Radioactive Iodine–Related Clonal Hematopoiesis in Thyroid Cancer Is Common and Associated With Decreased Survival

Laura Boucai; John Falcone; Jenny Ukena; Catherine C. Coombs; Ahmet Zehir; Ryan Ptashkin; Michael F. Berger; Ross L. Levine; James A. Fagin


J Clin Endocrinol Metab. 2018;103(11):4216-4223. 

In This Article


Human Subjects

The study population included patients with thyroid carcinoma and no other concomitant hematologic malignancy treated at Memorial Sloan-Kettering Cancer Center (MSKCC) who underwent matched tumor and blood sequencing using the Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) cancer panel[20] on an institutional prospective tumor sequencing protocol ( NCT01775072). This study was approved by the MSKCC Institutional Review Board. Patients with thyroid carcinoma undergoing MSK-IMPACT testing typically harbored recurrent or metastatic disease and were considered potentially to benefit from targeted therapies in the near future. Associations with any systemic cancer treatments or radiation therapy received before genotyping were ascertained by review of electronic medical records. Patients were excluded from analysis if they had an active hematologic cancer or precursor condition, such as monoclonal gammopathy of uncertain significance or monoclonal B cell lymphocytosis at the time of blood sequencing. Clinical characteristics were analyzed in all patients with thyroid cancer for whom MSK-IMPACT testing was performed between 16 May 2014 and 20 July 2017 (n = 309). Patients were prospectively followed for development of hematologic cancers and for OS through 31 January 2018.

Next-generation Sequencing Assay

All patients underwent next-generation sequencing using MSK-IMPACT, a hybridization capture-based next-generation sequencing assay encompassing all protein-coding exons of 468 cancer-associated genes.[20] MSK-IMPACT is validated and approved for clinical use by the New York State Department of Health Clinical Laboratory Evaluation Program and authorized by the Food and Drug Administration and is used to sequence patients with advance-stage cancer at MSKCC. DNA is extracted from deparaffinized formalin-fixed paraffin-embedded tumor tissue and patient-matched blood sample using the chemagic STAR instrument (Hamilton) with magnetic beads (PerkinElmer). Extracted DNA samples were normalized in Tris-EDTA buffer and sheared on the Covaris instrument. KAPA Biosystems library preparation kit was used to prepare barcoded DNA molecules on the Biomek FXP instrument. Libraries were pooled, and DNA fragments were captured using custom-designed biotinylated probes (NimbleGen). Further details have been previously described.[20,21]

Determination of CH Events

We identified CH mutations in all genes captured in MSK-IMPACT through a two-tiered filtering schema, where the variant allele fraction (VAF) in the blood was greater than twice the VAF in the tumor after removing false positives and germline mutations. The following criteria were used to retain mutations:

  • Mutations present in one of the curated leukemia/lymphoma-related gene list (ASXL1, CBL, DNMT3A, GNAS, JAK2, NRAS, SF3B1, TP53, U2AF1, IDH2, BCOR, PPM1D, TET2, IDH1, IDH2, SRSF2, RUNX1, SH2B3, ZRSR2, STAT3, KRAS, MYD88, ATM, CALR, CEBPA, ETV6, EZH2, FLT3, KIT, MPL, NPM1, STAG2, WT1, SETD2, CREBBP), where VAF was ≥2%, and at least eight reads supported the alternate allele.

  • Mutations present in nonleukemia/lymphoma genes where VAF is ≥5%, and at least eight reads supported the alternate allele.

  • In samples where one CH mutation was identified, we also looked for the presence of additional mutations at lower variant frequencies, where mutations with VAF ≥1% in leukemia/lymphoma genes and VAF ≥3% for nonleukemia/lymphoma genes were retained.

  • For mutations in leukemia/lymphoma genes where the variant frequency was >35% in the blood sample but <35% in the tumor tissue, which could conceivably be considered as germline events with loss of allele in tumor tissue, we included only the mutations that were present in the Catalogue for Somatic Mutations in Cancer (COSMIC) database with >10 occurrences in the ''hematopoietic and lymphoid'' category into the pool of CH mutations.

Determination of CH-PD Events

Variants were considered for CH-PD if they had a VAF ≥10% in the blood sample and satisfied at least one of the following criteria:

  • Any variant in Catalogue for Somatic Mutations in Cancer occurring in the ''hematopoietic and lymphoid'' category greater than or equal to five times

  • Any damaging variant in the DNMT3A gene within exons 7 to 23

  • Any damaging variant in the following genes: ASXL1, TET2, PPM1D, TP53, RAD21, STAG2, ATM, NF1

  • Any inactivating mutation in CALR exon 9

  • JAK2 V617F variant

  • CBL E366K, C384Y, C404S, and C416S variants

  • SETD2 R1625C variant

  • MPL W515S variant

  • Any oncogenic variant in the Papaemmanuil et al. study[22] that did not satisfy the above criteria

For detailed methods on determination of CH and CH-PD, refer to Coombs et al.[19] and Supplemental Table 1.

Survival Determination

Survival data were obtained through internal MSKCC databases based on deaths while admitted to an MSKCC hospital or by Death Notification forms submitted from physicians' offices. Last known follow-up information was also electronically updated for all patients with activity at any MSKCC site. Additionally, vital status was updated electronically by using the Social Security Death Index matched against the MSKCC patient database. The Social Security Death Index update is run on a monthly basis. If there is no update or activity on a patient's account over a 15-month period, then the Omnipro Medicare Database is queried to determine vital status and/or data of last account activity.

Statistical Analysis

χ 2 Tests, Student t tests, and Wilcoxon-rank sum tests were used to compare patient- and clinical-care characteristics among patients with and without CH. Logistic regression analysis was used to build multivariate models to examine the association between RAI and CH. The incidence of hematologic cancer, defined as the time from matched normal blood sampling to pathologically confirmed hematologic cancer development, was visually displayed using cumulative-incidence functions, whereas death in the absence of a hematologic malignancy was considered a competing event. OS, defined as the time from sample collection to death or last follow-up, was displayed using Kaplan-Meier curves. Cox-proportional hazards models were used to examine the association between CH and CH-PD and death. All statistical analyses were performed using STATA Statistical Software v12 (StataCorp).