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 10
and 3 in 10
, 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,
= 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 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 (
= 24) and whole exome sequencing (WES;
= 13) of primary tumors and matched normal tissue, WES (
= 10) of matched primary/metastatic/normal samples and RNA sequencing (
= 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
(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
(42%) and
(50%) aberrations. These findings set the stage for understanding OS development in dogs and humans, and establish genomic contexts for future comparative analyses.
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.
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 2 patients with aAA 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 patients with aAA for somatic HLA class I loss. We found somatic HLA loss in 11 patients (17%), with 13 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 1 mutation targeting the same HLA allele. Interestingly, HLA-B*14:02 and HLA-B*40:02 were significantly overrepresented in patients with aAA compared with 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 immunogenetics and clonal evolution of patients with aAA.
•Somatic HLA class I gene mutations are frequent in aAA and define HLA class I restricted autoimmunity in aAA.•HLA alleles targeted by inactivating mutations are overrepresented in aAA and correlate with poor therapy response and clonal evolution.
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.
Summary
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.
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.
Microalgae contributed 60% of the total biomass in the extremely hostile (pH 2 and metal-rich waters) environment of Rio Tinto (which is used as a model for the astrobiology of Mars). These algae are ...closely related to nonextreme lineages, suggesting that adaptation to Rio Tinto water (RTW) must occur rapidly. Fitness from both the microalga Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa was inhibited when they were cultured in RTW. After further incubation for several weeks, D. chlorelloides survived, as a result of the growth of a variant that was resistant to RTW, but RTW-resistant cells did not appear in M. aeruginosa. A Luria-Delbrück fluctuation test revealed that D. chlorelloides RTW-resistant cells arose randomly by rare spontaneous mutations before the RTW exposure (1.38 x 10⁻⁶ mutants per cell division). The mutants with a diminished fitness are maintained in nonextreme waters as the result of a balance between new RTW-resistant cells arising by mutation and RTW-resistant mutants eliminated by natural selection (equilibrium at c. 15 RTW-resistant per 10⁷ wild-type cells). Rapid adaptation of eukaryotic algae to RTW could be the result of selection of RTW-resistant mutants occurring spontaneously in nonextreme populations that arrived fortuitously at the river in the past, or in the present continuously.
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