Precision oncology in AML: validation of the prognostic value of the knowledge bank approach and suggestions for improvement

Recently, a novel knowledge bank (KB) approach to predict outcomes of individual patients with acute myeloid leukemia (AML) was developed using unbiased machine learning. To validate its prognostic value, we analyzed 1612 adults with de novo AML treated on Cancer and Leukemia Group B front-line trials who had pretreatment clinical, cytogenetics, and mutation data on 81 leukemia/cancer-associated genes available. We used receiver operating characteristic (ROC) curves and the area under the curve (AUC) to evaluate the predictive values of the KB algorithm and other risk classifications. The KB algorithm predicted 3-year overall survival (OS) probability in the entire patient cohort (AUCKB = 0.799), and both younger (< 60 years) (AUCKB = 0.747) and older patients (AUCKB = 0.770). The KB algorithm predicted non-remission death (AUCKB = 0.860) well but was less accurate in predicting relapse death (AUCKB = 0.695) and death in first complete remission (AUCKB = 0.603). The KB algorithm’s 3-year OS predictive value was higher than that of the 2017 European LeukemiaNet (ELN) classification (AUC2017ELN = 0.707, p < 0.001) and 2010 ELN classification (AUC2010ELN = 0.721, p < 0.001) but did not differ significantly from that of the 17-gene stemness score (AUC17-gene = 0.732, p = 0.10). Analysis of additional cytogenetic and molecular markers not included in the KB algorithm revealed that taking into account atypical complex karyotype, infrequent recurrent balanced chromosome rearrangements and mutational status of the SAMHD1, AXL and NOTCH1 genes may improve the KB algorithm. We conclude that the KB algorithm has a high predictive value that is higher than those of the 2017 and 2010 ELN classifications. Inclusion of additional genetic features might refine the KB algorithm.

Classical, Molecular, and Genomic Cytogenetics of the Pig, a Clinical Perspective

The chromosomes of the domestic pig (Sus scrofa domesticus) are known to be prone to reciprocal chromosome translocations and other balanced chromosome rearrangements with concomitant fertility impairment of carriers. In response to the remarkable prevalence of chromosome rearrangements in swine herds, clinical cytogenetics laboratories have been established in several countries in order to screen young boars for chromosome rearrangements prior to service. At present, clinical cytogenetics laboratories typically apply classical cytogenetics techniques such as giemsa-trypsin (GTG)-banding to produce high-quality karyotypes and reveal large-scale chromosome ectopic exchanges. Further refinements to clinical cytogenetics practices have led to the implementation of molecular cytogenetics techniques such as fluorescent in-situ hybridization (FISH), allowing for rearrangements to be visualized and breakpoints refined using fluorescently labelled painting probes. The next-generation of clinical cytogenetics include the implementation of DNA microarrays, and next-generation sequencing (NGS) technologies such as DNA sequencing to better explore tentative genome architecture changes. The implementation of these cytogenomics techniques allow the genomes of rearrangement carriers to be deciphered at the highest resolution, allowing rearrangements to be detected; breakpoints to be delineated; and, most importantly, potential gene implications of those chromosome rearrangements to be interrogated. Clinical cytogenetics has become an integral tool in the livestock industry, identifying rearrangements and allowing breeders to make informed breeding decisions.

Whole-genome mate-pair sequencing of apparently balanced chromosome rearrangements reveals complex structural variations: two case studies

BACKGROUND: Apparently balanced chromosome rearrangements (ABCRs) in non-affected individuals are well-known to possess high reproductive risks such as infertility, abnormal offspring, and pregnancy loss. However, caution should be exercised in genetic counseling and reproductive intervention because cryptic unbalanced defects and genome structural variations beyond the resolution of routine cytogenetics may not be detected. CASE PRESENTATION: Here, we studied two familial cases of ABCRs were recruited in this study. In family 1, the couple suffered two abortions pregnancies and underwent labor induction. Single nucleotide polymorphism (SNP) array analysis of the aborted sample from the second pregnancy revealed a 10.8 Mb heterozygous deletion at 10q26.13q26.3 and a 5.5 Mb duplication at 19q13.41-q13.43. The non-affected father was identified as a carrier of three-way complex chromosomal rearrangement [t(6;10;19)(p22;q26;q13)] by karyotyping. Whole-genome mate-pair sequencing revealed a cryptic breakpoint on the derivative chromosome 19 (der19), indicating that the karyotype was a more complex structural rearrangement comprising four breakpoints. Three genes, FAM24B, CACNG8, and KIAA0556, were disrupted without causing any abnormal phenotype in the carrier. In family 2, the couple suffered from a spontaneous miscarriage. This family had an affected child with multiple congenital deformities and an unbalanced karyotype, 46,XY,der(11)t(6;11)(q13;p11.2). The female partner was identified as a balanced translocation carrier with the karyotype 46,XX,t(6;11)(q13;p11.2)dn. Further SNP array and fluorescent in situ hybridization (FISH) indicated a cryptic insertion between chromosome 6 and chromosome 11. Finally, whole-genome mate-pair sequencing revealed an extremely complex genomic structural variation, including a cryptic deletion and 12 breakpoints on chromosome 11, and 1 breakpoint on chromosome 6 .CONCLUSIONS: Our study investigated two rare cases of ABCRs and demonstrated the efficacy of whole-genome mate-pair sequencing in analyzing the genome complex structural variation. In case of ABCRs detected by conventional cytogenetic techniques, whole genome sequencing (WGS) based approaches should be considered for accurate diagnosis, effective genetic counseling, and correct reproductive intervention to avoid recurrence risks.

Whole-genome mate-pair sequencing of apparently balanced chromosome rearrangements reveals complex structural variations: two case studies

BACKGROUND: Apparently balanced chromosome rearrangements (ABCRs) in non-affected individuals are well-known to have high reproductive risks such as infertility, abnormal offspring, and pregnancy loss. However, caution should be exercised in genetic counseling and reproductive intervention because cryptic unbalanced defects and genome structural variations beyond the resolution of routine cytogenetics may get omitted.CASE PRESENTATION: Two cases of ABCRs were recruited in this study. In family 1, the couple suffered two terminated pregnancies and underwent labor induction. Single nucleotide polymorphism (SNP) array analysis of the aborted sample from the second pregnancy showed a 10.8 Mb heterozygous deletion at 10q26.13q26.3 and a 5.5 Mb duplication at 19q13.41-q13.43. The non-affected father was diagnosed as a carrier of three-way complex chromosomal rearrangement [t(6;10;19)(p22;q26;q13)] by karyotyping. Whole-genome mate-pair sequencing revealed a cryptic breakpoint on derivative (der) chromosome 19 indicating that the karyotype was a more complex structural rearrangement including four breakpoints. Three genes, FAM24B, CACNG8, and KIAA0556, were disrupted without causing any abnormal phenotype in the carrier. In family 2, the couple suffered from a spontaneous miscarriage. They had an affected child with multiple congenital malformations and an unbalanced karyotype, 46,XY,der(11)t(6;11)(q13;p11.2). The female partner was considered a balanced translocation carrier with the karyotype 46,XX,t(6;11)(q13;p11.2)dn. Further SNP array and fluorescent in situ hybridization (FISH) indicated a cryptic insertion between chromosome 6 and chromosome 11. Finally, whole-genome mate-pair sequencing revealed an extremely complex genomic structural variation, including a cryptic deletion and 12 breakpoints on derivative chromosome 11[der(11)], and 1 breakpoint on derivative chromosome 6 [der(6)].CONCLUSIONS: Our study investigated two rare cases of ABCRs and demonstrated that whole-genome mate-pair sequencing is a powerful approach to analyze the genome complex structural variation. In case of ABCRs detected by conventional cytogenetic techniques, whole genome sequencing (WGS) based approaches should be considered for accurate diagnosis, effective genetic counseling, and correct reproductive intervention to avoid recurrence risks.

Non-Random Distribution of Reciprocal Translocation Breakpoints in the Pig Genome

Balanced chromosome rearrangements are one of the main etiological factors contributing to hypoprolificacy in the domestic pig. Amongst domestic animals, the pig is considered to have the highest prevalence of chromosome rearrangements. To date over 200 unique chromosome rearrangements have been identified. The factors predisposing pigs to chromosome rearrangements, however, remain poorly understood. Nevertheless, here we provide empirical evidence which sustains the notion that there is a non-random distribution of chromosomal rearrangement breakpoints in the pig genome. We sought to establish if there are structural chromosome factors near which rearrangement breakpoints preferentially occur. The distribution of rearrangement breakpoints was analyzed across three level, chromosomes, chromosome arms, and cytogenetic GTG-bands (G-banding using trypsin and giemsa). The frequency of illegitimate exchanges (e.g., reciprocal translocations) between individual chromosomes and chromosome arms appeared to be independent of chromosome length and centromere position. Meanwhile chromosome breakpoints were overrepresented on some specific G-bands, defining chromosome hotspots for ectopic exchanges. Cytogenetic band level factors, such as the length of bands, chromatin density, and presence of fragile sites, were associated with the presence of translocation breakpoints. The characteristics of these bands were largely similar to that of hotspots in the human genome. Therefore, those hotspots are proposed as a starting point for future molecular analyses into the genomic landscape of porcine chromosome rearrangements.

Therapy-Related Myeloid Neoplasms with Balanced Chromosome Rearrangements Frequently Arise from Pre-Existing Clonal Haematopoiesis

Introduction Therapy-related myeloid neoplasms (tMN) are an increasing healthcare problem resulting from rising long-term survival from primary cancers. Approximately 80% of cases are associated with an adverse karyotype and have a dismal prognosis; pre-existing clonal haematopoiesis (CH) appears to be a predisposing factor and mutations in genes including TP53, PPM1D and DNMT3A have been detected prior to chemo- or radiotherapy exposure. In contrast ~20% of tMN are characterised by balanced chromosome rearrangements and have a relatively favourable outcome; chemotherapy agents targeting topoisomerase II have been implicated in generating these rearrangements however the involvement of CH has not previously been described. Methods and results We performed whole exome sequencing (WES) of samples taken at diagnosis (D) and molecular complete remission (mCR) followed by targeted capture and error-corrected deep sequencing (ECS) on ≥2 mCR samples for 11 patients with therapy-related acute promyelocytic leukemia (tAPL) and 20 patients with de novo APL (dnAPL). All patients had t(15;17) / PML-RARA at diagnosis with no additional cytogenetic abnormality. The median age of patients with tAPL was 46.7y (range 30-78); primary cancer types were breast (4), colorectal (2), lymphoma (3), CNS (1) and testicular (1). Ten patients had received chemotherapy and 4 radiotherapy. The median latency between primary cancer treatment and tAPL diagnosis was 3.9y (range 2.2-6.1). Patients received a mixture of chemotherapy- and arsenic-based treatments. The median age of patients with dnAPL was 40.3y (range 18-69); all patients were treated with the AIDA regimen and none developed tMN subsequently. There were no significant differences between the number or type of mutations between dnAPL and tAPL however disease associated somatic mutations were detectable in mCR samples by ECS in 4/11 tAPL samples compared to 0/20 dnAPL samples (p=0.04). Mutations detected in mCR were UPN1: PPM1D exon (e) 6 1bp deletion (del, variant allele fraction, VAF, D 32% mCR 11.3%); UPN2: DNMT3A e8 1bp del (VAF D 40.2% mCR 0.48%); UPN3: DNMT3A e10 1bp del (VAF D 35.4% mCR 2.9%); UPN4: MYCN e3 6bp insertion (ins, VAF D 38% mCR 0.37%). We screened samples from these patients and a further 39 dnAPL patients for a panel of genes with known CH associated mutations (CH-M) using ECS and detected additional mutations in 2 tAPL patients (UPN4, DNMT3A G104R, VAF D 0% mCR 3% and UPN5 DNMT3A R693H VAF D 2% mCR 25%, who subsequently developed tMN with complex karyotype). We did not detect CH-M in diagnostic samples from any patient with dnAPL and publicly available NGS datasets encompassing 220 patients only showed 1 APL case with a CH-M (DNMT3A e8 ins, prior cytotoxic exposure unknown). We detected treatment-emergent CH clones in mCR by ECS in 6/59 dnAPL patients treated with AIDA (DNMT3A n=3, PPM1D n=2, TP53 n=1, SF3B1 n=1). Applying the same techniques to six patients with therapy-related core-binding factor AML, we identified a persistent CH-M in mCR in one (DNMT3A e15 del, VAF D 38%, mCR 11.8%). DNA samples taken at the time of primary cancer diagnosis were available from UPN 1-3. In UPN1 we detected the PPM1D mutation in a lymph node (LN) involved with T-cell lymphoma (LN) (VAF 2.1%) and an uninvolved staging bone marrow (VAF 3.3%). In UPN3 the DNMT3A mutation was detected in a breast biopsy (VAF 0.7%) and LN involved with carcinoma (VAF 0.9%). In UPN2 the DNMT3A mutation was not detected in the LN biopsy diagnostic for Hodgkin lymphoma. We used FACS to isolate T, B, monocyte, granulocyte and CD34+ cells from complete remission samples from UPN1-4 with >99% purity and detected the persistent mutations in each cell compartment e.g. UPN3 VAF: T cell 1.3%, B cell 8.6%, monocyte 4%, granulocyte 3.7%, CD34+ 3.9%. Diagnostic material from UPN1 and 4 was injected into irradiated NSG mice. After 12 weeks we detected multilineage human engraftment in both samples by FACS in 1/3 mice from UPN1 and 2/3 mice from UPN4. We detected the PPM1D and MYCN mutations respectively in bone marrow samples from each engrafting mouse. Conclusions Together these findings indicate that tMN with balanced chromosome rearrangements can occur on a background of non-malignant CH. Using ECS we observed this phenomenon in 5/17 (29%) patients with therapy-related APL or CBF AML. This has important implications for planning curative therapy, notably for tAPL where effective cytotoxic-free regimens are available. Disclosures Russell: Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau; Daiichi Sankyo: Consultancy. Hills:Daiichi Sankyo: Consultancy, Honoraria.

Structural disruption of genomic regions containing ultraconserved elements is associated with neurodevelopmental phenotypes

The development of the human brain and nervous system can be affected by genetic or environmental factors. Here we focus on characterizing the genetic perturbations that accompany and may contribute to neurodevelopmental phenotypes. Specifically, we examine two types of structural variants, namely, copy number variation and balanced chromosome rearrangements, discovered in subjects with neurodevelopmental disorders and related phenotypes. We find that a feature uniting these types of genetic aberrations is a proximity to ultraconserved elements (UCEs), which are sequences that are perfectly conserved between the reference genomes of distantly related species. In particular, while UCEs are generally depleted from copy number variant regions in healthy individuals, they are, on the whole, enriched in genomic regions disrupted by copy number variants or breakpoints of balanced rearrangements in affected individuals. Additionally, while genes associated with neurodevelopmental disorders are enriched in UCEs, this does not account for the excess of UCEs either in copy number variants or close to the breakpoints of balanced rearrangements in affected individuals. Indeed, our data are consistent with some manifestations of neurodevelopmental disorders resulting from a disruption of genome integrity in the vicinity of UCEs.