Comprehensive Genomic and DNA Methylation Analysis in Twins with ETV6-RUNX1 Acute Lymphoblastic Leukemia

Abstract DNA methylation as a source for epigenetic variability has been implicated in a variety of different cancer types. Often these studies are confounded by inter-individual differences in the epigenetic profiles. The pattern of epigenetic marks can be altered by factors like age, nutrition, behavior or other environmental factors, which are difficult to control. We had the unique opportunity to study DNA methylation profiles in a pair of monozygotic twin boys who developed ETV6-RUNX1 B-progenitor acute lymphoblastic leukemia at 2 years of age within 3 weeks of each other. ETV6-RUNX1 ALL is characterized by a high frequency of recurring genetic alterations, but the full complement of genomic and epigenetic alterations contributing to leukemogenesis is unknown. For these twin cases, environmental influences upon epigenetic variation are largely eliminated. We used a mass spectrometry-based quantitative DNA methylation analysis technique (Sequenom’s® EpiTYPER™ application) to investigate 597 amplicons covering the promoter regions of 190 genes. The genomic target regions were selected to be enriched for genes involved in transcriptional regulation (n=130) and/or genes known to be targeted by recurring DNA copy number alterations in childhood leukemia (n= 60). Methylation analysis were performed on DNA extracted from cryopreserved, Ficoll enriched bone leukemic blasts obtained from diagnostic bone marrow aspirates, and non-leukemic peripheral blood leukocytes obtained at remission. We also examined DNA copy number alterations (CNAs) and loss-of- heterozygosity (LOH) using Affymetrix single nucleotide polymorphism (SNP) 6.0 arrays, which examine over 1.8 million loci, in both tumor and normal tissue for both twins. Analysis of SNP array data identified different somatic CNAs in the tumor samples of the two twins involving 9p21.3 (the CDKN2A/B tumor suppressor locus), 12p13.2 (ETV6) and trisomy 21, indicating that the shared ETV6-RUNX1 positive pre-leukemic clone acquired different secondary genetic alterations during leukemogenesis in each twin. Despite these genetic differences, the methylation profiles of the tumor samples were remarkably similar. Unsupervised two-dimensional clustering of quantitative methylation data revealed that the tumor samples clustered separately from the control samples. Based on these findings we calculated the methylation differences in each genomic target region. A total of 51 genomic regions were significantly differentially methylated between tumor and control samples (paired t-test P<0.001, and an average methylation difference > 10%). Within the differentially methylated genomic regions, a subset of approximately 20 exhibited strong regional differences, indicating that DNA methylation changes can be limited to certain areas of the promoter. In the group of genes known to be involved in transcriptional regulation, 32% were differentially methylated, including the HOXA, HOXB, HOXC and HOXD regions, while in the remaining genes only 15% were differentially methylated. This enrichment is significant on the level of 0.05 (Fisher’s exact test, odds ratio: 2.7). This represents the first study comparing genomic and epigenetic alterations in B-precursor ALL involving monozygotic twins. Notably, different DNA copy number alterations are acquired in each twin during leukemogeneis. In contrast, the tumor samples exhibit similar methylation patterns that are strikingly different to control samples obtained from the same individuals. These results indicate that combined genomic and epigenetic analyses will be important to characterize the full repertoire of genomic alterations in acute lymphoblastic leukemia.

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Identification of Novel Recurring Mutations in Relapsed Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.703.703 ◽

2009 ◽

Vol 114 (22) ◽

pp. 703-703

Author(s):

Charles G. Mullighan ◽

Jinghui Zhang ◽

Letha A Phillips ◽

Ching-Hon Pui ◽

James R. Downing

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Transcriptional Regulators ◽

Genetic Alterations ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Sequence Variations ◽

Dna Copy Number Alterations ◽

Risk Of Relapse

Abstract Abstract 703 Relapse occurs across the spectrum of cytogenetic subtypes of acute lymphoblastic leukemia (ALL), and the biologic factors influencing risk of relapse are poorly understood. Previous studies have demonstrated substantial evolution in the complement of DNA copy number alterations from diagnosis to relapse, with the majority of cases acquiring new lesions at relapse that are not present at diagnosis, and also losing lesions present in the predominant clone at diagnosis. Moreover, fingerprinting and backtracking of deletions indicate a common ancestral origin of the diagnosis and relapse clones in most cases, suggesting that multiple, genetically distinct clones are present at diagnosis, and that specific genetic alterations influence risk of relapse. At present, detailed examination of DNA sequence variations in relapsed ALL has not been performed. In this study, we have performed genomic resequencing of 238 genes in leukemic samples obtained at diagnosis and relapse in 23 childhood ALL cases. The cohort comprised cases with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), MLL-rearrangement (N=3), BCR-ABL1 (N=3), and cases with low hyperdiploid, pseudodiploid, normal, and miscellaneous karyotypes (N=10). All samples had over 80% blasts or were flow sorted to high purity prior to DNA extraction. Whole genome amplification of DNA was performed prior to sequencing. We selected genes targeted by recurring copy number alterations in ALL, genes in key pathways targeted by genetic alterations in ALL (e.g. lymphoid development, tumor suppression, cell cycle regulation and apoptosis), known cancer genes and tyrosine kinases. The complete coding region of each gene was sequenced in both diagnosis and relapse sample in all cases. Validation of putative variants was performed by sequencing of matched normal DNA. 248 putative protein changing variations were identified. After removal of variants also identified in matched normal DNA, 55 variants in 32 genes were identified in 20 cases (mean 2.5 variants per case, range 0-5). Eleven genes were mutated in multiple patients (ASMTL, CREBBP, ERG, FLT3, KRAS, NF1, NRAS, PAX5, PTPN11, TP53 and TUSC3). We identified tumor-acquired variations in genes previously known to be mutated in acute leukemia, including ETV6 (1 case) JAK1 (1), NRAS (5), KRAS (2), NF1 (3) PTPN11 (2), PAX5 (2), FBXW7 (1), and TP53 (2). In addition, we identified recurring mutations in genes not previously known to be mutated in cancer, including the transcriptional regulators CREBBP (N=4), NCOR1 (N=2), the tumor suppressor candidate gene TUSC3 (N=2), and the ETS family transcription factor ERG (N=2). Single mutations were also identified in transcriptional regulators (THADA, SPI1 (PU.1), TCF4, TCF7L2), the histone gene HIST1H2BG, and additional genes also targeted by copy number alterations in ALL (ARMC2, ATP10A, PLEKHG1, STIM2). The patterns of evolution of sequence variations between diagnosis and relapse were similar to those previously reported for DNA copy number alterations. Four patients had identical sequence mutations at diagnosis and relapse. Twelve cases had the some sequence variations identified at both diagnosis and relapse, but either acquired additional mutations at relapse (N=9), lost mutations present at diagnosis (N=2), or both acquired new lesions at relapse and lost variants at diagnosis (N=1). An additional three cases had variants detected at either diagnosis or relapse with no commonality between the two samples. Notably, eight (35%) cases had lesions resulting in constitutive RAS activation at diagnosis or relapse (NRAS in 3 cases, FLT3 in 2, PTPN11 in 2, NF1 in 2, and KRAS in 1), with 5 cases harboring mutations at diagnosis only (NF1 in 2 cases, NRAS in 2, and FLT3 in 1), and three cases harboring mutations at relapse only (FLT1, PTPN11 and NRAS 1 case each). In several cases, relapse-acquired mutations were identified as minor subclones at diagnosis. These data have identified novel targets of somatic mutation in ALL, and suggest that sequence variation is important determinant of risk of relapse. Identification of mutations in multiple cases suggests that several of these variants are driver mutations. These findings also indicate that resequencing of the entire coding genome of relapsed ALL will be essential to identify all lesions influencing response to therapy. Disclosures: No relevant conflicts of interest to declare.

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Tiling-resolution array-CGH reveals the pattern of DNA copy number alterations in acute lymphoblastic leukemia with 21q amplification: the result of telomere dysfunction and breakage/fusion/breakage cycles?

Leukemia ◽

10.1038/sj.leu.2404628 ◽

2007 ◽

Vol 21 (6) ◽

pp. 1327-1330 ◽

Cited By ~ 20

Author(s):

E Kuchinskaya ◽

A Nordgren ◽

M Heyman ◽

J Schoumans ◽

M Corcoran ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Array Cgh ◽

Lymphoblastic Leukemia ◽

Telomere Dysfunction ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Dna Copy Number Alterations

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Identification of distinct protein Signatures Associated with genetic Abnormalities In Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.1313.1313 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1313-1313

Author(s):

Roland P. Kuiper ◽

Blanca Scheijen ◽

Agata Pastorczak ◽

Simon V. van Reijmersdal ◽

Deborah A. Thomas ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Protein Expression ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Chromosomal Translocation ◽

Genetic Alterations ◽

Chromosomal Translocations ◽

Integrated Analysis ◽

Copy Number Alterations ◽

Protein Signatures

Abstract Background Treatment outcome in acute lymphoblastic leukemia (ALL) has improved over the past 30 years, with overall survival rates of ∼45% in adults and ∼85% in children. Gross cytogenetic abnormalities, including numerical changes and chromosomal translocations, are of considerable prognostic value in both pediatric and adult ALL. In addition, we and others have recently identified novel molecular markers associated with a poor outcome in ALL, including deletions of the lymphoid transcription factor IKZF1. In order to identify downstream signaling events associated with these genetic alterations, we performed an integrated analysis of genomic abnormalities, including copy number alterations, sequence mutations and chromosomal translocations, with alterations in protein expression and modification. Methods A cohort of 91 precursor B-ALL cases treated at M.D. Anderson Cancer Center in Houston, USA, including 82 newly diagnosed cases and 5 diagnosis-relapse pairs was used for this study. The cohort consisted of 6 children (age 1-6), 30 young adults (age 15-39) and 45 adults (age>39), and 20 patients carried a BCR-ABL1 chromosomal translocation. Copy number alterations in eight genes frequently deleted in ALL (IKZF1, PAX5, EBF1, RB1, CRLF2, CDKN2A/2B, BTG1, and ETV6) were determined by multiplex ligation-dependent probe amplification analysis. IKZF1 deletions were associated with relapse (Pearson's chi-square test, p=0.009), and the presence of BCR-ABL1 translocation (p=0.032). Protein expression and modification levels were determined by probing Reverse Phase Protein Arrays (RPPA) containing protein lysates of all above samples with 128 rigorously validated antibodies including 34 phospho-specific antibodies. Hierarchical clustering analysis was used to determine which (phospho)proteins are differently expressed in genetic subsets of ALL. The significance of correlations was determined using two-sample t-test, with correction for multiple testing (Beta-Uniform Mixture model). Results We identified clustering of cases with a BCR-ABL1 chromosomal translocation (p=0.01; false discovery rate (FDR)=0.1), IKZF1-deletions (p=0.01, FDR=0.072), RB1-deletions (p=0.03, FDR=0.43) and EBF1 deletions (p=0.05, FDR=0.63). As expected RB1 deletion positive cases were characterized by decreased levels of (phospho)-RB1 and increased levels of cyclin E, illustrating the validity of our approach. EBF1-deleted cases showed relatively high levels of SHIP1, SSBP2 and phospho-STAT5, and lower levels of FAK and LYN. The protein signatures of BCR-ABL1-positive cases and IKZF1-deletion positive cases largely overlapped, and were characterized by elevated levels of (phospho)PKCα, SMAD1, phospho-STAT3, and phospho-STAT5 and lower levels of LYN and cyclinD3 (Figure 1). In total 70% of the BCR-ABL1-positive cases carried an IKZF1 deletion and several BCR-ABL1-negative cases with similar RPPA signature could be identified, all of which were IKZF1-deletion positive. These cases may represent the “BCR-ABL1-like” cases that were previously identified using gene expression signatures (Mullighan et al. 2009, NEJM 360:470-480; Den Boer et al. 2009, Lancet Oncol. 10:125-134), and could reflect activation of cAbl or other cellular tyrosine kinases. Together, we conclude that integrated analysis of genetic and proteomic aberrations identified protein signatures downstream of recurrent mutational events in ALL, a strategy that promises to facilitate the discovery of novel therapeutic targets in ALL and may aid in the identification of (high risk) patients that would benefit from tyrosine kinase inhibition. Disclosures: No relevant conflicts of interest to declare.

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Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma

Haematologica ◽

10.3324/haematol.2020.274258 ◽

2021 ◽

Author(s):

Man Chun John Ma ◽

Saber Tadros ◽

Alyssa Bouska ◽

Tayla Heavican ◽

Haopeng Yang ◽

...

Keyword(s):

B Cell ◽

Copy Number ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Genetic Alterations ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Cross Sectional ◽

Disease Etiology ◽

Dna Copy Number Alterations

B-cell non-Hodgkin’s lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

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Hidden DNA Copy Number Alterations and Mutations in IKZF1, TP53, CRLF2 and JAK2 Genes Are Associated with a Poor Prognosis in B-Progenitor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.1082.1082 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1082-1082

Author(s):

Jesús María Hernández-Rivas ◽

Maribel Forero ◽

Cristina Robledo ◽

Rocio Benito ◽

María Hernández ◽

...

Keyword(s):

Overall Survival ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Integrative Analysis ◽

Dna Copy Number ◽

Copy Number Aberrations ◽

Dna Copy Number Aberrations

Abstract Background: In B-progenitor acute lymphoblastic leukemia (B-ALL) the identification of additional genetic alterations associated with treatment failure is still a challenge. Aims: 1.To identify genomic gains and losses in B-ALL at the time of diagnosis and to correlate these abnormalities with the genetic characteristics and the patients outcome. 2.To assess the prevalence and prognostic impact of genetic lesions in IKZF1, TP53, CRLF2, IL7R, PAX5, JAK2 and LEF1 genes in B-ALL. Methods: A total of 215 B-ALL patients were eligible for this study. 115 (53.5%) had less than <18 years. In all cases oligonucleotide microarrays (aCGH) were carried out. For genetic gain and losses analysis, the NimbleGen CGH 12x135K array (Roche) was used. The data analysis was performed with GISTIC and aCGHweb software. In 118 of these cases (65 children and 53 adults), an integrative analysis of aCGH and NGS was performed to identify genetic lesions in genes associated with B-ALL: JAK2 (Exon 12-Exon 16), PAX5 (E2-E3), LEF1 (E2-E3), CRLF2 (E6), IL7R (E5), IKZF1 (E2-E8) and TP53 (E4-E11). The 454 GS Junior system (Roche) was used. The variant analysis was performed using Amplicon Variant Analyzer (AVA-Roche 454) and Sequence Pilot (JSI Medical Systems) software. Results: DNA copy number aberrations (CNAs) were observed in 96.5% of cases. Gains on chromosomes 4 (10%), 6 (10%), 10 (12%), 14 (13%), 21 (31%) and X (18%) were more common in children, whereas losses in 7p (13%) and 9p21 (24%) were frequent in adults. In children, gains on chromosomes 6, 10, 14q, 17, 18, 21 and X were associated with longer overall survival (OS) while loss on 7p and 17p were associated with shorter OS in adults (Table 1). The integrative analysis of aCGH and NGS showed that 60% of patients carried at least one alteration (deletion and/or mutation) in the seven genes analyzed. Focal deletions were common in IKZF1, CRLF2, PAX5, and LEF1 genes while broad deletions were more frequent in TP53, JAK2 and IL7R. Forty-one percent of patients harbored IKZF1 alterations (IKZF1+), 22.9% in PAX5, 18.6% in JAK2 and 11% had TP53 or CRLF2 alterations. LEF1 and IL7R genomic lesions were only present in 4.2% and 2.5% of the B-ALL, respectively. JAK2 and CRLF2 mutations were associated (p=0.01). Moreover abnormalities in IKZF1+ were associated with alterations in JAK2 (p=0.004), TP53 (p=0.04) and PAX5 (p=0.03). The presence of alterations was most frequent in high-risk (HR) B-ALL (38% vs 18%, p=0.02), although 8% low-risk (LR) childhood patients showed genetic alterations. Fourteen B-ALL patients carried mutations in TP53, CRLF2 and/or JAK2 genes: all of them were Ph-, and 11 were classified as HR. Of note, 3 cases were children. In the HR group the presence of mutation in TP53, CRLF2 and/or JAK2 was related to a shorter OS (3-year OS: 56% vs. 36%; P=0.034) and event-free survival (3-year EFS: 56% vs. 11%; P<0.002) (Figure 1), due to an increased cumulative incidence of relapse (3-year CIR: 38% vs. 88%; P<0.004). The presence of IKZF1 alterations stratified HR-Ph- B-ALL cases. Thus HR-Ph- patients with normal IKZF1 were associated with longer EFS compared to HR-Ph- B-ALL IKZF1+ (3-year EFS: 60% vs. 33%; P=0.005) (Figure 2). The presence of IKZF1 deletion was associated with shorter EFS (89% vs. 67%, P= 0.04) and increased CIR (3-year CIR: 12% vs. 33%; P=0.05) in children. Summary/Conclusions: CNAs are frequent in B-ALL and are associated with genetic subtype, age and overall survival. The integrative analysis by aCGH and NGS techniques demonstrated that alterations in IKZF1 gene and mutations in TP53, CRLF2 and/or JAK2 genes could stratify high-risk Ph- B-ALL patients. Subvention:FP7/2007-13, Nº306242-NGS-PTL; HUS272U13, JCyL,Consejería de Educación; BIO/SA31/13 Gerencia Regional de Salud, SACYL, Spain Table 1. DNA copy number aberrations (CNAs) associated with overall survival in 215 B-ALL patients Association CNA n Median (months) P value Short overall survival 7p-ChildrenAdults 19613 9615 0.05 17p-ChildrenAdults 1257 694 0.029 +19ChildrenAdults 351421 Not reached10 0.001 22q+ChildrenAdults 261214 Not reached7 <0.0001 Longoverall survival +6ChildrenAdults 26197 Not reached7 <0.0001 +10ChildrenAdults 25196 Not reached7 0.001 +14qChildrenAdults 26188 Not reached4 <0.0001 +17ChildrenAdults 24177 Not reached60 0.012 +18ChildrenAdults 25187 Not reachedNot reached <0.0001 21q+ChildrenAdults 473116 Not reached8 <0.0001 XChildrenAdults 522725 Not reached9 <0.0001 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Multivariate Boosting for Integrative Analysis of High-Dimensional Cancer Genomic Data

Cancer Informatics ◽

10.4137/cin.s16353 ◽

2014 ◽

Vol 13s7 ◽

pp. CIN.S16353

Author(s):

Lie Xiong ◽

Pei-Fen Kuan ◽

Jianan Tian ◽

Sunduz Keles ◽

Sijian Wang

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Copy Number ◽

Regression Models ◽

Genomic Data ◽

High Dimensional ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Boosting Method ◽

Dna Copy Number Alterations

In this paper, we propose a novel multivariate component-wise boosting method for fitting multivariate response regression models under the high-dimension, low sample size setting. Our method is motivated by modeling the association among different biological molecules based on multiple types of high-dimensional genomic data. Particularly, we are interested in two applications: studying the influence of DNA copy number alterations on RNA transcript levels and investigating the association between DNA methylation and gene expression. For this purpose, we model the dependence of the RNA expression levels on DNA copy number alterations and the dependence of gene expression on DNA methylation through multivariate regression models and utilize boosting-type method to handle the high dimensionality as well as model the possible nonlinear associations. The performance of the proposed method is demonstrated through simulation studies. Finally, our multivariate boosting method is applied to two breast cancer studies.

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Sex-dependent differences in DNA copy number alterations in hepatocellular carcinoma

Academic Journal of Second Military Medical University ◽

10.3724/sp.j.1008.2012.00005 ◽

2012 ◽

Vol 32 (1) ◽

pp. 5-9 ◽

Cited By ~ 1

Author(s):

Bing-ji WEN ◽

Wen-ming CONG ◽

Ai-zhong WANG ◽

Song-qin HE ◽

Hong-mei JIANG ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Copy Number ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Dna Copy Number Alterations

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From Benign Inflammatory Dermatosis to Cutaneous Lymphoma. DNA Copy Number Imbalances in Mycosis Fungoides versus Large Plaque Parapsoriasis

Medicina ◽

10.3390/medicina57050502 ◽

2021 ◽

Vol 57 (5) ◽

pp. 502

Author(s):

Georgiana Gug ◽

Caius Solovan

Keyword(s):

Mycosis Fungoides ◽

Copy Number ◽

Snp Array ◽

Chromosome 1 ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Large Plaque ◽

Inflammatory Dermatosis ◽

Fresh Frozen ◽

Dna Copy Number Alterations

Background and Objectives: Mycosis fungoides (MF) and large plaque parapsoriasis (LPP) evolution provide intriguing data and are the cause of numerous debates. The diagnosis of MF and LPP is associated with confusion and imprecise definition. Copy number alterations (CNAs) may play an essential role in the genesis of cancer out of genes expression dysregulation. Objectives: Due to the heterogeneity of MF and LPP and the scarcity of the cases, there are an exceedingly small number of studies that have identified molecular changes in these pathologies. We aim to identify and compare DNA copy number alterations and gene expression changes between MF and LPP to highlight the similarities and the differences between these pathologies. Materials and Methods: The patients were prospectively selected from University Clinic of Dermatology and Venereology Timișoara, Romania. From fresh frozen skin biopsies, we extracted DNA using single nucleotide polymorphism (SNP) data. The use of SNP array for copy number profiling is a promising approach for genome-wide analysis. Results: After reviewing each group, we observed that the histograms generated for chromosome 1–22 were remarkably similar and had a lot of CNAs in common, but also significant differences were seen. Conclusions: This study took a step forward in finding out the differences and similarities between MF and LPP, for a more specific and implicitly correct approach of the case. The similarity between these two pathologies in terms of CNAs is striking, emphasizing once again the difficulty of approaching and differentiating them.

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