Exome sequencing of lymphomas from three dog breeds reveals somatic mutation patterns reflecting genetic background

Lymphoma is the most common hematological malignancy in developed countries. Outcome is strongly determined by molecular subtype, reflecting a need for new and improved treatment options. Dogs spontaneously develop lymphoma, and the predisposition of certain breeds indicates genetic risk factors. Using the dog breed structure, we selected three lymphoma predisposed breeds developing primarily T-cell (boxer), primarily B-cell (cocker spaniel), and with equal distribution of B- and T-cell lymphoma (golden retriever), respectively. We investigated the somatic mutations in B- and T-cell lymphomas from these breeds by exome sequencing of tumor and normal pairs. Strong similarities were evident between B-cell lymphomas from golden retrievers and cocker spaniels, with recurrent mutations in TRAF3-MAP3K14 (28% of all cases), FBXW7 (25%), and POT1 (17%). The FBXW7 mutations recurrently occur in a specific codon; the corresponding codon is recurrently mutated in human cancer. In contrast, T-cell lymphomas from the predisposed breeds, boxers and golden retrievers, show little overlap in their mutation pattern, sharing only one of their 15 most recurrently mutated genes. Boxers, which develop aggressive T-cell lymphomas, are typically mutated in the PTEN-mTOR pathway. T-cell lymphomas in golden retrievers are often less aggressive, and their tumors typically showed mutations in genes involved in cellular metabolism. We identify genes with known involvement in human lymphoma and leukemia, genes implicated in other human cancers, as well as novel genes that could allow new therapeutic options.Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous, aggressive form of non-Hodgkin lymphoma (NHL) and the most prevalent form of B-cell NHL in people. Approximately 30,000 DLBCL cases are diagnosed each year in the United States, of which only half are curable (Abramson and Shipp 2005). Molecular subclassifications of DLBCL are highly predictive of treatment outcome (Alizadeh et al. 2000). NHL can also arise from T cells, making up 10%–15% of all NHL cases. T-cell lymphomas are highly heterogeneous and several subtypes exist (Evens and Gartenhaus 2003; Iqbal et al. 2010).Dogs have previously been proven useful in determining predisposing genetic markers for human diseases due to the breed structure caused by artificial breeding for phenotypic factors (Dodman et al. 2010; Wilbe et al. 2010; Shearin et al. 2012; Karlsson et al. 2013; Tang et al. 2014). A recent lymphoma study in dogs identified a gene (TRAF3) as being commonly mutated in both dog and human B-cell lymphomas (Bushell et al. 2015). However, this study did not separate canine tumors based on breed, preventing the discovery of somatic mutations reflecting genetic background. Since dog breeds represent genetic isolates, comparing dog breeds with differential predispositions to cancer can indicate the role of the genetic background and allow for better detection of mutations influenced by that genetic background. In dogs, malignant lymphoma is the most common tumor treated with chemotherapy, affecting dogs of all ages and breeds (Valli et al. 2013); however, the high rate of lymphoma in certain breeds and preferential cells of origin among different breeds indicates genetic risk factors (Modiano et al. 2005).Approximately 70% of all canine lymphomas arise from B cells (Modiano et al. 2005; Ponce et al. 2010). The most common form of canine B-cell lymphoma is the clinical and histological equivalent of human DLBCL (Vail and MacEwen 2000; Modiano et al. 2005; Valli et al. 2011; Ito et al. 2014). As in humans, the CHOP-based chemotherapy protocols are the most effective treatment for canine B-cell lymphomas. T-cell lymphomas are less common in dogs, and the most aggressive types have a higher risk of relapse and early death (Ruslander et al. 1997; Dobson et al. 2001). Many T-cell lymphomas also have a low long-term survival frequency in humans.Exome sequencing efforts in human lymphomas have shown that certain mutations are specific for lymphoma subtypes or shared between only a few subtypes (Zhang et al. 2014). For example, DLBCL typically have mutations affecting B-cell receptor signaling or subunits, e.g., CD79A/B, and in histone modifiers like EZH2 and MLL2, PRDM1, TP53, CARD11, and MYD88 (Lenz et al. 2008; Davis et al. 2010; Mandelbaum et al. 2010; Morin et al. 2010; Ngo et al. 2011; Lohr et al. 2012). MYD88 is also recurrently mutated in other B-cell lymphomas like primary central nervous system lymphoma (PCNSL) (Gonzalez-Aguilar et al. 2012), and MLL2 and TP53 mutations have been reported for mantle cell lymphoma (MCL) (Zhang et al. 2014). The two main human DLBCL subtypes—activated B cell (ABC) and germinal center B cell (GCB)—which can be distinguished based on differential gene expression and prognosis (Alizadeh et al. 2000), also have unique recurrent mutations. ABC DLBCLs are characterized by mutations in B-cell differentiation genes and often show constitutive activation of NF-κB signaling, whereas BCL2 and MYC translocations are characteristic of GCB DLBCLs (summarized in Pasqualucci et al. 2011b). Canine B-cell lymphomas can also be separated into germinal and post-germinal center types using gene expression data, sharing pathways with their human counterparts (Richards et al. 2013), although more studies are needed to fully elucidate this. T-cell lymphomas are much less studied compared with B-cell lymphomas, particularly DLBCL.The treatment outcome of canine lymphoma is predictive of the human response to the same treatment (Honigberg et al. 2010; Marconato et al. 2013; London et al. 2014), and canine clinical trials, although highly regulated, are easier to complete compared with human trials. Hence, a better understanding of canine lymphoma offers great potential to accelerate development of new treatments for human patients. Here, we have compared the somatic mutations of B- and T-cell canine lymphoma in three dog breeds with different lymphoma immunophenotype predispositions. The identified breed-specific patterns are a good opportunity to study interventions targeting the significant mutations.     

Cardiac adaptation to chronic high-altitude hypoxia: beneficial and adverse effects

This review deals with the capability of the heart to adapt to chronic hypoxia in animals exposed to either natural or simulated high altitude. From the broad spectrum of related issues, we focused on the development and reversibility of both beneficial and adverse adaptive myocardial changes. Particular attention was paid to cardioprotective effects of adaptation to chronic high-altitude hypoxia and their molecular mechanisms. Moreover, interspecies and age differences in the cardiac sensitivity to hypoxia-induced effects in various experimental models were emphasized.     

Effect of adaptation to high-altitude hypoxia on the microcirculation in the rat mesentery

After prolonged adaptation of rats to high-altitude hypoxia (in a pressure chamber at an altitude of 6500 m, for 6 h daily for 30–40 days) considerable hyperemia of the intestinal wall and mesentery was observed; the number of functioning capillaries was several times greater than in the control animals but the blood flow in the dilated microvessels was slowed and its structure was disturbed. Besides obvious hemoconcentration in the arteries and veins, hemodilution was observed in the capillaries. Definite hemorheological changes evidently connected with the polycythemia and increased hematocrit index were noted.Laboratory of Pathophysiology of Extremal States, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Ésperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 528–530, May, 1977.     

Effect of adaptation to high-altitude hypoxia in early ontogeny on higher nervous activity

Newborn male and female Wistar rats were adapted to hypoxia in a pressure chamber. Adaptation began at an altitude of 1000 m for 1 h daily, after which the duration and intensity of exposure were gradually increased so that, starting from the 17th day, the animals were adapted to an altitude of 5000 m for 5 h on 5 days a week. After adaptation for two months, a conditioned active avoidance reflex was produced in the animals. In the adapted males a tendency was observed for the reflex to be formed more rapidly and for it to be preserved to a much greater degree than in the control animals. In females adapted to hypoxia under similar conditions no changes were observed in the formation and preservation of the reflex.Laboratory of Pathophysiology of the Heart, Institute of General Pathology and Pathophysiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 902–903, August, 1976.     

Effect of adaptation to high-altitude hypoxia and subsequent disadaptation on conditioned reflex retention

After adaptation to high-altitude hypoxia the degree of retention of a conditioned active avoidance reflex is considerably increased. Only half as many combinations of the conditioned and unconditioned stimuli were required to reach the necessary level when the retention of the reflex was tested 24 h after its formation in adapted rats as in the control. This level of adaptation was produced 1 and 5 days after the end of a course of exposures to hypoxia. During disadaptation, the increase in the degree of retention of the reflex which had been achieved disappeared 10 and 27 days after the end of exposure to hypoxia.Laboratory of Pathophysiology of the Heart, Institute of General Pathology and Pathophysiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 526–527, May, 1977.     

Morphofunctional adaptation of the placenta under conditions of natural high-altitude hypoxia

Twenty-six III trimester placentae from Kirghiz puerperas residents of high-altitude regions (2200-3000 m above the sea level) and 22 such placentae from female residents of the plains (group I and II, respectively) were subjected to ultrasonic placentometry and organo-stereohistometry. Group I placentae appeared to fall behind in thickness on weeks 28, 32 and 36 as well as in development on the last 4 weeks which is coupled with synchronous reduction of weight of mature newborns and relevant placentae. Enhanced morphofunctional activity, excessive vasculature of villous network, increased epithelial volume of the villi and vascular-syncytial membranes, intensive growth of separate cotyledons were characteristic for placentae exposed to high-altitude factors, which also bore the signs of dysadaptation manifesting with more frequent infarctions, adherent villi, hemorrhages.     

Whole-genome sequencing of Campylobacter jejuni isolated from Danish routine human stool samples reveals surprising degree of clustering

ObjectivesOutbreaks of Campylobacter are traditionally considered to be rare; however, rather than being the true nature of the disease, this may reflect our present inability to detect them. The aim of this study was to determine the genetic and epidemiological degree of clustering among Campylobacter jejuni isolates from Danish patients.MethodsWhole-genome sequencing (WGS) was applied to 245 C. jejuni isolates from patients with domestically acquired infection over a 9-month period in 2015 and 2016.ResultsWGS demonstrated that 62 of the 245 isolates (25%) clustered genetically. In total, 21 genetic clusters were identified of which four (18%) consisted of five isolates or more. Seventeen (81%) of the 21 genetic clusters were clustered in space and/or time. Of the 245 isolates, 49 (20%) were part of a temporal and/or geographical cluster. The identified clusters included two outbreaks; one which had not been identified through the existing surveillance system.ConclusionsUsing WGS, we show that Campylobacter case clustering and even outbreaks appear to occur more often than previously assumed, providing important new insight into the relatively poorly understood epidemiology of the most important cause of bacterial gastroenteritis in the industrialized world.     

Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity.The presence of multiple cancer cell clones within a single tumor has been explained as a Darwinian process in which different clones compete for limited resources, and the most phenotypically fit cells eventually prevail (Greaves and Maley 2012; Yates and Campbell 2012; Aparicio and Caldas 2013). It has been suggested that such heterogeneity allows a tumor to respond to local and systemic selective pressures, such as those exerted by therapeutic interventions (Nowak and Sigmund 2004; Greaves and Maley 2012; Bozic et al. 2013). For example, the presence of subclonal driver mutations in cancer cells was indicative of rapid disease progression in chronic lymphocytic leukemia (Landau et al. 2013). Using single-cell sequencing or massively parallel sequencing, clonal architectures ranging from complex polyclonal structures to monoclonal tumors have been described in cancer lineages such as those of the breast, kidney, and blood (Navin et al. 2011; Ding et al. 2012; Shah et al. 2012; Landau et al. 2013; Gerlinger et al. 2014). Distinct subclonal tumor cell populations relating to mosaic amplification of receptor tyrosine kinases were reported in glioblastoma (GBM), suggesting a similarly dynamic architecture for this disease (Snuderl et al. 2011; Nickel et al. 2012; Szerlip et al. 2012; Sottoriva et al. 2013).GBM is the most common malignant brain tumor in adults (Van Meir et al. 2010; Dunn et al. 2012) and is standardly treated with surgical resection followed by concomitant radiotherapy and administration of the alkylating agent temozolomide (TMZ) (Stupp et al. 2005). Despite this aggressive treatment regimen, the median time to disease recurrence is 6.9 mo, with >90% of GBM tumors recurring at the original site (Wen and Kesari 2008). Therapy targeting the epidermal growth factor receptor variant III (EGFRvIII) led to an improved overall survival time among patients with GBM; however, 82% of these patients lost EGFRvIII expression when the tumor recurred, which suggests a competitive advantage for non-EGFRvIII expressing clones in these tumors (Sampson et al. 2010). Achieving a better understanding of the clonal structure of cancer cells is thus of vital importance and may inform the development of additional targeted therapies for rapidly lethal forms of cancer, such as GBM.Here, we analyzed genomic profiles of 252 GBM samples from The Cancer Genome Atlas (TCGA) (Brennan et al. 2013), and 60 biopsies taken from 23 pairs of pre- and post-treatment GBMs, to understand (1) the intratumoral clonal compositions of primary GBM; and (2) how GBM responds to therapeutic intervention. Our results provide a molecular portrait of GBM recurrence.     

Derived variants at six genes explain nearly half of size reduction in dog breeds

Selective breeding of dogs by humans has generated extraordinary diversity in body size. A number of multibreed analyses have been undertaken to identify the genetic basis of this diversity. We analyzed four loci discovered in a previous genome-wide association study that used 60,968 SNPs to identify size-associated genomic intervals, which were too large to assign causative roles to genes. First, we performed fine-mapping to define critical intervals that included the candidate genes GHR, HMGA2, SMAD2, and STC2, identifying five highly associated markers at the four loci. We hypothesize that three of the variants are likely to be causative. We then genotyped each marker, together with previously reported size-associated variants in the IGF1 and IGF1R genes, on a panel of 500 domestic dogs from 93 breeds, and identified the ancestral allele by genotyping the same markers on 30 wild canids. We observed that the derived alleles at all markers correlated with reduced body size, and smaller dogs are more likely to carry derived alleles at multiple markers. However, breeds are not generally fixed at all markers; multiple combinations of genotypes are found within most breeds. Finally, we show that 46%–52.5% of the variance in body size of dog breeds can be explained by seven markers in proximity to exceptional candidate genes. Among breeds with standard weights <41 kg (90 lb), the genotypes accounted for 64.3% of variance in weight. This work advances our understanding of mammalian growth by describing genetic contributions to canine size determination in non-giant dog breeds.Domestic dogs exhibit the greatest diversity in body size of any land mammal. Mastiffs can be 50 times heavier than Chihuahuas, and Great Danes five times taller than Pekingese. Dog breeds are all descended from the gray wolf (Wayne 1993; Lindblad-Toh et al. 2005) and are the product of artificial selection that began between 15,000 and 100,000 yr ago (Vilà et al. 1997; Sablin and Khlopachev 2002; Savolainen et al. 2002; Germonpré et al. 2009; Pang et al. 2009; Ovodov et al. 2011). However, the majority of the modern dog breeds were developed within the past 300 yr (American Kennel Club 1998; Parker et al. 2004). More than 400 breeds now exist worldwide, including 175 that are recognized in the United States by the American Kennel Club (AKC; www.akc.org).Modern domestic dog breeds are codified by standards, which apply persistent selective pressure on fixed phenotypes that are often breed defining, such as coat color, skull shape, leg length, and body size. This pressure reduces phenotypic and genetic heterogeneity within breeds, yet enormous phenotypic diversity exists across breeds (Parker et al. 2004, 2007; vonHoldt et al. 2010). These factors, along with the genetic isolation of breeds, have established domestic dog breeds as an excellent genetic system for the study of complex traits, including skeletal size and shape variation (Chase et al. 2002; Shearin and Ostrander 2010).Loci determining size have strong signatures of selection (Akey et al. 2010; Boyko et al. 2010; Vaysse et al. 2011). The first association studies of canine body size found an influential locus in spite of sparse marker density (Chase et al. 2002; Jones et al. 2008). Chase et al. (2002) used genotypes at ∼500 microsatellites to analyze the genetic basis for canid morphological variation in Portuguese water dogs, a breed with significant variation in skeletal size (Chase et al. 2002), and identified multiple quantitative trait loci (QTLs) related to canine body size. A locus on canine chromosome 15 (CFA15) was observed to be highly associated with measures of skeletal size. Further investigation by our collaborative group led to the identification of a single haplotype composed of 20 single-nucleotide polymorphisms (SNPs) that was shared among all small breeds (<9 kg [20 lb]), but was nearly absent from giant breeds (>30 kg [66 lb]) (Sutter et al. 2007). The haplotype spans the insulin-like growth factor 1 (IGF1) gene, which is known to regulate skeletal size in both mice and humans (Baker et al. 1993; Woods et al. 1996).A subsequent study by Jones et al. (2008) extended these findings and pioneered the use of breed-defined phenotypes (“stereotypes”) to identify associated markers, a method which is also used in the present study. Jones et al. (2008) tested the association of genotypes in 2801 dogs representing 147 breeds at 1536 SNPs with several breed stereotypes including weight, limb length, and height. They identified several new body size loci as well as replicating findings from previous studies (Chase et al. 2002, 2005), thus further supporting the use of breed standard measures, rather than individual measurements on each dog, in genetic studies of canine morphology.Subsequent studies performed by our collaborative group on a much larger data set of 915 dogs from 80 breeds genotyped using 60,968 SNPs (the “CanMap project”) highlighted a number of phenotype-associated loci (Boyko et al. 2010). Among these were loci important in body size, some of which had been previously identified (Chase et al. 2002, 2005; Jones et al. 2008). Associations at four of the size-associated loci were replicated in data released by a subsequent study of 509 dogs from 46 breeds genotyped with 170,000 SNPs (Vaysse et al. 2011). Finally, the CanMap data set was used by Hoopes et al. (2012) to identify a new dog body size locus on CFA3 at the insulin-like growth factor 1 receptor gene (IGF1R).Here we describe the combinatorial effects of genetic variation at six loci on determining body size in dog breeds. At four autosomal loci previously found to be associated with canine body size (Boyko et al. 2010), the critical intervals resulting from our fine-mapping revealed excellent candidate genes, including growth hormone receptor (GHR), high mobility group AT-hook 2 (HMGA2), stanniocalcin 2 (STC2), and SMAD family member 2 (SMAD2). We genotyped the most highly associated marker(s) at each locus, together with highly associated markers from the IGF1 and IGF1R genes in a large set of dogs representing the entire range of canine body size. The resulting analysis shows that approximately half of the variance of the weights of dog breeds can be explained by polymorphisms at just these six loci.     

Developmental responses to high-altitude hypoxia in Bolivian children of European ancestry: A test of the developmental adaptation hypothesis

The developmental adaptation hypothesis (DAH) proposes that highlanders adapt to their hypobaric hypoxic environment during growth and development. This report utilizes data on children (9.0–19.9 yr) of European ancestry residing in Santa Cruz and La Paz, Bolivia, to test selected aspects of the DAH. Previous tests of this hypothesis have been hampered by difficulties in controlling for socioeconomic and genetic influences. However, due to their high socioeconomic status and their relatively short history of residence at high altitudes, these factors can be reasonably well controlled in studies of European children. The data on European children are consistent with some but not all aspects of the DAH. First, examination of the available data on V?O₂max (ml/kg/min) in European boys suggests that chronic exposure to hypobaric hypoxia results in an enhancement of the overall functional capacity of the oxygen transport system during adolescence, as predicted by the DAH. Second, chronic exposure to hypobaric hypoxia results in a delay in linear growth and maturation in European children, as well as in an enhancement of their lung volumes, also as predicted by the DAH. However, the effects of chronic hypoxia on linear growth, after controlling for health and nutritional status, are not as large as previously believed and, contrary to the expectations of the DAH, they need not be acquired through a distinctive pattern of growth during late childhood and adolescence. Instead, it appears that these patterns are established in European highlanders prior to 9 years of age and are then maintained, rather than accentuated, during later developmental periods.     

Effect of adaptation to continuous and intermittent hypoxia on heart resistance to ischemic and reperfusion arrhythmias

The effect of adaptation to intermittent hypoxia in a pressure chamber (5.000 m daily for 6 hours for 30 days) on arrhythmias occurring in occlusion of the coronary artery and subsequent reperfusion in rats was compared with the effect of adaptation to continuous hypoxia at middle-mountain altitudes. Adaptation to intermittent hypoxia reduced the duration of severe ventricular arrhythmias in acute ischemia and reperfusion to a great measure. Adaptation to continuous hypoxia at middle-mountain altitudes has a similar effect only in ischemic arrhythmias but at the same time promotes the development of reperfusion arrhythmias. The possible mechanisms of these effects are discussed.     

Cytological changes in the myocardium of rats adapted to high-altitude hypoxia

During adaptation of rats to high-altitude hypoxia evidence of stimulation of both formative and lytic processes was observed, and either one process or the other was predominant in different cells. The ratios between volumes of mitochondria and myofibrils were substantially unchanged. The relative volume of the microcirculation was increased on account of dilatation of the small vessels with no increase in their number.Laboratory of Pathomorphology, Institute of Cytology and Genetics, Siberian Branch, Academy of Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1380–1382, November, 1976.     

Changes to the hepatocytes and muscle fibers of the diaphragm during adaptation to cold under normal conditions and in combination with high-altitude hypoxia

Comparative morphometric analysis of muscular fibres of the diaphragm and hepatocytes of albino rats was carried out in adaptation to cold (0-2 degrees C for 42 d) and to combined effect of cold and high-mountain hypoxia (2-5 degrees C temperature, 4000 m height, 40 d duration of the experiment). It was demonstrated that in adaptation to cold in normobaric conditions muscular fibres of the diaphragm develop moderate hypertrophy of mitochondria, accumulation of lipid inclusions with glycogen content remaining close control level. In combined effect of cold and high-mountain hypoxia direction of these changes was remained while their manifestation was increased. Relative volume of mitochondria was increased in hepatocytes in both experiments while volumetric indexes of rough endoplasmic reticulum and glycogen grew smaller. On one hand, the peculiarities of the dynamics of quantitative parameters of diaphragm muscular fibres and hepatocytes revealed in conditions of the long-term effect of the cold and in its combination with high mountain hypoxia indicate different efficiency of adaptive processes in the organ depending on the character of the effect and their organ-specificity on the other.