZAREM, IMPROVE). RESULTS: 657 patients (65.3% with GPA) in RCTs were compared to 437 in cohorts (90.6% with GPA). RCT patients were older at diagnosis than the cohort patients (56.6 ± 13.9 vs. 46.8 ± 17.3 years), had higher Birmingham vasculitis activity score (19.5 ± 9.1 vs. 16.9 ± 7.4), and more frequent kidney disease (84.0% vs. 54.9%) but fewer ear, nose, and throat symptoms (56.8% vs. 72.2%). At 56 months post-diagnosis, mortality and relapse rates, adjusted for age and renal function, were higher for patients with GPA in RCTs vs. cohorts (10.7% vs. 2.5% [p=0.001] and 22.5% vs. 15.6% [p=0.03], respectively) but similar for patients with MPA (6.2% vs. 6.6% [p=0.92] and 16.6% vs. 10.1% [p=0.39], respectively). CONCLUSIONS: Patients with GPA or MPA in RCTs and those in observational cohorts show important differences that should be remembered when interpreting results based on these study populations.

Outcomes of nonsevere relapses in antineutrophil cytoplasmic antibody-associated vasculitis treated with glucocorticoids.

OBJECTIVE: Nonsevere relapses are more common than severe relapses in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but their clinical course and treatment outcomes remain largely unexamined. We undertook this study to analyze the outcomes of patients with nonsevere relapses in the Rituximab in ANCA-Associated Vasculitis (RAVE) trial who were treated with prednisone according to a prespecified protocol. METHODS: RAVE was a randomized, double-blind, placebo-controlled trial comparing rituximab (RTX) to cyclophosphamide (CYC) followed by azathioprine (AZA) for induction of remission. Patients who experienced nonsevere relapses between months 1 and 18 were treated with a prednisone increase without a concomitant change in their nonglucocorticoid immunosuppressants, followed by a taper. RESULTS: Forty-four patients with a first nonsevere relapse were analyzed. In comparison to the 71 patients who maintained relapse-free remission over 18 months, these patients were more likely to have proteinase 3-ANCAs, diagnoses of granulomatosis with polyangiitis (Wegener's), and a history of relapsing disease at baseline. A prednisone increase led to remission in 35 patients (80%). However, only 13 patients (30%) were able to maintain second remissions through the followup period (mean 12.5 months); 31 patients (70%) had a second disease relapse, 14 of them with severe disease. The mean time to second relapse was 9.4 months (4.7 months in the group treated with RTX versus 13.7 months in the group treated with CYC/AZA; P < 0.01). Patients who experienced nonsevere relapses received more glucocorticoids than those who maintained remission (6.7 grams versus 3.8 grams; P < 0.01). CONCLUSION: Treatment of nonsevere relapses in AAV with an increase in glucocorticoids is effective in restoring temporary remission in the majority of patients, but recurrent relapses within a relatively short interval remain common. Alternative treatment approaches are needed for this important subset of patients.

γ134.5-deleted HSV-1-expressing human cytomegalovirus IRS1 gene kills human glioblastoma cells as efficiently as wild-type HSV-1 in normoxia or hypoxia.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however, the efficacy of γ(1)34.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric human cytolomegalovirus (HCMV)/HSV-1 virus, which expresses the human CMV protein kinase R evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared with the γ(1)34.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase p38 and its substrate heat-shock protein 27 (Hsp27) was seen after viral infection in normoxia compared with hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery, indicating that the p38 pathway has a role in the reduced efficacy of the γ(1)34.5-deleted virus in hypoxia. Taken together, these findings demonstrate that chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Clinical outcomes of remission induction therapy for severe antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To evaluate the reasons that complete remission is not achieved or maintained with original treatment in some patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) treated with rituximab (RTX) or with cyclophosphamide/azathioprine (CYC/AZA). METHODS: The Rituximab in AAV trial was a randomized, double-blind, placebo-controlled trial comparing the rate of remission induction among patients treated with RTX (n = 99) and patients treated with CYC followed by AZA (n = 98). Glucocorticoids were tapered over a period of 5 months. The primary outcome measure was lack of disease activity without glucocorticoid treatment at 6 months. To determine the most important reason for failure to achieve the primary outcome, 7 hierarchical categories of reasons were defined retrospectively (uncontrolled disease, adverse event leading to therapy discontinuation, severe flare, limited flare, Birmingham Vasculitis Activity Score for Wegener's Granulomatosis >0, prednisone treatment at any dosage, and other). RESULTS: Although remission (lack of disease activity) was achieved in 170 of the 197 patients (86%) in the first 6 months, the primary outcome measure was not achieved in 42%. There were 3 deaths. Twenty-four percent of the patients failed to achieve the primary end point due to active disease: 10 (5%) experienced uncontrolled disease in the first month and 37 (19%) experienced flares after initial improvement. In the majority of such patients, treatment with blinded crossover or according to best medical judgment led to disease control. Ninety-one percent of patients who had uncontrolled disease or experienced a severe flare had proteinase 3 (PR3)-ANCA. When patients with uncontrolled disease were excluded from analysis, those who were PR3-ANCA positive were found to experience fewer flares when treated with RTX compared to CYC/AZA (8 of 59 [14%] versus 20 of 62 [32%]; P = 0.02). Neither ANCA titers nor B cell counts predicted disease flare. CONCLUSION: Current treatment regimens are largely successful in controlling AAV, but in approximately one-fourth of patients, active disease persists or recurs in the first 6 months despite treatment. PR3-ANCA positivity is a risk factor for recurrence or persistence of severe disease. ANCA titers and B cell detectability are poor predictors of both disease relapse and disease quiescence in the first 6 months.

Cyclophosphamide as induction therapy for Wegener's granulomatosis and microscopic polyangiitis.

The introduction of cyclophosphamide (CyP) as a treatment for Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) has been among the most significant contributions in vasculitis. Prior to the introduction of CyP, WG was a uniformly fatal disease, with mortality occurring within 5-12 months from pulmonary or renal failure or from infection due to glucocorticoids. In 1973 Fauci and Wolff, at the National Institutes of Health, published their experience with a regimen that combined CyP and prednisone in which disease remission was seen in 12 of 14 patients. Long-term experience with CyP provided even greater evidence for its efficacy in which an 80% survival rate was seen, with 91% of patients having significant improvement and 75% achieving complete remission. However, extended follow-up also demonstrated that disease relapse occurred in at least 50% of patients and that 42% experienced morbidity solely as a result of treatment. These observations showed that while CyP was life-saving, it did not prevent relapse and was associated with significant toxicity such that safer means to induce remission needed to be pursued. Strategies aimed at reducing exposure to CyP have included intermittent administration, induction-maintenance regimens and avoidance of CyP for non-severe disease. Recently, the introduction of rituximab has raised important questions regarding the place of CyP in the treatment of WG/MPA. This paper examines the past, present and future of CyP through a review of its efficacy and safety.

High-resolution longitudinal assessment of flow and permeability in mouse glioma vasculature: Sequential small molecule and SPIO dynamic contrast agent MRI.

The poor prognosis associated with malignant glioma is largely attributable to its invasiveness and robust angiogenesis. Angiogenesis involves host-tumor interaction and requires in vivo evaluation. Despite their versatility, few studies have used mouse glioma models with perfusion MRI approaches, and generally lack longitudinal study design. Using a micro-MRI system (8.5 Tesla), a novel dual bolus-tracking perfusion MRI strategy was implemented. Using the small molecule contrast agent Magnevist, dynamic contrast enhanced MRI was implemented in the intracranial 4C8 mouse glioma model to determine K(trans) and v(e), indices of tumor vascular permeability and cellularity, respectively. Dynamic susceptibility contrast MRI was subsequently implemented to assess both cerebral blood flow and volume, using the macromolecular superparamagnetic iron oxide, Feridex, which circumvented tumor bolus susceptibility curve distortions from first-pass extravasation. The high-resolution parametric maps obtained over 4 weeks, indicated a progression of tumor vascularization, permeability, and decreased cellularity with tumor growth. In conclusion, a comprehensive array of key parameters were reliably quantified in a longitudinal mouse glioma study. The syngeneic 4C8 intracerebral mouse tumor model has excellent characteristics for studies of glioma angiogenesis. This approach provides a useful platform for noninvasive and highly diagnostic longitudinal investigations of anti-angiogenesis strategies in a relevant orthotopic animal model.

A genome assembly-integrated dog 1 Mb BAC microarray: a cytogenetic resource for canine cancer studies and comparative genomic analysis.

Molecular cytogenetic studies have been instrumental in defining the nature of numerical and structural chromosome changes in human cancers, but their significance remains to be fully understood. The emergence of high quality genome assemblies for several model organisms provides exciting opportunities to develop novel genome-integrated molecular cytogenetic resources that now permit a comparative approach to evaluating the relevance of tumor-associated chromosome aberrations, both within and between species. We have used the dog genome sequence assembly to identify a framework panel of 2,097 bacterial artificial chromosome (BAC) clones, selected at intervals of approximately one megabase. Each clone has been evaluated by multicolor fluorescence in situ hybridization (FISH) to confirm its unique cytogenetic location in concordance with its reported position in the genome assembly, providing new information on the organization of the dog genome. This panel of BAC clones also represents a powerful cytogenetic resource with numerous potential applications. We have used the clone set to develop a genome-wide microarray for comparative genomic hybridization (aCGH) analysis, and demonstrate its application in detection of tumor-associated DNA copy number aberrations (CNAs) including single copy deletions and amplifications, regional aneuploidy and whole chromosome aneuploidy. We also show how individual clones selected from the BAC panel can be used as FISH probes in direct evaluation of tumor karyotypes, to verify and explore CNAs detected using aCGH analysis. This cytogenetically validated, genome integrated BAC clone panel has enormous potential for aiding gene discovery through a comparative approach to molecular oncology.

Arteriovenous malformation of the brain mimicking primary central nervous system vasculitis.

In the diagnosis of primary central nervous system (CNS) vasculitis, it is crucial to rule out clinical, angiographic, and pathological mimics. We report a case of arteriovenous malformation (AVM) mimicking primary CNS vasculitis. A young male presented with intracerebral haemorrhage and no other clinical, laboratory, or angiographic features suggesting vasculitis. Cerebral biopsy showed perivascular inflammation and slight infiltration of the muscular layer of cerebral vessels by chronic inflammatory cells close to the haemorrhagic areas. These findings led to a diagnosis of CNS vasculitis. The patient was initially treated with corticosteroids, but 10 months after the discovery and surgical repair of the AVM, the patient is not receiving any immunosuppressant and has not developed any features of cerebral or systemic vasculitis.

Anti-tumour necrosis factor therapy in patients with refractory Takayasu arteritis: long-term follow-up.

OBJECTIVE: To assess the efficacy of anti-tumour necrosis factor (TNF) therapy to induce remission in patients with Takayasu arteritis (TAK) refractory to other immunosuppressive therapies. METHODS: Retrospective single-centre study of 25 patients with refractory TAK. RESULTS: Patients were treated with infliximab (IFX) or etanercept (ETA) for up to 7 years; 21 with IFX (median 28 months (range 2-84)) and 9 with ETA (median 28 months (range 4-82)); 5 patients initially treated with ETA subsequently switched to IFX. Following anti-TNF therapy, remission was achieved and prednisone was discontinued in 15 patients (60%) and successfully tapered below 10 mg/day in an additional 7 patients (28%). Of 18 patients treated with other immunosuppressive agents concurrent with anti-TNF therapy, 9 (50%) could taper or discontinue the additional agent. Major relapses occurred in four patients that initially achieved stable remission. Four patients suffered adverse events, including one with opportunistic infections and one with breast cancer. CONCLUSIONS: In this group of patients with refractory TAK, anti-TNF therapy was associated with remission in a majority of patients, facilitating dose reduction or discontinuation of prednisone and other immunosuppressive therapy. These findings strengthen the rationale for the conducting of a randomised controlled trial of anti-TNF therapy in TAK.

1p36 is a preferential target of chromosome 1 deletions in astrocytic tumours and homozygously deleted in a subset of glioblastomas.

Astrocytic, oligodendroglial and mixed gliomas are the commonest gliomas in adults. They have distinct phenotypes and clinical courses, but as they exist as a continuous histological spectrum, differentiating them can be difficult. Co-deletions of total 1p and 19q are found in the majority of oligodendrogliomas and considered as a diagnostic marker and a prognostic indicator. The 1p status of astrocytomas has not yet been thoroughly examined. Using a chromosome 1 tile path array, we investigated 108 adult astrocytic tumours for copy number alterations. Total 1p deletions were rare (2%), however partial deletions involving 1p36 were frequently identified in anaplastic astrocytomas (22%) and glioblastomas (34%). Multivariate analysis showed that patients with total 1p deletions had significantly longer survival (P=0.005). In nine glioblastomas homozygous deletions at 1p36 were identified. No somatic mutations were found among the five genes located in the homozygously deleted region. However, the CpG island of TNFRSF9 was hypermethylated in 19% of astrocytic tumours and 87% of glioma cell lines. TNFRSF9 expression was upregulated after demethylation of glioma cell lines. Akt3 amplifications were found in four glioblastomas. Our results indicate that 1p deletions are common anaplastic astrocytomas and glioblastomas but are distinct from the 1p abnormalities in oligodendrogliomas.

Epigenetic identification of ADAMTS18 as a novel 16q23.1 tumor suppressor frequently silenced in esophageal, nasopharyngeal and multiple other carcinomas.

Tumor suppressor genes (TSGs) often locate at chromosomal regions with frequent deletions in tumors. Loss of 16q23 occurs frequently in multiple tumors, indicating the presence of critical TSGs at this locus, such as the well-studied WWOX. Herein, we found that ADAMTS18, located next to WWOX, was significantly downregulated in multiple carcinoma cell lines. No deletion of ADAMTS18 was detected with multiplex differential DNA-PCR or high-resolution 1-Mb array-based comparative genomic hybridization (CGH) analysis. Instead, methylation of the ADAMTS18 promoter CpG Island was frequently detected with methylation-specific PCR and bisulfite genome sequencing in multiple carcinoma cell lines and primary carcinomas, but not in any nontumor cell line and normal epithelial tissue. Both pharmacological and genetic demethylation dramatically induced the ADAMTS18 expression, indicating that CpG methylation directly contributes to the tumor-specific silencing of ADAMTS18. Ectopic ADAMTS18 expression led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells lacking the expression. Thus, through functional epigenetics, we identified ADAMTS18 as a novel functional tumor suppressor, being frequently inactivated epigenetically in multiple carcinomas.

Identification of a novel tumor transforming gene GAEC1 at 7q22 which encodes a nuclear protein and is frequently amplified and overexpressed in esophageal squamous cell carcinoma.

By comparative DNA fingerprinting, we identified a 357-bp DNA fragment frequently amplified in esophageal squamous cell carcinomas (ESCC). This fragment overlaps with an expressed sequence tag mapped to 7q22. Further 5' and 3'-rapid amplification of cDNA ends revealed that it is part of a novel, single-exon gene with full-length mRNA of 2052 bp and encodes a nuclear protein of 109 amino acids ( approximately 15 kDa). This gene, designated as gene amplified in esophageal cancer 1 (GAEC1), was located within a 1-2 Mb amplicon at 7q22.1 identified by high-resolution 1 Mb array-comparative genomic hybridization in 6/10 ESCC cell lines. GAEC1 was ubiquitously expressed in normal tissues including esophageal and gastrointestinal organs; with amplification and overexpression in 6/10 (60%) ESCC cell lines and 34/99 (34%) primary tumors. Overexpression of GAEC1 in 3T3 mouse fibroblasts caused foci formation and colony formation in soft agar, comparable to H-ras and injection of GAEC1-transfected 3T3 cells into athymic nude mice formed undifferentiated sarcoma in vivo, indicating that GAEC1 is a transforming oncogene. Although no significant correlation was observed between GAEC1 amplification and clinicopathological parameters and prognosis, our study demonstrated that overexpressed GAEC1 has tumorigenic potential and suggest that overexpressed GAEC1 may play an important role in ESCC pathogenesis.

Array CGH profiling of favourable histology Wilms tumours reveals novel gains and losses associated with relapse.

Despite the excellent survival of Wilms tumour patients treated with multimodality therapy, approximately 15% will suffer from tumour relapse, where response rates are markedly reduced. We have carried out microarray-based comparative genomic hybridisation on a series of 76 Wilms tumour samples, enriched for cases which recurred, to identify changes in DNA copy number associated with clinical outcome. Using 1Mb-spaced genome-wide BAC arrays, the most significantly different genomic changes between favourable histology tumours that did (n = 37), and did not (n = 39), subsequently relapse were gains on 1q, and novel deletions at 12q24 and 18q21. Further relapse-associated loci included losses at 1q32.1, 2q36.3-2q37.1, and gain at 13q31. 1q gains correlated strongly with loss of 1p and/or 16q. In 3 of 11 cases with concurrent 1p(-)/1q(+), a breakpoint was identified at 1p13. Multiple low-level sub-megabase gains along the length of 1q were identified using chromosome 1 tiling-path arrays. One such recurrent region at 1q22-q23.1 included candidate genes RAB25, NES, CRABP2, HDGF and NTRK1, which were screened for mRNA expression using quantitative RT-PCR. These data provide a high-resolution catalogue of genomic copy number changes in relapsing favourable histology Wilms tumours.

A comprehensive study of chromosome 16q in invasive ductal and lobular breast carcinoma using array CGH.

We analysed chromosome 16q in 106 breast cancers using tiling-path array-comparative genomic hybridization (aCGH). About 80% of ductal cancers (IDCs) and all lobular cancers (ILCs) lost at least part of 16q. Grade I (GI) IDCs and ILCs often lost the whole chromosome arm. Grade II (GII) and grade III (GIII) IDCs showed less frequent whole-arm loss, but often had complex changes, typically small regions of gain together with larger regions of loss. The boundaries of gains/losses tended to cluster, common sites being 54.5-55.5 Mb and 57.4-58.8 Mb. Overall, the peak frequency of loss (83% cancers) occurred at 61.9-62.9 Mb. We also found several 'minimal' regions of loss/gain. However, no mutations in candidate genes (TRADD, CDH5, CDH8 and CDH11) were detected. Cluster analysis based on copy number changes identified a large group of cancers that had lost most of 16q, and two smaller groups (one with few changes, one with a tendency to show copy number gain). Although all morphological types occurred in each cluster group, IDCs (especially GII/GIII) were relatively overrepresented in the smaller groups. Cluster groups were not independently associated with survival. Use of tiling-path aCGH prompted re-evaluation of the hypothetical pathways of breast carcinogenesis. ILCs have the simplest changes on 16q and probably diverge from the IDC lineage close to the stage of 16q loss. Higher-grade IDCs probably develop from low-grade lesions in most cases, but there remains evidence that some GII/GIII IDCs arise without a GI precursor.