The significance of genetic mutations and their prognostic impact on patients with incidental finding of isolated del(20q) in bone marrow without morphologic evidence of a myeloid neoplasm

ABSTRACT Patients with a sole del(20q) chromosomal abnormality and without morphologic features of a myeloid neoplasm (MN) have shown variable clinical outcomes. To explore the potential

risk stratification markers in this group of patients, we evaluated their genetic mutational landscape by a 35-gene MN-focused next-generation sequencing (NGS) panel and examined the

association of mutations to progression of MNs. Our study included 56 patients over a 10-year period with isolated del(20q), of whom 23 (41.1%) harbored at least one mutation. With a median

follow-up of 32.6 months (range: 0.1−159.1), 9 of 23 patients with mutation(s) progressed to MNs, while all 33 patients without mutations did not progress to MN. Kaplan−Meier survival

analysis demonstrated the presence of mutation(s) as a significant risk factor for progression to MN (_P_ _<_ 0.0001). MN progression was strongly associated with the presence of

non-_DNMT3A/TET2/ASXL1_ epigenetic modifiers and nonspliceosome mutations (_P_ _=_ 0.003). There was no significant difference among patients with and without MN progression with respect to

the number of mutations, variant allele frequency, percentage of del(20q), and other clinical/laboratory variables. This study illustrates the underlying genetic heterogeneity and complexity

of isolated del(20q), and underscores the prognostic value of NGS mutational analysis in these cases. SIMILAR CONTENT BEING VIEWED BY OTHERS MUTATIONAL SPECTRUM AND PROGNOSIS IN

NRAS-MUTATED ACUTE MYELOID LEUKEMIA Article Open access 22 July 2020 PROGNOSTIC IMPACT OF THE ADVERSE MOLECULAR-GENETIC PROFILE ON LONG-TERM OUTCOMES FOLLOWING ALLOGENEIC HEMATOPOIETIC STEM

CELL TRANSPLANTATION IN ACUTE MYELOID LEUKEMIA Article 25 March 2021 REARRANGEMENTS INVOLVING 11Q23.3/_KMT2A_ IN ADULT AML: MUTATIONAL LANDSCAPE AND PROGNOSTIC IMPLICATIONS – A HARMONY STUDY

Article Open access 04 July 2024 INTRODUCTION Deletion of the long arm of chromosome 20q [del(20q)] is a recurring chromosomal abnormality identified in a variety of myeloid neoplasms

(MNs), including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS/MPN, and acute myeloid leukemia (AML). Del(20q) may occur as a sole abnormality or in the setting of

other cytogenetic alterations, either as an early or late event in patients with MNs. Irrespective of the variable MN subcategories, bone marrow samples from MN patients with del(20q)

characteristically show morphologic abnormalities in erythroid precursors and megakaryocytes1, including the unique features of neutrophilic erythrophagocytosis and prominent megakaryocytic

emperipolesis in MDS2,3. Del(20q) as a sole chromosomal abnormality identified in a bone marrow specimen, which shows no morphologic diagnostic features of MNs (referred to isolated del(20q)

in this study), may be incidentally encountered in patients evaluated for nonmyeloid malignancies or unexplained cytopenia(s). According to the WHO classification (fourth edition)4,

isolated del(20q) is not considered a definitive evidence for MDS in patients with unexplained cytopenia in the absence of morphologic evidence for MDS5, which sometimes results in a

diagnostic and therapeutic dilemma. Studies have shown variable clinical outcomes in patients with isolated del(20q) and about 10−25% of patients ultimately evolve into various MNs including

AML6,7. However, the underlying pathogenic mechanisms associated with MN progression are largely unknown in isolated del(20q) patients. In this study, we investigated the mutational

landscape in this patient cohort and explored its association to progression into MN(s). METHODS CASE SELECTION We performed a 10-year retrospective review (January 1, 2005 to September 30,

2015) of the Mayo Clinic cytogenetic database and found 4428 Mayo Clinic patients with cytogenetic abnormalities in bone marrow karyotype analysis. Among these patients, a total of 242 had

sole del(20q) observed in at least 2 of 20 metaphases. Bone marrow pathology reports were reviewed and indicated 72 patients had no morphologic diagnostic features of involvement by an MN,

thus meeting the inclusion criteria of isolated del(20q) for this study. The remaining 170 patients fulfilled the criteria for MPN (93), MDS (51), AML (14), and MDS/MPN (12) and were

excluded. Archived bone marrow aspirate cell pellets with sufficient material for next-generation sequencing (NGS) analysis were available in 56 of 72 cases (Fig. 1). Clinical information

was obtained from medical charts for the final study cohort of 56 patients. Progression to MN was determined based on deteriorating clinical/laboratory findings and subsequent confirmation

by a bone marrow biopsy. This study was approved by the Mayo Clinic Institutional Review Board. CONVENTIONAL CHROMOSOME ANALYSIS Cells from the bone marrow aspirate were cultured

(unstimulated) for 24 and 48 h, harvested and G-banded using standard cytogenetic techniques. A total of at least 20 metaphases were fully analyzed and reviewed for each sample when

available. A del(20q) was identified in at least 2 of 20 metaphases to be considered a clone. MYELOID NEOPLASM-FOCUSED NGS TEST DNA was extracted from the available bone marrow cytogenetic

cell pellets stored in 3:1 methanol:acetic acid fixative from the 56 patients using Qiagen EZI (Qiagen, Germantown, MD). NGS testing was performed using a targeted OncoHeme panel, which

interrogated 35 genes recurrently mutated in MNs, including _ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88,

NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TERT, TET2, TP53, U2AF1, WT1_, and _ZRSR2_. Two-hundred nanogram sheared DNA was target-enriched with a custom

hybridization-capture reagent (SureSelectXT, Agilent, Santa Clara, CA) and sequenced on the MiSeq or HiSeq platforms (Illumina, San Diego, CA) at the Mayo Clinic Clinical Genome Sequencing

Laboratory. NGS data were processed through a proprietary bioinformatics analysis pipeline (Mayo NGS Workbench) and genetic variants were classified and annotated in our clinical Molecular

Hematopathology laboratory following the American College of Medical Genetics and Genomics (ACMG) five-tier system, the ACMG standards and guidelines for the interpretation of sequencing

variants, and the Association for Molecular Pathology (AMP)/ACMG approach to somatic mutation characterization8. STATISTICAL METHODS Statistical analyses were performed using JMP Pro

software version 14 (SAS Institute Inc., Cary, NC). Continuous variables are reported as median (range) or mean (standard deviation (SD); range) and categorical variables as number

(percentage). When the normality assumption was violated, we used nonparametric tests such as the Fisher’s exact test, chi-square test, or Wilcoxon rank-sum/Mann−Whitney _U_ test, as

appropriate, for test variables. Statistical significance was based on a two-sided significance level of 0.05. The main end point of the study was progression to a myeloid neoplasm. The

median follow-up was calculated from the time of isolated del(20q) detection to progression to MN or last available clinical follow-up for those without progression to MN. The overall

survival and time to progression were analyzed using Kaplan−Meier survival curves to account for differences in follow-up per patient, and group comparisons were performed using log-rank

tests. RESULTS Fifty-six patients with isolated del(20q) were included in this study, with 42 (75%) male and 14 (25%) female patients. The average age at isolated del(20q) detection was 67.9

years (SD ± 11.0; range: 44−90). At initial diagnosis, the average percentage of del(20q) was 38.5% (SD ± 29.2). The mean hemoglobin (Hb), absolute neutrophil count (ANC), and platelet

count (Plt) were 11.7 g/dL (SD ± 1.7; range: 8.3−15.1), 3.2 × 109/L (SD ± 1.8; range: 0.6−9.4) and 149 × 109/L (SD ± 84.4; range: 16−392), respectively. The indications for bone marrow

examination were variable and included evaluation for cytopenia(s) (_n_ = 17), monoclonal gammopathy (_n_ = 3), amyloidosis (_n_ = 2), allogeneic stem-cell transplant donor evaluation (_n_ =

 1), and staging for multiple myeloma (_n_ = 17), non-Hodgkin lymphoma (_n_ = 14), and chronic lymphocytic leukemia (CLL, _n_ = 2). At a median follow-up of 32.6 months (range: 0.1−159.1),

nine progressed to a myeloid neoplasm. The clinical and laboratory features of patients with and without progression are described in Table 1. At the time of isolated del(20q) detection, NGS

revealed mutations in 23 of the 56 (41.1%) patients, while the remaining 33 (58.9%) patients did not show mutations (Fig. 2). Mutations were detected in the following genes: _TET2 (8),

ASXL1 (7), SRSF2 (3), SF3B1 (3), DNMT3A (3), PHF6 (2), CBL (2), U2AF1 (2), IDH1 (1), IDH2 (1), BCOR (1), JAK2 (1), PTPN11 (1), TP53 (1)_, and _RUNX1 (1)_. The variant allele fraction (VAF)

of mutated genes ranged from 5.2 to 53.4%. Overall, 15 patients harbored one mutation, two patients harbored two mutations, six patients harbored three mutations, and no patient showed more

than three mutations (Fig. 2). Nine of the 23 (39.1%) patients with mutation(s) progressed to an MN including five MDS, two AML, and two MDS/MPN unclassifiable, at a median follow-up of 36.7

months (range: 1.9−57.4). At the time of isolated del(20q), among these nine patients, five had one mutation (_BCOR, TP53_, _DNM3TA, CBL, IDH2_) with VAF ranging from 5.2 to 53.4%, one had

two mutations (_SF3B1/CBL)_ each with VAF of 25.3%, and three had three mutations (_PHF6/TET2/TET2, ASXL1/IDH1/SRSF2, DNM3TA/PTPN11/TET2_) with VAF ranging from 24.7 to 46.4% (Table 2). Five

of the nine patients had subsequent NGS performed at the time of MN progression (Table 2, patients #1−4, and #7). Four patients (#1, 2, 4, 7) acquired additional mutations during disease

progression; two of these four patients (#1, 4) also acquired additional cytogenetic abnormalities. One patient (#3) did not acquire additional mutations, but the VAF of the pre-existing

mutation(s) slightly increased over the interval. Patients #5 and #9 did not have NGS performed at progression; however, they acquired complex cytogenetics around the time of progression.

Interestingly, del(20q) disappeared in two patients (#5, 7) and decreased in four patients (#1, 2, 3, 9) during disease progression. Among these six patients who had disappearance/decrease

of del(20q) during disease progression, some gained another complex clone (patients #1, 5, 9), some obtained an additional mutation in a different gene (patients #2, 7), one had an increase

in the allele burden of the existing mutation (patient #3) (Table 2). These findings indicated that the del(20q) clone had survival/proliferation disadvantage in comparison to other

aggressive clones. Of the remaining 14 patients with mutations, none progressed to an MN at a median follow-up of 24.9 months (range: 0.1−82.7). Ten patients had one mutation (_ASXL1, U2AF1,

TET2, SRSF2, SF3B1, ASXL1, TET2, PHF6_), one patient had two mutations (_ASXL1/ASXL1_), and three patients had three mutations (_ASXL1/RUNX1/SRSF2, TET2/U2AF1/ASXL1, JAK2/TET2/TET2_; Table

3). One patient (#4, Table 3) had subsequent NGS performed at 71.8 months since isolated del(20q) detection and showed disappearance of the initial low level truncating _ASXL1_ mutations

(p.R404* and p.Q512*), but with acquisition of a new frameshift ASXL1 mutation (p. G646Yfs*12) at 24.6% VAF. Both bone marrow examinations of this patient showed no diagnostic features of an

MN. The follow-up intervals for the three patients (#2, 6, 12, Table 3) with three mutations were 40.9, 8.4 and 0.2 months, respectively. Evaluation of the functional pathways of the

identified mutations in patients with and without MN progression revealed that non-DTA epigenetic modifier/nonspliceosome mutations, including _TP53, CBL, IDH1, IDH2, PHF6, BCOR, PTPN11,

RUNX1, JAK2_, occurred significantly more frequently in patients with MN progression (8 of 9) in comparison to patients without MN progression (3 of 14) (_P_ = 0.003). However, no

significant differences in the frequency of the mutations involving DTA epigenetic modifiers (_DNMT3A, TET2, ASXL1_) or spliceosome genes (_SF3B1, U2AF1, SRSF2_) among the two groups, with

DTA epigenetic modifiers seen in 4/9 versus 9/14 patients (_P_ = 0.42), and mutations involving spliceosome genes in 2/9 versus 6/14 patients (_P_ = 0.40), respectively. As _ASXL1_ gene is

located on chromosome 20q11.21, we examined whether del(20q) affects _ASXL_1 allele burden if an _ASXL1_ mutation is identified. Six patients carried _ASXL1_ mutation(s) in our cohort, but

no relationship could be established regarding the _ASXL1_ VAF and the percentage of del(20q) (Supplemental Fig. 1). This could be explained by the fact that the _ASXL1_ gene is not

necessarily deleted when del(20q) is encountered. The breakpoints of del(20q) are heterogeneous that could be located more towards centromere regions or more towards telomere regions9. Since

_ASXL1_ is located close to centromere regions, this gene may not be disturbed in a 20q deletion. This is supported by a study that evaluated chromosomal microarray in 30 MDS patients with

del(20q), and found 2/3 of patients had intact _ASXL1_ gene and only 1/3 of patients had partial/entire _ASXL1_ deletion10. At initial diagnosis of isolated del(20q) in the 23 patients with

mutations, the average percentage of del(20q) was 36.4% (SD ± 28.9%). The mean Hb, ANC and Plt count were 12.2 g/dL (SD ± 1.8; range: 8.3−14.8), 3.5 × 109/L (SD ± 2.3, range: 0.6−9.4) and

126.9 × 109/L (SD ± 80.9, range: 32−392), respectively. Statistical significance was not demonstrated with respect to age, sex, Hb, ANC, Plt, % VAF, prior history of cytotoxic chemotherapy,

or % del(20q) metaphases among those with progression (_n_ = 9) versus those without progression (_n_ = 14) to an MN (Supplemental Table 1). For the 33 patients without mutation, none of

them progressed to an MN at a median follow-up of 33.9 months (range: 0.1−159.1). Statistical significance was not demonstrated on comparing patients with mutation(s) (_n_ = 23) versus those

without mutation (_n_ = 33) with respect to % del(20q) metaphases, age, sex, prior history of cytotoxic chemotherapy, and other laboratory variables including Hb, ANC, and Plt (Supplemental

Table 2). Four of these 33 patients had NGS performed on a follow-up bone marrow examination at a median follow-up of 18.7 months (range: 12.1−63.8), and none acquired mutations. Overall,

at a median follow-up of 32.6 months (range: 0.1−159.1), 9 of the 56 patients progressed to MNs. On Kaplan−Meier survival analysis, the time to progression and/or death among patients with

mutation(s) was 32.8 months and among those without mutation was 50.5 months (Log-rank _P_ = 0.03, Fig. 3a). When the event of interest was progression to MN only, the presence of

mutation(s) was associated with a significantly higher risk of disease progression into MNs (Log-rank _P_ < 0.0001, Fig. 3b). At 5 years, at least 76% patients with mutation(s) progressed

to a myeloid neoplasm (95% CI: 40.9−93.5, Fig. 3b). However, Kaplan−Meier analysis did not demonstrate statistical significance in the number of mutations (1 versus >1) associated with

progression to MN (Log-rank _P_ = 0.74, Fig. 3c). DISCUSSION Isolated del(20q) is associated with broad clinical presentations including neoplastic and nonneoplastic conditions, and can be

seen in MNs such as MDS, MPN, MDS/MPN, and AML. Patients with MDS or MPN commonly have an indolent clinical course when del(20q) is the sole chromosomal alteration11,12,13, especially when

del(20q) occurs at initial presentation14. However, the late occurrence of del(20q) in MDS, MPN or AML is usually associated with an unfavorable prognosis14,15. In addition to overt MNs,

isolated del(20q) can be incidentally identified in patients without evident bone marrow morphologic features of involvement by an MN. Based on the presence or absence of cytopenia(s), these

patients belong to either a nonneoplastic or preneoplastic category encompassing clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia(s) of undetermined

significance16,17. The clinical outcome in patients with isolated del(20q) is generally indolent, but 10−25% ultimately develop MDS, MPN, MDS/MPN or AML6,7,18. The association of del(20q)

with such a wide range of diseases and variable clinical outcomes clearly indicates the underlying genetic and biologic heterogeneity. It further implies that additional abnormalities

(second hit) are required for the development of an MN in patients with unremarkable bone marrow morphology at the time of isolated del(20q) detection. In our study, mutations were detected

in 41.1% of isolated del(20q) patients, of whom 16.1% progressed into MNs during the course of follow-up. All progressed patients harbored one or more pathogenic mutations at the time of

isolated del(20q) diagnosis and the presence of mutation is a statistically significant risk factor for MN progression. Additionally, among the 23 patients harboring mutations, the presence

of non-DTA epigenetic modifier/spliceosome mutations were strongly associated with MN progression (8/9 versus 3/14, _P_ = 0.003), whereas mutations occurring in the DTA epigenetic modifiers

and spliceosome genes showed no statistically significant differences in patient with and without progression. The mutations involving non-DTA epigenetic modifiers include _IDH1, IDH2_ and

_BCOR_, kinases _CBL, PTPN11_ and _JAK2_, tumor suppressors _TP53 and PHF6_, and transcription factor _RUNX1_. No statistically significant association with MN progression was observed in

the percentages of VAF or del(20q), patient age, gender, Hb, ANC, Plt, or prior history of chemotherapy. Certain mutations may play a critical role in the pathogenesis, evolution and disease

progression of MNs. The strong association of presence of non-DTA epigenetic modifier/nonspliceosome mutations with MN progression observed in our study (8/9 verse 3/14, _P_ = 0.003) was

also in keeping with the documented pathogenic role of mutations in signaling pathway kinases, transcription factors, tumor suppressors and non-DTA epigenetic modifiers. They have been shown

to be associated with disease progression in MDS and cooperate with earlier events of DTA epigenetic modifier and spliceosome mutations to drive disease progression. In contrast, mutations

of epigenetic modifiers _DNMT3A, TET2_ and _ASXL1_ (DTA mutations) are commonly seen in MNs, they are also relatively frequently mutated in healthy aging individuals and represent the most

common mutations of CHIP16,19. Isolated mutations in _DNMT3A, TET2_ or _ASXL1_, particularly those with a low VAF, showed a low positive predictive value for MN development20. Detection of

DTA mutations in AML patients in complete remission is also not associated with an increased risk of relapse, supporting the premalignant nature of these mutations21. Our findings were

consistent with previous studies showing no differences in DTA mutation frequency among isolated del(20q) patients with or without MN progression (4/9 verse 9/14, _P_ = 0.42). Of note,

_ASXL1_ is located on chromosome 20q11.21, and _ASXL1_ gene is partially/completely deleted in approximately 1/3 of patients with del(20q)10. Therefore, del(20q) sometimes “pheno-copy”

_ASXL1_ mutation to give rise to clonal hematopoiesis. This study broadens the current concept of clonal hematopoiesis and implies that clonal hematopoiesis is resulted from not only the

genetic mutation(s), but also cytogenetic abnormality. Some patterns of mutations identified as determining factors for the progression to MN in patients with unexplained cytopenia(s)20 were

not detected in our cohort. For instance, it has been shown that mutations in the RNA spliceosome factors (_SF3B1, SRSF2_, _U2AF1_, and _ZRSR2_), or ≥2 mutations, or those with mutation

associated with high VAF, had a high positive predictive value for the development of an MN in patients with unexplained cytopenia(s)20. However, we did not find a clear association of

spliceosome mutations, number of mutations or %VAF with MN progression in isolated del(20q) patients. The observed discrepancies with previous reports may be attributable to the presence of

the distinct genetic background of del(20q), the relatively small sample size and the relatively short follow-up for some patients in our study cohort. Moreover, factors other than mutations

may contribute to the development of an MN, such as immune dysregulation, chromosomal instability and detrimental marrow microenvironment22. We did not encounter _ZRSR2_ mutations in our

patients, conforming to the rarity and exclusion of _ZRSR2_ from the list of CHIP genes which include other more common spliceosome genes _SF3B1, U2AF1 and SRSF2_ (ref. 23). Interestingly,

six patients had a decrease/disappearance of del(20q) during disease progression in our cohort. Three patients gained complex cytogenetics, two gained an additional mutation in a different

gene and one had increased VAF of the pre-existing mutations. This finding indicates the survival/proliferative disadvantage of del(20q) clone compared to other aggressive clones. The

presence of a 20q deletion may be an initial risk factor that provides milieu for the growth of other aggressive clones. Our study reveals the genetic changes in MNs are heterogeneous and it

demonstrates dynamics of clonal evolution in the progression of a myeloid neoplasm. In our cohort, all 33 patients without mutations did not develop an MN. This finding indicates the lack

of mutations had a low risk of MN progression in patients with isolated del(20q) and it further implies the presence of sole del(20q) is insufficient for the development of an MN. Consistent

with our finding, the absence of mutations is a negative predictor for MN progression among patients with unexplained cytopenia20,24. Additionally, the advent of large-scale sequencing

analyses have shown the vast majority of MDS patients are associated with somatic mutations25,26,27, providing further evidence of a high negative predictive value for the lack of

identifiable mutations to MN development. In summary, at the time of isolated del(20q) detection, patients without mutations had a very low risk for progression to an MN, while approximately

one-third of those patients with mutations ultimately developed an MN. The subsequent development of MNs was significantly associated with the presence of mutation(s), but not with the

number of mutations, %VAF, % del(20q), age, gender, Hb, ANC, Plt, or prior history of chemotherapy. The presence of non-_DTA_ epigenetic modifier/nonspliceosome mutations was significantly

associated with MN progression in our study cohort. Overall, these data support genetic mutation analysis as a valuable complement to the current diagnostic evaluation of patients with

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references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Hematopathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Aishwarya Ravindran, 

Rong He, Dong Chen, Jennifer L. Oliveira, Phuong L. Nguyen, David S. Viswanatha, Kaaren K. Reichard, James D. Hoyer & Min Shi * Division of Laboratory Genetics and Genomics, Mayo Clinic,

Rochester, MN, USA Rhett P. Ketterling * Division of Hematology, Mayo Clinic, Rochester, MN, USA Majd D. Jawad & Ronald S. Go Authors * Aishwarya Ravindran View author publications You

can also search for this author inPubMed Google Scholar * Rong He View author publications You can also search for this author inPubMed Google Scholar * Rhett P. Ketterling View author

publications You can also search for this author inPubMed Google Scholar * Majd D. Jawad View author publications You can also search for this author inPubMed Google Scholar * Dong Chen View

author publications You can also search for this author inPubMed Google Scholar * Jennifer L. Oliveira View author publications You can also search for this author inPubMed Google Scholar *

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inPubMed Google Scholar * Kaaren K. Reichard View author publications You can also search for this author inPubMed Google Scholar * James D. Hoyer View author publications You can also

search for this author inPubMed Google Scholar * Ronald S. Go View author publications You can also search for this author inPubMed Google Scholar * Min Shi View author publications You can

also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Min Shi. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of

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mutations and their prognostic impact on patients with incidental finding of isolated del(20q) in bone marrow without morphologic evidence of a myeloid neoplasm. _Blood Cancer J._ 10, 7

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