DNA Sequence of the Cancer Genome of a Patient with Therapy-Related Acute Myeloid Leukemia
Authors: Schuettpelz Laura , Link Daniel C. , Shen Dong , Walter Matthew J. , Koboldt Daniel C , Dooling David J. , Fulton Robert S. , Schmidt Heather , Maupin Rachel , O'Laughlin Michelle , Chen Ken , McLellan Michael D. , Kulkarni Shashikant , Ivanovich Jennifer , Lebeau Michelle , Varghese Nobish , Nagarajan Rakesh , Ding Li , Graubert Timothy A. , Ley Timothy J , Wilson Richard K. , Mardis Elaine R. 
Published: October 2019

Abstract
Abstract 580
Therapy-related acute myeloid leukemia/myelodysplasia (t-AML/t-MDS) accounts for 10–20% of all cases of AML, and its incidence is rising. Treatment options are limited and the prognosis very poor, highlighting the need for new therapies in t-AML/t-MDS. However, the genetic mutations contributing to transformation in t-AML/t-MDS are largely unknown, limiting the development of novel targeted therapeutics. Our group previously reported the sequence of the first two cancer genomes, both in patients with de novo AML (Nature 456:66, 2008; NEJM 361:1058, 2009). Herein, we report the sequence of the cancer genome of a patient with t-AML. The patient presented with early-onset breast, then ovarian cancer (age <40), and was treated with surgery, radiation and combination chemotherapy (cytoxan, etoposide, adriamycin, carboplatinum and taxol). Clinical sequencing of BRCA1 and BRCA2 revealed no mutations. Four years later, recurrence of her ovarian cancer was detected and she again was treated with chemotherapy. Two months after completing this chemotherapy, she presented with t-AML and respiratory failure, and she died 8 days after presentation. Typical of t-AML, the karyotype of this leukemia was complex, with -7, del(5q), and several marker chromosomes that could not be resolved with standard cytogenetic analysis. Bone marrow and a skin biopsy were obtained after informed consent and analyzed in the following ways: 1) whole genome sequencing of leukemic bone marrow and skin DNA on the Illumina platform using paired end reads with an average read length of 75 bp; 2) SNP genotyping on the Affymetrix 6.0 array (on leukemic and skin DNA) to detect copy number alterations and uniparental disomy; 3) RNA expression profiling using the Affymetrix Exon 1.0 array; 4) spectral karyotyping. For the leukemic sample, a total of 115 Gb of sequence was obtained (28.7X haploid coverage). Based on SNP genotyping, >96% of heterozygous SNPs were detected. Similar data were obtained for the skin sample.
A total of 27 validated somatic single nucleotide variants or indels were detected in coding sequences. None of these mutations have been previously reported in de novo AML. Eight novel chromosomal translocations were identified and the breakpoints defined. One translocation, t(3;4)(q27.3;p15.32), resulted in the production of an in frame fusion transcript of DGKG (diacylglycerol kinase gamma) with BST1 (bone marrow cell stromal antigen 1). Studies are underway to characterize the effect of this fusion gene on hematopoietic cell growth and differentiation. In addition to -7 and del(5q), somatic copy number alterations on chromosome 3 and 12 were identified. There is controversy whether haploinsufficiency of genes on chromosomes 7 and 5q is sufficient to contribute to transformation, or whether further mutations lead to loss of heterozygosity of one or more genes in these regions. In the present case, careful review of the sequence and array data revealed no ‘homozygous' somatic single nucleotide variants, indels, or copy number alterations of coding genes on the remaining copy of chromosome 5 or 7. The patient's clinical presentation strongly suggested genetic cancer susceptibility. Analysis of the skin genome of this patient identified a heterozygous deletion of exons 7–9 of TP53, likely contributing to the early onset of her breast cancer; a uniparental disomy event resulted in the deletion being homozygous in the leukemia sample. Interestingly, the mutant TP53 allele is expressed, and it is predicted to produce a truncated p53 protein lacking most of its DNA binding domain. Functional studies of the mutant p53 protein are underway. Of note, based on a detailed family history and genotyping of the patient's mother, we suspect that the TP53 deletion occurred spontaneously. Ongoing whole genome sequencing studies in a large number of t-AML samples should identify novel somatic mutations and germline variants that contribute to t-AML.

Disclosures:
No relevant conflicts of interest to declare.