Jane E. Churpek

Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens

Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

Genomic analysis of germ line and somatic variants in familial myelodysplasia/acute myeloid leukemia

Familial clustering of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) can be caused by inherited factors. We screened 59 individuals from 17 families with 2 or more biological relatives with MDS/AML for variants in 12 genes with established roles in predisposition to MDS/AML, and identified a pathogenic germ line variant in 5 families (29%). Extending the screen with a panel of 264 genes that are recurrently mutated in de novo AML, we identified rare, nonsynonymous germ line variants in 4 genes, each segregating with MDS/AML in 2 families. Somatic mutations are required for progression to MDS/AML in these familial cases. Using a combination of targeted and exome sequencing of tumor and matched normal samples from 26 familial MDS/AML cases and asymptomatic carriers, we identified recurrent frameshift mutations in the cohesin-associated factor PDS5B, co-occurrence of somatic ASXL1 mutations with germ line GATA2 mutations, and recurrent mutations in other known MDS/AML drivers. Mutations in genes that are recurrently mutated in de novo AML were underrepresented in the familial MDS/AML cases, although the total number of somatic mutations per exome was the same. Lastly, clonal skewing of hematopoiesis was detected in 67% of young, asymptomatic RUNX1 carriers, providing a potential biomarker that could be used for surveillance in these high-risk families.

Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies

Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

Inherited mutations in cancer susceptibility genes are common among survivors of breast cancer who develop therapy‐related leukemia

Risk factors for the development of therapy‐related leukemia (TRL), an often lethal late complication of cytotoxic therapy, remain poorly understood and may differ for survivors of different malignancies. Survivors of breast cancer (BC) now account for the majority of TRL cases, making the study of TRL risk factors in this population a priority.

Familial myelodysplastic syndrome/acute leukemia syndromes: a review and utility for translational investigations.

The familial myelodysplastic (MDS)/acute leukemia (AL) predisposition syndromes are inherited disorders that lead to significantly increased lifetime risks of MDS and AL development. At present, four recognized syndromes have Clinical Laboratory Improvement Amendments—certified testing for their respective germ‐line mutations: telomere biology disorders due to mutation of TERC or TERT, familial acute myeloid leukemia (AML) with mutated CEBPA, familial MDS/AML with mutated GATA2, and familial platelet disorder with propensity to myeloid malignancy. These disorders are heterogeneous with regard to their causative genetic mutations, clinical presentation, and progression to MDS/AL. However, as a group, they all share the unique requirement for a high index of clinical suspicion to allow appropriate genetic counseling, genetic testing, and mutation‐specific clinical management. In addition, translational investigations of individuals and families with these syndromes provide a rare opportunity to understand key pathways underlying susceptibility and progression to MDS/AL and allow the possibility of novel strategies for the prevention and treatment of both familial and sporadic forms of MDS/AL.

Proposal for the clinical detection and management of patients and their family members with familial myelodysplastic syndrome/acute leukemia predisposition syndromes

As with most genetic cancer predisposition syndromes, inherited susceptibility to myelodysplastic syndrome (MDS) and acute leukemia (AL) is likely to be more common than previously appreciated. As next-generation sequencing technologies become integrated into clinical practice, we anticipate that the number of cases of familial MDS/AL identified will increase. Although the existence of syndromes predisposing to MDS/AL has been known for some time, clinical guidelines for the screening and management of suspected or confirmed cases do not exist. Based on our collective experience caring for families with these syndromes, we propose recommendations for genetic counseling, testing, and clinical management. We welcome discussion about these proposals and hope that they will catalyze an ongoing dialog leading to optimal medical and psychosocial care for these patients.

The evolving challenge of therapy-related myeloid neoplasms

Therapy-related myelodysplastic syndrome (t-MDS) and therapy-related acute myeloid leukemia (t-AML) are late complications of cytotoxic therapies used to treat malignant, and increasingly, non-malignant conditions. Although distinct clinical, morphologic, and genetic features can be recognized, these disorders should be seen as part of a single disease spectrum recognized by the WHO in a singular classification, therapy-related myeloid neoplasms (t-MNs). Etiologic factors for t-MNs remain elusive, but ongoing research has characterized risk factors which vary between patient subgroups and exposures. Agents that damage DNA directly, interfere with DNA repair, and suppress the immune systems ability to detect malignant cells increase the risk of t-MNs. As in primary MDS and de novo AML, prognosis and treatment strategies rely on patient characteristics as well as cytogenetics. However, the overall outcome for patients with t-MNs remains poor. Here we review our current understanding of t-MNs as they are most often encountered by the practicing clinician.

How I diagnose and manage individuals at risk for inherited myeloid malignancies.

Although inherited hematopoietic malignancies have been reported clinically since the early twentieth century, the molecular basis for these diseases has only recently begun to be elucidated. Growing utilization of next-generation sequencing technologies has facilitated the rapid discovery of an increasing number of recognizable heritable hematopoietic malignancy syndromes while also deepening the fields understanding of the molecular mechanisms that underlie these syndromes. Because individuals with inherited hematopoietic malignancies continue to be underdiagnosed and are increasingly likely to be encountered in clinical practice, clinicians need to have a high index of suspicion and be aware of the described syndromes. Here, we present the methods we use to identify, test, and manage individuals and families suspected of having a hereditary myeloid malignancy syndrome. Finally, we address the areas of ongoing research in the field and encourage clinicians and researchers to contribute and collaborate.