G-CSF receptor (CSF3R) mutations in X-linked neutropenia evolving to acute myeloid leukemia or myelodysplasia

X-linked neutropenia (XLN) is a rare form of Congenital Neutropenia(CN) caused by inherited gain-of-function mutations of WAS.Here we report 2 cases of the original L270P X-linked neutropeniakindred that evolved to MDS or AML, with acquisition of G-CSFR(CSF3R) mutations and monosomy 7. Thus, leukemic transformationwith acquisition of CSF3R mutations and monosomy 7 is not restrictedto classical congenital neutropenia with autosomal inheritance,but can also occur in other genotypes of inherited neutropenia.

Key words: G-CSF, X-linked neutropenia, acute myeloid leukemia.

Introduction

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Introduction

In 2001, we described X-linked neutropenia (XLN) as a novelsubtype of congenital neutropenia (CN) in a three-generationfamily with 5 affected males and with a g.810T>C WAS mutation,resulting in a p.L270P gain-of-function mutation in the GTPasebinding domain (GBD) of the Wiskott-Aldrich Syndrome protein(WASP).^1,4 In 2006, 2 more XLN cases were described, one witha p.S272P and one with a p.I294T mutation, the latter reportedlypresenting with myelodysplasia (MDS).² In 2008, we reporteda large XLN kindred with 10 affected males and 8 female carrierswith a p.I294T WASP mutation, which also results in a gain-offunction. CN is a heterogeneous disease with mostly autosomalinheritance patterns. Autosomal dominant CN is caused by ELA2mutations in 60% of cases.⁵ Autosomal recessive CN or Kostmann’sdisease is caused by HAX1 mutations.⁶ Other rare subtypes ofthe disease include autosomal dominant mutations of GFI1⁷ andautosomal recessive mutations of G6PC3.⁸ The diagnosis of CNis usually made at birth or in the first months of life withrecurrent, severe infections and neutrophil counts below 0.510⁹/L. Monocyte and eosinophil counts are elevated in CN.^7,9In contrast, XLN has an X-linked inheritance pattern and althoughalso characterized by severe neutropenia, it is often diagnosedat a later age, due to the infectious phenotype which is relativelymild considering the degree of neutropenia. Monocytopenia andlow NK cell numbers are typical hematologic features of XLN.Low B-cell counts, low-normal platelets, inversion of the CD4/CD8ratio and low-normal IgA levels can be present.³

Autosomal dominant and recessive CN carry an increased riskof myeloid transformation with a cumulative incidence of upto 25% at 20 years.⁹ This leukemic conversion is often associatedwith the development of monosomy 7 and mutations in the G-CSFreceptor gene.^6,9–14 Whether a risk of malignant transformationin XLN exists has not yet been reported to date. Here we reportfor the first time 2 cases with XLN, who developed MDS-RAEBand AML, both with monosomy 7 and CSF3R mutations in leukemicsamples.

Design and Methods

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Design and Methods

Patient material
Blood and bone marrow samples were obtained according to theguidelines of the local IRB at the University Hospitals Leuven.

Detection and quantification of CSF3R mutations in XLN patients
DNA was extracted from viably frozen blood, bone marrow samplesor from fixed cells. Exon 17 of CSF3R^11,13,15 was amplifiedusing the primers: CSF3R-F 5’-accctttgtgttccaccagt-3’and CSF3R-R 5’-ttggtcctttcttcctccct-3 (GeneAmp PCR system2400, Applied Biosystems, Foster City, California). PCR productswere verified with 6% PAGE-gels and sequenced in both directions,using BigDye^® Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems). Sequencing results were analyzed using SequenceScanner (Applied Biosystems).

For the detection of the g.2425T>G (p.Y729) mutation by qPCR,we used primers DF 5’-ccagcgatcaggtcctttag-3’ asallele specific forward primer together with 5’-gggcac-tatctccgctgtga-3’as reverse primer. QPCR was performed in a Real Time PCR quantificationAB7000 using p53 as a control.

Results and Discussion

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Results and Discussion

Case II.3 (Figure 1) was diagnosed with neutropenia with neutrophilcounts between 0.15–0.9 10⁹/L in his late twenties. Beforethat time his medical history consisted of frequent staphylococcalskin infections, recurrent bronchitis, pulmonary tuberculosis,a sacral fistula and peritonitis of unknown origin (possiblyTBC). The bone marrow karyotype was normal. At age 63, a marrowaspirate showed a poorly represented myeloid lineage with neutrophilmaturation arrest and discrete dysplasia in the red lineageand a normal karyotype. From age 63 onwards, G-CSF (filgrastim)was initiated at 0.3 mg/day subcutaneously. At age 65, the diagnosisof MDS type RAEB¹⁶ was made and monosomy 7 was found in 10/10evaluated metaphases.^17–18 At age 65, he was admittedwith fever and a leukocyte count of 17.9 10⁹/L of which 31%were blasts. After discontinuation of G-CSF, the leukocyte numberand circulating blasts decreased and the cytological diagnosiswas revised to MDS-RAEB. A stable clinical and hematologic remissionwas achieved after 4 courses of decitabine. Although the bonemarrow blastosis remained at 10%, this was interpreted as evidenceof maturation arrest and not of MDS, as repeated marrow karyotypesremained normal. A truncating CSF3R mutation g.2390C>T (p.Q718),previously described by other authors,^11,13,15 was identifiedin a heterozygous pattern in a marrow sample obtained at thetime of RAEB (Figure 2). The same mutation could also be documentedat lower levels in a sample 2.5 years after treatment, duringstable remission (Figure 2). He died at age 68, due to an unrelatedcause.

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Figure 1. Updated L270P Pedigree. Filled squares: males with documented neutropenia; circles with a black dot: carrier females; open symbols: unaffected individuals; patients with MDS/AML are indicated in red squares.

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Figure 2. Sequencing profile of patients II.3 and III.6 at different time points.

In case III.6 (Figure 1), neutropenia was first diagnosed atage 27, with a neutrophil count of 0.3 10⁹/L and a medical historyconsisting of frequent herpes labialis and anal fistulas. Hereportedly had a hypocellular bone marrow aspirate with lownumbers of granulocyte precursors. At age 31, on the occasionof neutropenic fever and a staphylococcal abscess in the rightthigh, a bone marrow aspirate showed a maturation arrest inthe myeloid lineage and monosomy 7 in 2 of 11 metaphases. Hewas not on G-CSF at that time. Interfase FISH was also consistentwith monosomy 7 in a minor subpopulation (7%). However, twosubsequent marrows six months and five years later revealeda normal karyotype. At age 33, supportive treatment with G-CSFwas started at 0.3 mg SC 3 x/wk. At age 37, monosomy 7 reappearedin the bone marrow (7/10 metaphases)¹⁹. One year later he developeda refractory AML with monosomy 7 (10/10) as sole karyotypicabnormality. CSF3R mutation screening in a bone marrow sampleat this time showed a heterozygous g.2425T>G (p.Y729) mutationin approximately all cells. The same mutation was also identifiedby qPCR in ~34% of cells in a pre-leukemic sample obtained atage 37 (Figure 2).

In this article we describe 2 cases of XLN with evolution toAML/MDS with acquired monosomy 7 and CSF3R mutations. Monosomy7 is among the most common chromosomal aberrations in AML andMDS(20). Acquired loss of 7 or of 7q is associated with an arrayof congenital bone marrow failure syndromes, including CN, Shwachman-DiamondSyndrome and Fanconi anemia and is considered a poor prognosticfactor.^17,20

CN is well known as a pre-leukemic condition.^{6,9,13,21–23}Evolution to acute myeloid leukemia has been best documentedin autosomal dominant CN (with ELA-2 mutations).^9,13 The cumulativeincidence of malignant transformation in CN is reportedly morethan 25% after 20 years of observation.⁹ In CN, developmentof MDS/AML is significantly associated with the acquisitionof truncating CSF3R mutations. The frequency of CSF3R mutationsin CN patients with leukemic transformation is approximately80% in both ELA2 and HAX1 positive patients, whereas in CN patientswithout leukemia, the frequency of CSF3R mutations is only 30%.⁹CSF3R mutations have also been described in one of 4 membersof the original Kostmann family with a presumable HAX1 mutationwithout clear malignant evolution.²⁴ In addition, 3 other patientswith a HAX1-mutation have been described with a CSF3R mutation,one of whom developed myelodysplasia.⁶ Moreover, Ward et al.described a young patient with an ELA2 mutation and a constitutiveextracellular G-CSF-R mutation, leading to hyporesponsivenessto G-CSF treatment. This patient later developed consecutiveacquired (intracellular) CSF3R mutations, leading to restorationof CSF3R responsiveness, but so far without malignant conversion.²⁵

The g.2390C>T (p.Q718) and g.2425T>G (p.Y729) CSF3R mutationsthat were observed here, have already been reported in CN andwere found to be associated with malignancy.^11,13,15 Thus, XLNalso encompasses an increased risk of conversion to a myeloidmalignancy, as does classical CN. At the time of overt leukemia,both patients exhibited a heterozygous CSF3R mutation in approximately100% of analyzed cells. In case III.6 we could show that theCSF3R mutation preceded the leukemic transformation by approximately21 months. In patient II.3, no antecedent samples were availablebut the mutational burden was decreased in two remission bonemarrow samples 1.5 and 2.5 years later. Thus, the CSF3R mutationalburden was found to correlate with the leukemic burden, as wasalso described in CN.¹³ Given the context of congenital neutropeniaand administration of G-CSF in both cases, it is unclear towhat extent the chronic neutropenia and the administration ofG-CSF contributed to the malignant transformation. Patient II.3had received high doses of G-CSF for two years and patient III.6had received only supportive G-CSF during the five years precedingthe development of AML. Although the duration of G-CSF is nota risk factor for leukemia, a G-CSF dose-effect has been suggestedin CN patients requiring high doses of G-CSF. Whether this isdue to the severity of the disease, the apparent resistanceto G-CSF or the high doses of G-CSF is still under debate.¹⁴

CSF3R mutations often precede the leukemic transformation andcan be present for years in patients who never develop leukemia.Therefore, CSF3R mutations are considered an early event inleukemogenesis, occurring prior to malignant transformation,as an adaptive response of a defective stem cell pool in CNand leading to activation of anti-apoptotic signaling cascades.¹³In line with this is the observation that mutant ELA2 leadsto increased apoptosis of myeloid progenitors and that thispro-apoptotic effect could be overridden by CSF3R mutations,leading to enhanced survival and proliferation of hematopoieticprogenitor cells.²⁶ According to this model, G-CSF therapy maycause genomic instability of the progenitor cells due to increasedpressure on proliferation. Long-term G-CSF therapy could leadto preferential clonal expansion of CSF3R mutant cells thatmay already be more susceptible to additional aberrations, possiblyleading to leukemia.^9,27

Non-truncating CSF3R mutations have also been documented inde novo MDS and AML and aberrant expression of G-CSF and itsreceptor have been observed in solid tumors like ovarian cancer,bladder cancer and squamous cell carcinoma.²⁸ Although G-CSFis frequently administered in these malignancies to overcomechemotherapy-induced neutropenia, this could be a reason forconcern.

So far, no cases of hematologic malignancies have been diagnosedin the large Irish p.I294T kindred that we recently reported,nor have we detected major sub-populations with CSF3R mutationsusing direct sequencing. However, most cases in the latter familyare younger than in the first family; the oldest p.I294T XLNcase in the Irish family is now 48 years old (range 10–48;mean 35 years) and G-CSF is used more sparingly in this family.

We conclude that XLN is a pre-leukemic condition, similar toautosomally inherited CN. Acquisition of monosomy 7 and CSF3Rmutations appear to be a final common pathway associated withleukemic transformation, irrespective of the genetic defectunderlying the neutropenia. The benefits of G-CSF treatmentshould, therefore, be carefully weighed against the possibleeffects in leukemogenesis in pre-leukemic diseases, such ascongenital neutropenias. Based on our findings and on the mildinfectious phenotype of XLN, we recommend that G-CSF be usedsparingly in XLN and restricted to infectious episodes.

Acknowledgments

for clinical data gathering, we are grateful to Dr IsabelleMeyts, Emiel Baeten, Koen Devriendt and Hilde Demuynck.

Footnotes

Authorship and Disclosures

KB designed research, performed experiments, collected and analyzeddata, and wrote the article; PV designed research, analyzeddata and wrote the article.

This text presents research results of the Belgian program ofInteruniversity Poles of Attraction initiated by the BelgianState, Prime Minister’s Office, Science Policy Programming.The authors assume complete scientific responsibility.

The authors reported no potential conflicts of interest.

Funding: fund for Scientific Research-Flanders (Belgium) (FWOVlaanderen). KB is an aspirant researcher and PV is a seniorclinical investigator of the Fund for Scientific Research-Flanders(Belgium) (FWO Vlaanderen).

Received for publication March 20, 2009. Revision received May 13, 2009. Accepted for publication May 25, 2009.

References

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