Genomic landscapes of canine splenic angiosarcoma (hemangiosarcoma) contain extensive heterogeneity within and between patients.

Cancer genomic heterogeneity presents significant challenges for understanding oncogenic processes and for cancer's clinical management. Variation in driver mutation frequency between patients with the same tumor type as well as within an individual patients' cancer can shape the use of mutations as diagnostic, prognostic, and predictive biomarkers. We have characterized genomic heterogeneity between and within canine splenic hemangiosarcoma (HSA), a common naturally occurring cancer in pet dogs that is similar to human angiosarcoma (AS). HSA is a clinically, physiologically, and genomically complex canine cancer that may serve as a valuable model for understanding the origin and clinical impact of cancer heterogeneity. We conducted a prospective collection of 52 splenic masses from 43 dogs (27 HSA, 15 benign masses, and 1 stromal sarcoma) presenting for emergency care with hemoperitoneum secondary to a ruptured splenic mass. Multi-platform genomic analysis included matched tumor/normal targeted sequencing panel and exome sequencing. We found candidate somatic cancer driver mutations in 14/27 (52%) HSAs. Among recurrent candidate driver mutations, TP53 was most commonly mutated (30%) followed by PIK3CA (15%), AKT1 (11%), and CDKN2AIP (11%). We also identified significant intratumoral genomic heterogeneity, consistent with a branched evolution model, through multi-region exome sequencing of three distinct tumor regions from selected primary splenic tumors. These data provide new perspectives on the genomic landscape of this veterinary cancer and suggest a cross-species value for using HSA in pet dogs as a naturally occurring model of intratumoral heterogeneity.

Identification of Genetic Susceptibility Factors Associated with Canine Gastric Dilatation-Volvulus.

Canine gastric dilatation-volvulus (GDV) is a common life-threatening condition occurring primarily in large and giant breeds with a 3.9% to 36.7% lifetime risk. The genetic correlates of GDV have not previously been systematically explored. We undertook an inter-breed genome-wide association analysis (GWAS) of 253 dogs from ten breeds including 106 healthy dogs and 147 dogs with at least one GDV episode. SNP array genotyping followed by imputation was conducted on 241 samples to identify GDV-associated single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). A subset of 33 dogs (15 healthy dogs and 18 GDV patients from the three most represented breeds) was characterized by whole genome sequencing (WGS). After genome-wide Bonferroni correction, we identified a significant putatively protective intergenic SNP (rs851737064) across all breeds. The signal was most significant in Collies, German Shorthaired Pointers, and Great Danes. Subsequent focused analysis across these three breeds identified 12 significant additional putatively protective or deleterious SNPs. Notable significant SNPs included those occurring in genes involved in gastric tone and motility including VHL, NALCN, and PRKCZ. These data provide important new clues to canine GDV risk factors and facilitate generation of hypotheses regarding the genetic and molecular underpinnings this syndrome.

Identification of Recurrent Activating HER2 Mutations in Primary Canine Pulmonary Adenocarcinoma.

PURPOSE: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC). EXPERIMENTAL DESIGN: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response. RESULTS: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2 V659E vs. IC50 > 2,500 nmol/L in HER2 WT). CONCLUSIONS: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer.

Longitudinal analysis of circulating tumor DNA (ctDNA) has shown promise for monitoring treatment response. However, most current methods lack adequate sensitivity for residual disease detection during or after completion of treatment in patients with nonmetastatic cancer. To address this gap and to improve sensitivity for minute quantities of residual tumor DNA in plasma, we have developed targeted digital sequencing (TARDIS) for multiplexed analysis of patient-specific cancer mutations. In reference samples, by simultaneously analyzing 8 to 16 known mutations, TARDIS achieved 91 and 53% sensitivity at mutant allele fractions (AFs) of 3 in 104 and 3 in 105, respectively, with 96% specificity, using input DNA equivalent to a single tube of blood. We successfully analyzed up to 115 mutations per patient in 80 plasma samples from 33 women with stage I to III breast cancer. Before treatment, TARDIS detected ctDNA in all patients with 0.11% median AF. After completion of neoadjuvant therapy, ctDNA concentrations were lower in patients who achieved pathological complete response (pathCR) compared to patients with residual disease (median AFs, 0.003 and 0.017%, respectively, P = 0.0057, AUC = 0.83). In addition, patients with pathCR showed a larger decrease in ctDNA concentrations during neoadjuvant therapy. These results demonstrate high accuracy for assessment of molecular response and residual disease during neoadjuvant therapy using ctDNA analysis. TARDIS has achieved up to 100-fold improvement beyond the current limit of ctDNA detection using clinically relevant blood volumes, demonstrating that personalized ctDNA tracking could enable individualized clinical management of patients with cancer treated with curative intent.

Canine osteosarcoma genome sequencing identifies recurrent mutations in DMD and the histone methyltransferase gene SETD2.

Osteosarcoma (OS) is a rare, metastatic, human adolescent cancer that also occurs in pet dogs. To define the genomic underpinnings of canine OS, we performed multi-platform analysis of OS tumors from 59 dogs, including whole genome sequencing (n = 24) and whole exome sequencing (WES; n = 13) of primary tumors and matched normal tissue, WES (n = 10) of matched primary/metastatic/normal samples and RNA sequencing (n = 54) of primary tumors. We found that canine OS recapitulates features of human OS including low point mutation burden (median 1.98 per Mb) with a trend towards higher burden in metastases, high structural complexity, frequent TP53 (71%), PI3K pathway (37%), and MAPK pathway mutations (17%), and low expression of immune-associated genes. We also identified novel features of canine OS including putatively inactivating somatic SETD2 (42%) and DMD (50%) aberrations. These findings set the stage for understanding OS development in dogs and humans, and establish genomic contexts for future comparative analyses.

Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine malignant melanoma by integrated comparative genomic analysis.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Somatic HLA Mutations Expose the Role of Class I-Mediated Autoimmunity in Aplastic Anemia and its Clonal Complications.

Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by Human Leukocyte Antigen (HLA)-restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole exome sequencing (WES), we recently identified two aAA patients with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the Major Histocompatibility Complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping we screened 66 aAA patients for somatic HLA class I loss. We found somatic HLA loss in eleven patients (17%), with thirteen loss-of-function mutations in HLA-A*33:03, HLA-A*68:01, HLA-B*14:02 and HLA-B*40:02 alleles. Three patients had more than one mutation targeting the same HLA allele. Interestingly, HLA-B*14:02 and HLA-B*40:02 were significantly overrepresented in aAA patients, compared to ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA, and establishes a novel link between aAA patients' immunogenetics and clonal evolution.

Capturing tumor heterogeneity and clonal evolution in solid cancers using circulating tumor DNA analysis.

Circulating tumor DNA analysis has emerged as a potential noninvasive alternative to tissue biopsies for tumor genotyping in patients with metastatic cancer. This is particularly attractive in cases where tissue biopsies are contraindicated or repeat genotyping after progression on treatment is required. However, tissue and plasma analysis results are not always concordant and clinical interpretation of discordant results is not completely understood. Discordant results could arise due to analytical limits of assays used for tumor and plasma DNA analysis or due to low overall contribution of tumor-specific DNA in plasma. Once these factors are ruled out, tissue-plasma concordance and quantitative levels of somatic mutations in plasma can capture tumor heterogeneity. During longitudinal follow-up of patients, this feature can be leveraged to track subclonal evolution and to guide combination or sequential adaptive treatment. Here, we summarize recent results evaluating the opportunities and limitations of circulating tumor DNA analysis in the context of tumor heterogeneity and subclonal evolution in patients with advanced cancers.

Clonal hematopoiesis in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited telomeropathy most frequently caused by mutations in a number of genes all thought to be involved in telomere maintenance. The main causes of mortality in DC are bone marrow failure as well as malignancies including leukemias and solid tumors. The clinical picture including the degree of bone marrow failure is highly variable and factors that contribute to this variability are poorly understood. Based on the recent finding of frequent clonal hematopoiesis in related bone marrow failure syndromes, we hypothesized that somatic mutations may also occur in DC and may contribute at least in part to the variability in blood production. To evaluate for the presence of clonal hematopoiesis in DC, we used a combination of X-inactivation, comparative whole exome sequencing (WES) and single nucleotide polymorphism array (SNP-A) analyses. We found that clonal hematopoiesis in DC is common, as suggested by skewed X-inactivation in 8 out of 9 female patients compared to 3 out of 10 controls, and by the finding of acquired copy neutral loss-of-heterozygosity on SNP-A analysis. In addition, 3 out of 6 independent DC patients were found to have acquired somatic changes in their bone marrow by WES, including a somatic reversion in DKC1, as well as missense mutations in other protein coding genes. Our results indicate that clonal hematopoiesis is a common feature of DC, and suggest that such somatic changes, though commonly expected to indicate malignancy, may lead to improved blood cell production or stem cell survival. Am. J. Hematol. 91:1227-1233, 2016. © 2016 Wiley Periodicals, Inc.

Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia.

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies.

Single nucleotide polymorphism array analysis of bone marrow failure patients reveals characteristic patterns of genetic changes.

The bone marrow failure syndromes (BMFS) are a heterogeneous group of rare blood disorders characterized by inadequate haematopoiesis, clonal evolution, and increased risk of leukaemia. Single nucleotide polymorphism arrays (SNP-A) have been proposed as a tool for surveillance of clonal evolution in BMFS. To better understand the natural history of BMFS and to assess the clinical utility of SNP-A in these disorders, we analysed 124 SNP-A from a comprehensively characterized cohort of 91 patients at our BMFS centre. SNP-A were correlated with medical histories, haematopathology, cytogenetic and molecular data. To assess clonal evolution, longitudinal analysis of SNP-A was performed in 25 patients. We found that acquired copy number-neutral loss of heterozygosity (CN-LOH) was significantly more frequent in acquired aplastic anaemia (aAA) than in other BMFS (odds ratio 12·2, P < 0·01). Homozygosity by descent was most common in congenital BMFS, frequently unmasking autosomal recessive mutations. Copy number variants (CNVs) were frequently polymorphic, and we identified CNVs enriched in neutropenia and aAA. Our results suggest that acquired CN-LOH is a general phenomenon in aAA that is probably mechanistically and prognostically distinct from typical CN-LOH of myeloid malignancies. Our analysis of clinical utility of SNP-A shows the highest yield of detecting new clonal haematopoiesis at diagnosis and at relapse.

Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A-.

We describe a case of paroxysmal nocturnal hemoglobinuria (PNH) in a woman who is heterozygous for the glucose-6-phosphate dehydrogenase A-   ( G6PDA-) allele. PNH is associated with one or more clones of cells that lack complement inhibition due to loss of function somatic mutations in the PIGA gene.  PIGA encodes the enzyme phosphatidylinositol glycan anchor biosynthesis, class A, which catalyses the first step of glycosylphosphatidylinisotol ( GPI)  anchor synthesis. Two GPI anchored red cell surface antigens regulate complement lysis. G6PD catalyses the first step of the pentose phosphate pathway and enzyme variants, frequent in some populations have been selected because they confer resistance to malaria, are associated with hemolysis in the presence of oxidizing agents including several drugs. The patient had suffered a hemolytic attack after taking co-trimoxazole, a drug that precipitates hemolysis in G6PD deficient individuals. Since both G6PD and PIGA are X-linked we hypothesized that the PIGA mutation was on the X-chromosome carrying the G6PDA- allele. Investigations showed that in fact the PIGA mutation was on the X-chromosome carrying the normal G6PD B allele. We speculate that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells or that the patient may have had a PNH clone expressing G6PDA- at the time of the hemolytic episode.

Common polymorphic deletion of glutathione S-transferase theta predisposes to acquired aplastic anemia: Independent cohort and meta-analysis of 609 patients.

Acquired aplastic anemia (AA) is a rare life-threatening bone marrow failure syndrome, caused by autoimmune destruction of hematopoietic stem and progenitor cells. Epidemiologic studies suggest that environmental exposures and metabolic gene polymorphisms contribute to disease pathogenesis. Several case-control studies linked homozygous deletion of the glutathione S-transferase theta (GSTT1) gene to AA; however, the role of GSTT1 deletion remains controversial as other studies failed to confirm the association. We asked whether a more precise relationship between the GSTT1 null polymorphism and aplastic anemia could be defined using a meta-analysis of 609 aplastic anemia patients, including an independent cohort of 67 patients from our institution. We searched PubMed, Embase, and the Cochrane Database for studies evaluating the association between GSTT1 null genotype and development of AA. Seven studies, involving a total of 609 patients and 3,914 controls, fulfilled the eligibility criteria. Meta-analysis revealed a significant association of GSTT1 null genotype and AA, with an OR = 1.74 (95% CI 1.31-2.31, P < 0.0001). The effect was not driven by any one individual result, nor was there evidence of significant publication bias. The association between AA and GSTT1 deletion suggests a role of glutathione-conjugation in AA, possibly through protecting the hematopoietic compartment from endogenous metabolites or environmental exposures. We propose a model whereby protein adducts generated by reactive metabolites serve as neo-epitopes to trigger autoimmunity in aplastic anemia.

A family with Hoyeraal-Hreidarsson syndrome and four variants in two genes of the telomerase core complex.

We describe an African American family with Hoyeraal-Hreidarrson syndrome (HHS) in which 2 TERT mutations (causing P530L and A880T amino acid changes) and two in the DKC1 variants (G486R and A487A) were segregating. Both genes are associated with dyskeratosis congenita and HHS. It was important to determine the importance of these mutations in disease pathogenesis to counsel family members. From genetic analysis of family members, telomere length and X-inactivation studies we concluded that compound heterozygosity for the TERT mutations was the major cause of HHS and the DKC1 G486R variant is a rare African variant unlikely to cause disease.

Study of chromosomal region 5p13.1 in Crohn's disease, ulcerative colitis, and rheumatoid arthritis.

Chromosomal region 5p13 includes regulatory elements of the prostaglandin receptor EP4 (PTGER4) gene and is associated with inflammatory bowel disease (IBD) susceptibility. We aimed at corroborating the association of the PTGER4 risk variant in IBD. Given the proinflammatory activity of prostaglandin E(2) in rheumatoid arthritis (RA), the reduction in incidence and severity of collagen-induced arthritis observed in mice deficient in the prostaglandin receptor EP4, and a modest signal of association found in an RA genome-wide scan, we proposed to extend the investigation of this locus to RA patients. A total of 709 Crohn's disease (CD) patients, 662 ulcerative colitis (UC) patients, and 1369 control subjects were genotyped for rs17234657. This polymorphism was also analyzed in 605 RA patients, and rs6871834 was studied in the RA patient group. Replication of the previous finding in CD was achieved in our independent collections, although with a milder effect (odds ratios = 1.23) than that originally described. No further association of the previously mentioned polymorphisms was detected with either UC or RA patients. We validated this 5p13 signal as a genuine susceptibility factor for CD in Caucasian populations. Our data seem to rule out a major influence of these polymorphisms on UC or RA predisposition.

Evidence of epistasis between TNFRSF14 and TNFRSF6B polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE: Genetic variants located close to 2 genes codifying for members of the tumor necrosis factor receptor superfamily (TNFRSF), TNFRSF14 and TNFRSF6B, have recently been associated with rheumatoid arthritis (RA) and with inflammatory bowel disease susceptibility, respectively. The TNFRSF6B protein has been related to osteoclastic activity, apoptosis inhibition, and modulation of T cell activation and differentiation. Interestingly, peptides encoded by both genes bind a common ligand called LIGHT, which is overexpressed in RA synovium. The aim of this study was to investigate the combined effect of the TNFRSF14 rs6684865 and TNFRSF6B rs4809330 polymorphisms in RA predisposition. METHODS: TaqMan genotyping of these polymorphisms was conducted in 649 patients with RA and 553 ethnically matched control subjects (first study). To validate the results, an independent replication cohort with 211 patients and 255 control subjects was additionally studied (replication study). RESULTS: The frequency of the rs6684865 G allele in the RA subgroup with the rs4809330 GG susceptibility genotype was significantly higher than that in the other patients with RA (74% versus 65%; P = 0.002) or in control subjects (74% versus 67%; P = 0.003). Because no significant differences between the control and patient groups in the first and replication studies were observed, the data were pooled. When compared with control subjects overall, the effect of the rs6684865 G allele in the group with the rs4809330 GG genotype (odds ratio [OR] 1.49) was significantly different from the effect observed in the group carrying the rs4809330 A allele (OR 0.97; P = 0.0015 by Breslow-Day test of homogeneity). CONCLUSION: We have identified and replicated a novel gene-gene interaction between 2 polymorphisms of TNFRSF members in Spanish patients with RA, based on the hypothesis of shared pathogenic pathways in complex diseases.

6q23 polymorphisms in rheumatoid arthritis Spanish patients.

OBJECTIVE: The aim of this work is to replicate the role of two recently described RA genetic markers (rs10499194 and rs6920220) situated at 6q23 in the autoantibody-positive phenotype. METHODS: A case-control study (630 RA patients and 664 healthy blood donors, all white Spaniards) was performed with two single nucleotide polymorphisms (rs6920220 and rs10499194) situated at 6q23. Genotyping was performed by TaqMan technology; autoantibody-stratified analyses in RA patients were also undertaken to replicate the previously reported effect of these polymorphisms. RESULTS: No association was observed for rs10499194 even after autoantibody stratification. The minor allele frequency of rs6920220 was higher in anti-CCP or RF-positive patients than in controls (P = 0.014 and P = 0.015 respectively), thus replicating previous findings. CONCLUSIONS: Our data replicate the association of rs6920220 with autoantibody-positive RA disease, although not for rs10499194.