Acute myeloid leukemia with eosinophilia after cyclin‐dependent kinases 4/6 inhibitor treatment due to underlying clonal hematopoiesis of indeterminate potential
Authors: Kartik Anand, Praveen Kumar, Sai Ravi Pingali, Debananda Pati, Swaminathan P. Iyer
Journal: American Journal of Hematology (2018)

We report an unusual case of a patient who presented with acute myeloid leukemia (AML-M4Eo) after one dose of Ribociclib1 (an orally available FDA approved CDK 4/6 inhibitor [CDKi]) and subsequent granulocyte colony stimulating factor (G-CSF) for the ensuing neutropenia. Genetic and karyotype analysis showed loss of function mutations in the STAG2 gene that encodes cohesin complex protein,2 and inversion (16) (p13.1q22). We attempted to recreate the events using a cohesin mutant mouse model. Our results indicated that the mouse model with a single mutation in the cohesin gene could not explain the mechanism of leukemogenesis. Based on our analysis, we believe our patient likely had underlying clonal hematopoiesis of indeterminate potential (CHIP).3 We hypothesize that the silent cohesin mutated clone in the patient acquired additional hit and evolved into AML after which it was put into maturation arrest in G1/S by a combination of CKD4/6 inhibition and G-CSF which ultimately lead to AML. A 44-year-old previously healthy female participated in a healthy volunteer clinical trial of Ribociclib. Her blood counts 4 days prior to receiving study drug were normal with hemoglobin 12.4 g/dL, platelet 124k/μL, WBC of 4.2k/μL and ANC (absolute neutrophil-count) of 2400. Laboratory studies, as part of trial, showed neutropenia and she was referred to an oncologist. The ANC was 700 on day 14 and since it did not improve by day 18, she was started on G-CSF daily for 3 days. Subsequent blood work on day 67 showed that the WBC count was 5k/μL and ANC was 200. She received G-CSF daily for 3 days and her WBC and ANC improved to 20.9k/μL and 800, respectively. On day 4 of G-CSF her WBC jumped to 65k/μL with reported blasts. Due to concern of AML, she was sent to emergency room (day 72) and her labs reveled leukocytosis of 95k/μL, hemoglobin 10.5 g/dL and platelet count of 104k/μL (Figure 1A). Peripheral smear showed blasts and eosinophils. Patient denied any family history of leukemia and was on any medications. She received induction chemotherapy with Idarubicin and Cytrabine for AMLM4Eo. Her bone marrow (BM) biopsy was sent for FISH which was positive for CBFB-MYH11 fusion, and karyotype which showed inversion (16) (p13.1q22). Targeted Next Gen sequencing showed STAG2 c.1811G>A, pR604E and c.3133C.T, p.R1045term (variant allele frequency: 23%) - a Cohesin Complex protein.1 Patient achieved complete remission (CR) post induction therapy (BM biopsy: karyotype 46 XX & NGS: results non-conclusive because of technical issues) and remains in CR 15 months after subsequent consolidations with high dose Cytarabine. To confirm if CDK 4/6 inhibition followed by G-CSF causes AML due to underlying cohesin subunit gene mutation, we used Rad21 (a cohesin subunit gene) heterozygous mice as a model to recreate the findings in the patient with cohesin-Stag2 mutation. The rationale for using Rad21 mice is that Rad21 mutations are found in ~4% of de novo AML, Stag2 and Rad21 are core subunits of the cohesin structural complex, and Stag2 mutant mice are not currently available. For methods and design, see the supplementary material. Compared to wild type mice, there was a trend toward higher LT HSC (long-term hematopoietic stem cells) in the BM of Rad21 heterozygous mice after CKD4/6i injection (P > 0.05). However, no significant difference in the frequency of LT HSC was observed in the BM of Rad21 mice treated with CKD4/6i alone or with CKD 4/6i+G-CSF (Figure 1B). Compared to wild type mice, there was a trend toward higher CD11b+/Ly6g+myeloid cells in peripheral blood of Rad21 heterozygous mice but it did not reach statistical significance(P > 0.05) (Figure 1C). Cyclin-dependent kinases (CDKs) are regulatory proteins, which help transition and regulate different phases of cell cycle. CDK4 and CDK6 are of particular importance as they help in transition from G1 to S phase and are responsive to numerous growth regulatory signals.4 CDKi as therapeutic options have been of particular interest in different cancers particularly breast cancer.1 Ribociclib in combination with letrozole has shown improvement in progression free survival in hormone receptor positive, HER2-negative metastatic breast cancer.1 Neutropenia is the most common adverse event being reported in 74% of the patients and >59% of the patients in the trial had Grade 3 or higher neutropenia4 and therefore requires frequent blood count monitoring. Cohesin is a tripartite ring containing Smc1a, Smc3, Rad21, and the peripheral subunit Scc3 in yeast that in humans exists as two closely related functional paralogs: the abundant SA2 (Stag2) and the minor SA1 (Stag1). There are two types of cohesin rings either with peripheral SA1 or SA2 subunits. SA1/2 plays an important role in tethering the two cohesin rings entrapping one chromatid each in a handcuff configuration for chromosomal cohesion via interaction with two Rad21 molecules,5 and thereby regulating their movement through G1/S till their separation during anaphase. Somatic mutations in cohesin complex genes have been found to be present in approximately 10% of primary AML patient and 20% in patients with secondary AML in a cohort of myeloid malignancies.6 We queried AML-TCGA http://cancergenome.nih.gov/ for mutations in these 9 Cohesin related genes (SMC1A, SMC3, RAD21, STAG1, STAG2, ESCO1, NIPBL, ESCO1 and ESCO2) and found that 16% (23/144) of AML cases have Cohesin gene mutations. Jaiswal et al reported in a pooled whole exome sequencing datasets derived from 17 182 people in 22 genome-wide association study (GWAS) cohorts focusing on risk factors for diabetes mellitus; the rate of somatic mutations defined as CHIP was noted at 1.7% in study subjects <40 years of age (50/2894), however, was higher in age groups 70-79 (9.6%), 80-89 (11.7%), and ≥90 years (18.4%). Out of 95 gene panel used in the study, 5 were cohesion related genes (SMC1A, SMC3, RAD21, STAG1, STAG2), total of 10 variants in Cohesin related genes were detected with RAD21 being most commonly mutated (6 variants).3 We attempted to recreate the sequence of events in our patient in mice using Rad21 heterozygous mice; although there was a trend toward higher frequency of blasts in peripheral blood after CKD4/6i injection (P > 0.05), none of mice developed AML. Also, after dual treatment of CKD4/6i followed by G-CSF, we observed a decreasing trend in the frequency of blasts in peripheral blood in Rad21 heterozygous mice compared to WT group (data not shown). This proves that, merely having clonal hematopoiesis with cohesin gene complex mutations followed by CKD4/6i inhibitor treatment or dual treatment with CKD4/6 inhibitor followed by G-CSF is not enough to develop acute myeloid leukemia. Our mouse model is incomplete as it does not have second hit “inversion 16” to explain the mechanism of leukemogenesis after treatment with CKDi and G-CSF. Another limitation of our study is small number of mice in control and intervention group. We therefore hypothesize that our patient had “two hits” - cohesin gene complex mutation plus inversion 16 (p13.1q22), and treatment by CKD4/6i caused arrest of hematopoietic progenitor stem cells (HPSCs) in G1 phase. Subsequent exposure to G-CSF lead to increased proportion of non-dividing cells in S phase leading to maturation arrest. This is the first reported case of AML in a patient receiving CDK4/6i. Given the increased risk of neutropenia with CKD4/6i1 and emerging data about incidence of CHIP,3 long-term follow-up of patients receiving this class of drugs is needed. Nothing to report. The authors gratefully acknowledge support from the Simmons Family Foundation Mini-Collaborative Research Grant to S.P. Iyer (1RO1CA224304-01) and D. Pati, and Cancer Prevention and Research Institute of Texas Grant # RP160022 to D. Pati. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.