Clonal hematopoiesis, isolated cytopenias , and ...

Clonal hematopoiesis, isolated cytopenias , and differences with MDS

Pierre Fenaux Hôpital St Louis Université Paris 7

GFM Roma, Sept 2016

MDS or not MDS ? (A Jacobs)

Mankind is divided into 3 categories: •  MDS •  Not quite MDS •  Not yet MDS

MDS or not MDS ?

•  MDS •  ICUS: Idiopathic Cytopenias of Undetermined Significance

•  IDUS: Idiopathic Dysplasia of Undetermined significance

•  CHIP: Clonal Hematopoiesis of Indeterminate PotenIal

Leukemia Research 31 (2007) 727–736

Consensus report

Definitions and standards in the diagnosis and treatment of themyelodysplastic syndromes: Consensus statements and

report from a working conference

Peter Valent a,∗, Hans-Peter Horny b, John M. Bennett c, Christa Fonatsch d, Ulrich Germing e,Peter Greenberg f, Torsten Haferlach g, Detlef Haase h, Hans-Jochen Kolb i, Otto Krieger j,

Michael Loken k, Arjan van de Loosdrecht l, Kiyoyuki Ogata m, Alberto Orfao n,Michael Pfeilstocker o, Bjorn Ruter p, Wolfgang R. Sperr a,

Reinhard Stauder q, Denise A. Wells k

a Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna,Waehringer Guertel 18-20, A-1090 Vienna, Austria

b Klinikum Ansbach, Institute of Pathology, Ansbach, Germanyc James P. Wilmot Cancer Center, University of Rochester Medical Center, Rochester, NY, USA

d Institute of Human Genetics (KIMCL), Medical University of Vienna, Vienna, Austriae Department of Hematology Oncology and Clinical Immunology, Heinrich-Heine-University, Dusseldorf, Germany

f Stanford University Cancer Center, Stanford, CA, USAg Munich Leukemia Laboratory (MLL), Munich, Germany

h Department of Hematology and Oncology, Georg-August-University, Gottingen, Germanyi Department of Medicine III, University of Munich, GSF-National Research Centre for Environment and Health, Munich, Germany

j First Department of Internal Medicine, Elisabethinen Hospital, Linz, Austriak Hematologics, Inc., Fred Hutchinson Cancer Research Center, Seattle, WA, USA

l Department of Hematology, VU University Medical Center, Amsterdam, The Netherlandsm Division of Hematology, Department of Medicine, Nippon Medical School, Tokyo, Japan

n Servicio Central de Citometrıa, Centro de Investigacion del Cancer and Department of Medicine, Universidad de Salamanca, Spaino Ludwig-Boltzmann Institute for Leukemia Research and Hematology, Vienna, Austria

p Department of Hematology/Oncology, Albert-Ludwigs University (ALU), Freiburg, Germanyq Division of Hematology and Oncology, Innsbruck Medical University, Innsbruck, Austria

Accepted 14 November 2006Available online 25 January 2007

Abstract

The classification, scoring systems, and response criteria for myelodysplastic syndromes (MDS) have recently been updated and havebecome widely accepted. In addition, several new effective targeted drugs for patients with MDS have been developed. The current articleprovides a summary of updated and newly proposed markers, criteria, and standards in MDS, with special reference to the diagnostic interfaceand refinements in evaluations and scoring. Concerning the diagnostic interface, minimal diagnostic criteria for MDS are proposed, andfor patients with unexplained cytopenia who do not fulfill these criteria, the term ‘idiopathic cytopenia of uncertain significance’ (ICUS) issuggested. In addition, new diagnostic and prognostic parameters, histopathologic and immunologic determinants, proposed refinements inscoring systems, and new therapeutic approaches are discussed. Respective algorithms and recommendations should facilitate diagnostic andprognostic evaluations in MDS, selection of patients for therapies, and the conduct of clinical trials.© 2007 Elsevier Ltd. All rights reserved.

Keywords: Myelodysplastic syndromes; Criteria; Standardization; Patient selection; ICUS; Minimal diagnostic criteria; Targeted therapy

∗ Corresponding author. Tel.: +43 1 40400 5488; fax: +43 1 40400 4030.E-mail address: [email protected] (P. Valent).

0145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.leukres.2006.11.009

P. Valent et al. / Leukemia Research 31 (2007) 727–736 729

Table 1Minimal diagnostic criteria in MDSa

(A) Prerequisite criteriaConstant cytopenia in one or more of the following cell lineages:

erythroid (hemoglobin <11 g dL−1); neutrophilic(ANC < 1500 !L−1) or megakaryocytic (platelets <100,000 !L−1)

Exclusion of all other hematopoietic or non-hematopoietic disorders asprimary reason for cytopenia/dysplasiab

(B) MDS-related (decisive) criteriaDysplasia in at least 10% of all cells in one of the following lineages in

the bone marrow smear: erythroid; neutrophilic; or megakaryocyticor >15% ringed sideroblasts (iron stain)

5–19% Blast cells in bone marrow smearsTypical chromosomal abnormality (by conventional karyotyping or

FISH)c

(C) Co-criteriad (for patients fulfilling ‘A’ but not ‘B’, and otherwiseshow typical clinical features, e.g. macrocytic transfusion-dependentanemia)

Abnormal phenotype of bone marrow cells clearly indicative of amonoclonal population of erythroid or/and myeloid cells,determined by flow cytometry

Clear molecular signs of a monoclonal cell population in HUMARAassay, gene chip profiling, or point mutation analysis (e.g. RASmutations)

Markedly and persistently reduced colony-formation (±clusterformation) of bone marrow or/and circulating progenitor cells(CFU-assay)

a The diagnosis MDS can be established when both prerequisite criteriaand at least one decisive criterion are fulfilled. If no decisive criterion isfulfilled, but the patient is likely to suffer from a clonal myeloid disease,co-criteria should be applied and may help in reaching the conclusion thepatient has MDS or a condition called ‘highly suspective of MDS’.

b As more patients with two co-existing bone marrow neoplasms are diag-nosed it is important to state that in a few cases, MDS can be diagnosed evenif another co-existing disease potentially causing cytopenia was detected.

c Typical chromosome abnormalities are those that are recurrently foundin MDS (+8, −7, 5q−, 20q−, others). If the abnormal karyotype is the onlydecisive criterion, the condition should be considered as ‘highly suspectiveof MDS’.

d Co-criteria are not considered to be used as standard in the routine hema-tologic work up in all centers. If not available, questionable cases should bemonitored and repeated tests be performed to establish the diagnosis MDSin the follow-up.

the number of abnormalities found in such assays, but in theabsence of morphologic dysplasia or an increase in blasts(in the follow-up), the diagnosis MDS should not be estab-lished. If such assays (for co-criteria) are not available orshow negative results, the recommended standard is to fol-low the clinical course and to repeat diagnostic tests after a(in) certain time interval(s). Patients with an increase in blastsshould also be followed carefully after diagnosis as some ofthese patients may rapidly progress into AML.

3. Idiopathic cytopenia of uncertain (undetermined)significance (ICUS)

Cytopenia in one or more myeloid lineages (erythro-cytes, neutrophils, platelets) that is (i) constant (≥6 months),(ii) does not meet the (minimal) criteria of an MDS and

Table 2Idiopathic cytopenia of uncertain (undetermined) significance (ICUS)

(A) DefinitionCytopenia in one or more of the following cell lineages (for ≥ 6

months): erythroid (Hb < 11 g dL−1); neutrophilic (<1500 !L−1);platelet (<100,000 !L−1)

MDS excluded (see ‘B’ and ‘C’)All other causes of cytopenia also excluded (see ‘B’ and ‘C’)

(B) Initial investigations required to establish the diagnosis of ICUSDetailed case history (toxins, drugs, mutagenic events, etc.)Thorough clinical investigations including X-ray and sonography of

spleenDifferential blood count (microscopic) and complete serum chemistryBone marrow histology and immunohistochemistryBone marrow smear including an iron stainFlow cytometry of bone marrow and peripheral blood cellsChromosome analysis including FISHa

Molecular analysis where appropriate (e.g. T cell receptorrearrangement—neutropenia)

Exclusion of viral infections (HCV, HIV, CMV, EBV, others)

(C) Recommended investigations in the follow-upBlood count and differential count as well as serum chemistry in 1–6

months intervalsIn those in whom suspicion for MDS becomes evident: bone marrow

examinationa Proposed minimum standard panel: 5q31, CEP7, 7q31, CEP8, 20q,

CEPY, p53.

(iii) cannot be explained by any other hematologic or non-hematologic disease is termed ICUS (Table 2), a term thatwas also proposed by Dr. G. Mufti (8th International Sym-posium on MDS, Nagasaki, Japan, 2005). In some of thesepatients, the type of cytopenia (transfusion-dependent macro-cytic anemia) may point to the potential of an MDS or anMDS-prephase. These patients should then be carefully mon-itored and repeated tests be performed in the follow-up toconfirm or exclude MDS. If suspicion for MDS becomesevident from the routine follow-up, a repeat bm examinationshould be performed.

At initial presentation, it is standard to perform a suffi-cient hematologic investigation in all patients, including abm trephine biopsy with appropriate histology and immuno-histochemistry, bm smear-examinations with Romanowskystain and iron stain as well as cytogenetics (Table 2).

4. The bone marrow and peripheral blood smear

The examination of an appropriately prepared and stainedbm and peripheral blood smear remains the most impor-tant diagnostic approach in patients with (suspected) MDS[1–3]. For proper morphologic assessment, well-preparedthin films (bm films containing particles at the featherededge) are required. An excellent Romanowsky stain (MGGor WG stain) should be utilized that has a good balancebetween the azur dyes to identify the cytoplasmic gran-ules and basophilic cytoplasm. Overstaining on thick smearsresults in all cells resembling each other, and understainingprevents the separation of the major cell lines as well (and

WHO classifica+on of MDS 1)blood cytopenias and exclusion of reacIve or other nonhematopoieIc causes of those cytopenias 2) one of the following diagnosIc features -‐ excess blasts(>5%) with a myeloid phenotype -‐10% dysplasIc cells in 1 of the 3 myeloid lineages (erythroid, granulocyIc, megakaryocyIc) -‐15% ring sideroblasts -‐ evidence of clonality as manifested by an abnormal MDS-‐associated karyotype.

MDS defined by

Unexplained Cytopenias +/-‐ •  Marrow dysplasia •  Clonal cytogeneIc abnormaliIes •  SomaIc mutaIons •  (immunophenotypic abnormaliIes)

Are cytopenias with clonal chromosomal abnormaliIes without dysplasia MDS ?

•  Cytopenias without dysplasia + « typical » cytogeneIc abnormality (-‐7, del 5q, etc…) = MDS

•  3 rearrangements considered not be sufficient for diagnosis of MDS ?

–  Loss of Y chromosome

–  Trisomy 8

–  Del 20q

MDS or not MDS ?




P. Valent et al. / Leukemia Research 31 (2007) 727–736 729

Table 1Minimal diagnostic criteria in MDSa

(A) Prerequisite criteriaConstant cytopenia in one or more of the following cell lineages:

erythroid (hemoglobin <11 g dL−1); neutrophilic(ANC < 1500 !L−1) or megakaryocytic (platelets <100,000 !L−1)

Exclusion of all other hematopoietic or non-hematopoietic disorders asprimary reason for cytopenia/dysplasiab

(B) MDS-related (decisive) criteriaDysplasia in at least 10% of all cells in one of the following lineages in

the bone marrow smear: erythroid; neutrophilic; or megakaryocyticor >15% ringed sideroblasts (iron stain)

5–19% Blast cells in bone marrow smearsTypical chromosomal abnormality (by conventional karyotyping or

FISH)c

(C) Co-criteriad (for patients fulfilling ‘A’ but not ‘B’, and otherwiseshow typical clinical features, e.g. macrocytic transfusion-dependentanemia)

Abnormal phenotype of bone marrow cells clearly indicative of amonoclonal population of erythroid or/and myeloid cells,determined by flow cytometry

Clear molecular signs of a monoclonal cell population in HUMARAassay, gene chip profiling, or point mutation analysis (e.g. RASmutations)

Markedly and persistently reduced colony-formation (±clusterformation) of bone marrow or/and circulating progenitor cells(CFU-assay)

a The diagnosis MDS can be established when both prerequisite criteriaand at least one decisive criterion are fulfilled. If no decisive criterion isfulfilled, but the patient is likely to suffer from a clonal myeloid disease,co-criteria should be applied and may help in reaching the conclusion thepatient has MDS or a condition called ‘highly suspective of MDS’.

b As more patients with two co-existing bone marrow neoplasms are diag-nosed it is important to state that in a few cases, MDS can be diagnosed evenif another co-existing disease potentially causing cytopenia was detected.

c Typical chromosome abnormalities are those that are recurrently foundin MDS (+8, −7, 5q−, 20q−, others). If the abnormal karyotype is the onlydecisive criterion, the condition should be considered as ‘highly suspectiveof MDS’.

d Co-criteria are not considered to be used as standard in the routine hema-tologic work up in all centers. If not available, questionable cases should bemonitored and repeated tests be performed to establish the diagnosis MDSin the follow-up.

the number of abnormalities found in such assays, but in theabsence of morphologic dysplasia or an increase in blasts(in the follow-up), the diagnosis MDS should not be estab-lished. If such assays (for co-criteria) are not available orshow negative results, the recommended standard is to fol-low the clinical course and to repeat diagnostic tests after a(in) certain time interval(s). Patients with an increase in blastsshould also be followed carefully after diagnosis as some ofthese patients may rapidly progress into AML.

3. Idiopathic cytopenia of uncertain (undetermined)significance (ICUS)

Cytopenia in one or more myeloid lineages (erythro-cytes, neutrophils, platelets) that is (i) constant (≥6 months),(ii) does not meet the (minimal) criteria of an MDS and

Table 2Idiopathic cytopenia of uncertain (undetermined) significance (ICUS)

(A) DefinitionCytopenia in one or more of the following cell lineages (for ≥ 6

months): erythroid (Hb < 11 g dL−1); neutrophilic (<1500 !L−1);platelet (<100,000 !L−1)

MDS excluded (see ‘B’ and ‘C’)All other causes of cytopenia also excluded (see ‘B’ and ‘C’)

(B) Initial investigations required to establish the diagnosis of ICUSDetailed case history (toxins, drugs, mutagenic events, etc.)Thorough clinical investigations including X-ray and sonography of

spleenDifferential blood count (microscopic) and complete serum chemistryBone marrow histology and immunohistochemistryBone marrow smear including an iron stainFlow cytometry of bone marrow and peripheral blood cellsChromosome analysis including FISHa

Molecular analysis where appropriate (e.g. T cell receptorrearrangement—neutropenia)

Exclusion of viral infections (HCV, HIV, CMV, EBV, others)

(C) Recommended investigations in the follow-upBlood count and differential count as well as serum chemistry in 1–6

months intervalsIn those in whom suspicion for MDS becomes evident: bone marrow

examinationa Proposed minimum standard panel: 5q31, CEP7, 7q31, CEP8, 20q,

CEPY, p53.

(iii) cannot be explained by any other hematologic or non-hematologic disease is termed ICUS (Table 2), a term thatwas also proposed by Dr. G. Mufti (8th International Sym-posium on MDS, Nagasaki, Japan, 2005). In some of thesepatients, the type of cytopenia (transfusion-dependent macro-cytic anemia) may point to the potential of an MDS or anMDS-prephase. These patients should then be carefully mon-itored and repeated tests be performed in the follow-up toconfirm or exclude MDS. If suspicion for MDS becomesevident from the routine follow-up, a repeat bm examinationshould be performed.

At initial presentation, it is standard to perform a suffi-cient hematologic investigation in all patients, including abm trephine biopsy with appropriate histology and immuno-histochemistry, bm smear-examinations with Romanowskystain and iron stain as well as cytogenetics (Table 2).

4. The bone marrow and peripheral blood smear

The examination of an appropriately prepared and stainedbm and peripheral blood smear remains the most impor-tant diagnostic approach in patients with (suspected) MDS[1–3]. For proper morphologic assessment, well-preparedthin films (bm films containing particles at the featherededge) are required. An excellent Romanowsky stain (MGGor WG stain) should be utilized that has a good balancebetween the azur dyes to identify the cytoplasmic gran-ules and basophilic cytoplasm. Overstaining on thick smearsresults in all cells resembling each other, and understainingprevents the separation of the major cell lines as well (and

Leukemia Research 36 (2012) 1– 5

Contents lists available at SciVerse ScienceDirect

Leukemia Research

jo ur nal homep age: www.elsev ier .com/ locate / leukres

Invited review

Idiopathic cytopenia of undetermined significance (ICUS) and idiopathicdysplasia of uncertain significance (IDUS), and their distinction fromlow risk MDS

Peter Valenta,b,∗, Barbara J. Bainc, John M. Bennettd, Friedrich Wimazala,e, Wolfgang R. Sperra,b,Ghulam Mufti f, Hans-Peter Hornyg

a Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austriab Ludwig Boltzmann Cluster Oncology Vienna, Austriac Department of Haematology, Medicine, St Mary’s Hospital Campus of Imperial College, London, UKd Division of Hematopathology and Hematology/Oncology, Department of Pathology and the James P. Wilmot Cancer Center, University of Rochester Medical Center,Rochester, NY, USAe Department of Obstetrics and Gynecology, Medical University of Vienna, Austriaf Department of Haematology, Medicine, King’s College, London, UKg Institute of Pathology Ansbach, Ansbach, Germany

a r t i c l e i n f o

Article history:Received 3 May 2011Received in revised form 1 August 2011Accepted 18 August 2011Available online 13 September 2011

Keywords:MDSDiagnostic criteriaPre-malignant lesionsICUSIDUS

a b s t r a c t

It is now generally appreciated that hematologic neoplasms can develop over many years if not decades,often being initially occult or showing minimal (subdiagnostic) abnormalities. However, whereas suchearly neoplastic conditions have been defined in some detail in lymphoproliferative neoplasms, lit-tle is known about minimal lesions preceding the manifestation of an overt myeloid neoplasm, aboutunderlying mechanisms, the clinical course and outcome, and the prognostic significance of such condi-tions. Members of several groups have recently described two ‘premalignant’ myeloid conditions, namelyidiopathic cytopenia of undetermined significance (ICUS) and idiopathic bone marrow dysplasia of uncer-tain significance (IDUS). At least in some patients these are neoplastic conditions. Both conditions mayprogress to an overt myelodysplastic syndrome (MDS), but may also progress to another myeloid neo-plasm such as acute myeloid leukemia, a myeloproliferative neoplasm (MPN), or a mast cell disorder(mastocytosis). In ICUS the dysplasia is mild and does not fulfill the WHO criteria for MDS but cytope-nias can be severe. In IDUS the dysplasia is prominent but cytopenias, if detectable, are mild. In bothconditions it is possible that a neoplastic clone has already replaced most or all of normal bone marrowcells when ICUS or IDUS is detected, but evidence to support this possibility is not necessarily available.For both groups of patients we recommend a thorough hematologic follow up because of the potentialof disease-manifestation and the unpredictable form and time of progression. In the present review, wediscuss current concepts relating to ICUS and IDUS as well as diagnostic approaches and available criteria.

© 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction and historical overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Idiopathic cytopenia of undetermined significance—ICUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. Idiopathic dysplasia of uncertain significance—IDUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34. IDUS and ICUS are mutually exclusive conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45. Recommendations for clinical practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

∗ Corresponding author at: Department of Internal Medicine I, Division of Hematology & Hemostaseology & Ludwig Boltzmann Cluster Oncology, Medical Universityof Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel.: +43 1 40400 5488; fax: +43 1 40400 4030.

E-mail address: [email protected] (P. Valent).


Leukemia Research 36 (2012) 1– 5

Contents lists available at SciVerse ScienceDirect

Leukemia Research

jo ur nal homep age: www.elsev ier .com/ locate / leukres

Invited review

Idiopathic cytopenia of undetermined significance (ICUS) and idiopathicdysplasia of uncertain significance (IDUS), and their distinction fromlow risk MDS

Peter Valenta,b,∗, Barbara J. Bainc, John M. Bennettd, Friedrich Wimazala,e, Wolfgang R. Sperra,b,Ghulam Mufti f, Hans-Peter Hornyg

a Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austriab Ludwig Boltzmann Cluster Oncology Vienna, Austriac Department of Haematology, Medicine, St Mary’s Hospital Campus of Imperial College, London, UKd Division of Hematopathology and Hematology/Oncology, Department of Pathology and the James P. Wilmot Cancer Center, University of Rochester Medical Center,Rochester, NY, USAe Department of Obstetrics and Gynecology, Medical University of Vienna, Austriaf Department of Haematology, Medicine, King’s College, London, UKg Institute of Pathology Ansbach, Ansbach, Germany

a r t i c l e i n f o

Article history:Received 3 May 2011Received in revised form 1 August 2011Accepted 18 August 2011Available online 13 September 2011

Keywords:MDSDiagnostic criteriaPre-malignant lesionsICUSIDUS

a b s t r a c t

It is now generally appreciated that hematologic neoplasms can develop over many years if not decades,often being initially occult or showing minimal (subdiagnostic) abnormalities. However, whereas suchearly neoplastic conditions have been defined in some detail in lymphoproliferative neoplasms, lit-tle is known about minimal lesions preceding the manifestation of an overt myeloid neoplasm, aboutunderlying mechanisms, the clinical course and outcome, and the prognostic significance of such condi-tions. Members of several groups have recently described two ‘premalignant’ myeloid conditions, namelyidiopathic cytopenia of undetermined significance (ICUS) and idiopathic bone marrow dysplasia of uncer-tain significance (IDUS). At least in some patients these are neoplastic conditions. Both conditions mayprogress to an overt myelodysplastic syndrome (MDS), but may also progress to another myeloid neo-plasm such as acute myeloid leukemia, a myeloproliferative neoplasm (MPN), or a mast cell disorder(mastocytosis). In ICUS the dysplasia is mild and does not fulfill the WHO criteria for MDS but cytope-nias can be severe. In IDUS the dysplasia is prominent but cytopenias, if detectable, are mild. In bothconditions it is possible that a neoplastic clone has already replaced most or all of normal bone marrowcells when ICUS or IDUS is detected, but evidence to support this possibility is not necessarily available.For both groups of patients we recommend a thorough hematologic follow up because of the potentialof disease-manifestation and the unpredictable form and time of progression. In the present review, wediscuss current concepts relating to ICUS and IDUS as well as diagnostic approaches and available criteria.

© 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction and historical overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Idiopathic cytopenia of undetermined significance—ICUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. Idiopathic dysplasia of uncertain significance—IDUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34. IDUS and ICUS are mutually exclusive conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45. Recommendations for clinical practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

∗ Corresponding author at: Department of Internal Medicine I, Division of Hematology & Hemostaseology & Ludwig Boltzmann Cluster Oncology, Medical Universityof Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel.: +43 1 40400 5488; fax: +43 1 40400 4030.

E-mail address: [email protected] (P. Valent).


2 P. Valent et al. / Leukemia Research 36 (2012) 1– 5

6. Summary and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1. Introduction and historical overview

Myelodysplastic syndromes (MDS) are clonal disorders ofmyelopoietic progenitor cells. The disease is characterized by dys-plasia of blood and/or bone marrow cells, peripheral cytopenia, anda genetic instability with enhanced risk of transformation into sec-ondary acute myeloid leukemia (AML) [1–6]. Minimal diagnosticcriteria for MDS have been discussed in the past by several workinggroups [1,4,6–8]. It is generally appreciated that marked persis-tent cytopenia that cannot be explained by any other underlyingdisease, is an important feature and should thus count as a pre-requisite to establish the diagnosis of MDS, although there is anongoing discussion about cut-off levels (Table 1). In addition, mor-phologic signs of dysplasia in myeloid cells are generally requiredto establish the diagnosis of MDS [1,4,6–8]. According to the pro-posal of the International Working Group on Morphology of MDS(IWGM-MDS), and in accordance with WHO criteria, at least 10% ofall cells in a given lineage should show morphological signs of dys-plasia in bone marrow smears to count as a diagnostic criterion forMDS (Table 2) [4,8]. In the case of the granulocyte lineage, assess-ment may be more readily carried out on peripheral blood cells thanon bone marrow cells. When morphologic criteria for MDS are notmet, the diagnosis of MDS can still be established in a markedlycytopenic patient if a typical (MDS-related) karyotype anomaly isfound [4,6]. The minimum number of metaphases required to diag-nose MDS in a cytopenic patient without morphologic evidence ofdysplasia has not been explicitly defined in the past. Although thispoint is still under discussion, the demonstration of an overt clonebearing a chromosome anomaly, as defined by ISCN criteria, may bejudged ‘diagnostic’, provided that the anomaly is typically found inMDS as specified in the WHO classification. FISH analysis may assistto confirm the presence of the anomaly in these cases, especiallywhen the number of examined metaphases is low. In cases of ques-tionable results, poor material, no growth, or when karyotype andFISH results point at a very small clone-size, it may be advisable torepeat the bone marrow examination [6].

There are also other additional (albeit non-specific) featuresthat are commonly seen in MDS, such as macrocytosis, an abnor-mal (aberrant) immunophenotype of bone marrow cells, or adecrease in colony-forming progenitors (CFC) [6,9]. In addition, a

thorough hematopathological investigation including immunohis-tochemistry is important to exclude or confirm the diagnosis ofMDS [6,10–13]. However, sometimes it is difficult to establish thediagnosis, especially in older patients (often with comorbidity) inwhom only mild cytopenia is seen or no substantial dysplasia isdetectable. In other patients, cytopenia may be triggered by sev-eral different factors and may improve or worsen over time, whichis important when considering cut-off levels (Table 1).

Recently, two conditions where criteria of an MDS are not ful-filled, although either cytopenia or dysplasia is present, have beendefined, namely idiopathic cytopenia of undetermined significance(ICUS) and idiopathic dysplasia of uncertain significance (IDUS)[6–8,13–15]. The term ICUS has first been suggested by two of us,Barbara Bain and Ghulam Mufti, in several meetings of the Interna-tional Working Group on Morphology of MDS (IWGM-MDS) andat numerous conferences, including four MDS symposia, one inLisbon (2005), one in Nagasaki (2005), one in Amsterdam (2006)[7], and one in Florence (2007) [8]. In addition, the term was dis-cussed in a Working Conference organized in Vienna in 2006 [6].Finally, in the WHO 2008 Update, the term ICUS was included[4]. So far only small numbers of cases of ICUS have been pub-lished, and little is known about mechanisms and the prognosis.In one series of 10 patients recorded at the Medical University ofVienna, 2 patients progressed to an overt MDS after 2 and 3 years,respectively [14]. In one of these patients, a small clone carryinga monosomy 7 (1 out of 20 metaphases and 10% of interphasecells) was initially detectable, and later expanded to a predomi-nant clone [14]. More recently, Schroeder et al. analyzed 67 patientswith ICUS identified in the Duesseldorf registry. In their patientgroup, 8 (12%) progressed to AML [16]. Interestingly, in 2 of these8 patients, the HUMARA assay revealed a ‘clonal’ pattern duringthe ICUS phase of the disease [16]. In line with this observation, anabnormal immunophenotype of myeloid cells may sometimes berecorded in ICUS [17].

The term IDUS has recently been proposed in order to createa niche for those cases where dysplasia is clearly demonstra-ble but no or only mild cytopenia is detectable [13,15,18,19].Unlike ICUS, patients with IDUS show substantial dysplasia ina considerable number of bone marrow cells (>10% in one ormore lineages) with or without an MDS-related karyotype, but

Table 1Proposed diagnostic cut-off levels for anemia to count as minimal diagnostic criteria for MDS and ICUS: comparison to normal values.

Lower limit: source of criteria (proposed by) Proposed cut off levels (lower limit)

Hb (g/L) ANC (×109/L) PLT (×109/L)

Cut off levels defining the normal blood count (WHO definition) 120 (f) 1.8a 150130 (m)

Cut off levels defining MDS (or ICUS); by WHO criteriab 100b 1.8b 100b

Cut off levels defining MDS and ICUS proposed by the IWGM-MDS groupc 100 1.8 100Cut off levels defining MDS or ICUS; by the 2007 consensus groupd 110 1.5 100

MDS, myelodysplastic syndrome; ICUS, idiopathic cytopenia with undetermined significance; Hb, hemoglobin; ANC, absolute neutrophil count; PLT, platelet count; WHO,World Health Organization; IWGM-MDS, International Working Group on Morphology of MDS. Also, the WHO chapter did not explicitly state that cytopenia-cut off criteriaproposed for MDS would also count for ICUS (but this is proposed by us) [4].

a Reported lower limits of normal ANC in different studies vary from 1.5 to 1.8 × 109/L with levels significantly lower than these being observed in healthy individuals withAfrican ancestry.

b The WHO states that MDS can also be diagnosed in patients with less severe cytopenia if morphologic and cytogenetic evidence would clearly argue for MDS. Their cutoff levels are those used in the International Prognostic Scoring System (IPSS) [26].

c The proposal of the IWGM-MDS group was presented in various meetings [7,8].d The 2007-consensus group proposal was published in [6].

•  exclude dysplasia (10% cut-‐off) in any of the 3 major lineages •  cytopenia must be substanIal and must be recorded over a Ime period of at least 6 months

Unexplained cytopenias (ICUS: idiopathic cytopenias of undetermined significance)

P. Valent et al. / Leukemia Research 36 (2012) 1– 5 3

Table 2Key features discriminating between MDS, ICUS, and IDUS.

Features/findings MDS ICUS IDUS

Peripheral blood:Diagnostic cytopeniaa + + −Transfusion dependence +/− −/+ −Pseudo-Pelger-Huët Cells +/− − −/+Circulating blast cells −/+ − −BFU-E markedly reduced +/− −/+ −/+Clonality by HUMARA + −/+ n.k.Bone marrow:Ring sideroblasts ≥15% +/− − +/−Marked bone marrow dysplasiab + − +Blast cell increase ≥5% +/− − −Marked bone marrow fibrosis −/+ − −MDS-related Karyotype +/− − −/+Flow cytometry abnormalities +/− n.k. n.k.

MDS, myelodysplastic syndromes; ICUS, idiopathic cytopenia of undetermined sig-nificance; IDUS, idiopathic dysplasia of uncertain significance; BFU-E, burst formingunit erythroid progenitor cells; HUMARA, human androgen receptor X-chromosomeinactivation assay; n.k., not known.

a Diagnostic cytopenia: neutrophils <1.0 × 109/L or/and hemoglobin <110 g/Lor/and platelets <100 × 109/L.

b Erythroid dysplasia >10% or/and granulocyte dysplasia >10% or/and megakary-ocyte dysplasia >10%. +, seen in all patients; +/−, seen in a majority of patients; −/+,seen in a minority of patients; −, not seen.

are not suffering from persistent cytopenia [17]. It is importantto note that the ‘IDUS-state’ can be stable for many years or evendecades [19,20]. Use of the term MDS for these patients is inap-propriate as some of them subsequently develop another myeloidneoplasm rather than MDS, and the type of neoplasm that subse-quently occurs is unpredictable [19]. Moreover, some IDUS patientsshow a constant course and remain in an ‘IDUS-state’ for decades[19,20]. Finally, for neither ICUS nor IDUS is there proof that everypatient actually has or will develop a neoplastic process, whichis important in terms of patient management and therapeuticconsiderations.

2. Idiopathic cytopenia of undetermined significance—ICUS

The definition of ICUS is based on unexplained persistentcytopenia, and the failure to establish a diagnosis of MDS byaccepted criteria [4,6–8]. In each patient, it is important to studythe peripheral blood and bone marrow cytology and histology, andto exclude dysplasia (10% cut-off) in any of the 3 major lineages(Table 3). Based on the lineage(s) involved we propose to separatefour different variants of ICUS: idiopathic anemia of undeterminedsignificance (ICUS-A), idiopathic neutropenia of undetermined sig-nificance (ICUS-N), idiopathic thrombocytopenia of undeterminedsignificance (ICUS-T), and idiopathic bi/pancytopenia of undeter-mined significance (ICUS-BI/PAN) (Table 3). An important pointis that, as for MDS, cytopenia must be substantial and must berecorded over a time period of at least 6 months to count as ‘diag-nostic’ for ICUS [6–8]. Another important point is that a number ofinvestigations, including a thorough analysis of bone marrow sec-tions and aspirate material by standard cytological, cytochemical,histologic, and immunohistochemical techniques need to be per-formed before one can arrive at the diagnosis of ICUS [6–8]. Finally,the diagnosis of ICUS should lead to a hematologic follow up sim-ilar to that for low risk MDS. Notably, in some instances, ICUS willprogress to MDS or another myeloid disorder over time, with avariable latency period [6–8,14].

So far little is known about the biochemical defects andmechanisms underlying ICUS. Sometimes, clonal hematopoiesisis detectable by molecular studies or FISH [14,16]. These clonesare often small at diagnosis or are only transiently detectable. Inother cases, no karyotypic abnormality and no gene defects are

Table 3Proposed classification of idiopathic cytopenia of uncertain significance (ICUS).

Proposed term Suggestedabbreviation

Definition (ICUScriteria already meta)

Idiopathic anemia of uncertainsignificance

ICUS-A Hb <110 g/L

PLT ≥100 × 109/LANC ≥1.0 × 109/L

Idiopathic neutropenia ofuncertain significance

ICUS-N Hb ≥110 g/L

PLT ≥100 × 109/LANC <1.0 × 109/L

Idiopathic thrombocytopeniaof uncertain significance

ICUS-T Hb ≥110 g/L

PLT <100 × 109/LANC ≥1.0 × 109/L

Idiopathic bi/pancytopenia ofuncertain significance

ICUS-BI/PAN Hb <110 g/L and/orb

PLT <100 × 109/L and/orb

ANC <1.0 × 109/L

a ICUS criteria have already been fulfilled: the patient has constant cytopenia forat least 6 months, MDS criteria are not met, and no other reason/underlying diseasethat could explain cytopenia has been found.

b Two or three cytopenias required.

found even if an extensive search is performed [14–16]. In somepatients with ICUS, flow cytometry reveals an aberrant pheno-type of myeloid cells suggesting clonality [17]. Currently, genomics,deep sequencing studies, and other approaches are being appliedin investigational studies in order to learn more about molecularfeatures and lesions that may lead to ICUS. One important addi-tional factor may be the microenvironment, including stromal cells,immune cells, and membrane bound or soluble cytokines. Espe-cially the inadequate production of cytokines has been discussedas a potential mechanisms contributing to ICUS. Likewise, in casesof ICUS with anemia (ICUS-A), a suboptimal response in erythro-poietin (EPO) production by the kidney (even if renal function isotherwise normal) has been described and has been discussed as apotential reason for the development of anemia in these patients[18].

3. Idiopathic dysplasia of uncertain significance—IDUS

Idiopathic dysplasia is a term that was created to describepatients who have clonal myelopoiesis without diagnosticcytopenia (Table 1) [13,15,19]. Sometimes, chromosomal defectsindicative of MDS are detectable [19,20]. In other patients, ringsideroblasts are found and the diagnosis of RARS is establishedas soon as diagnostic cytopenia (Table 1) develops [19]. However,patients with IDUS may also progress to AML, a myeloproliferativeneoplasm (MPN), or an MDS/MPN overlap disease [19] (Table 4).The latency period between IDUS and MDS is variable. Sometimes,stable clonal myelopoiesis is recorded over many years or evendecades [19,20]. In other cases, a relatively fast evolution to MDSand AML is seen. Here, it is extremely important to note that IDUScan only be diagnosed when blood counts remain stable (abovediagnostic cut off levels) over at least 6 months. An important ques-tion is how blood cell production can remain normal (or almostnormal) in IDUS even if normal myelopoiesis in the bone marrowhas been completely replaced by the IDUS clone.

This phenomenon has three potential explanations: one is thatresidual normal stem cells are capable of keeping blood countswithin the normal or near-normal range. The second explana-tion is that clonal cells retain their ability to differentiate intomature hematopoietic cells, and retain (a certain) responsivenessto differentiation-inducing cytokines [18,20]. In both hypotheses, itis important that the cytokine-network is also acting and respond-ing (to imminent cytopenia) in an appropriate manner. Likewise, it

Idiopathic dysplasia of uncertain significance—IDUS

•  Dysplasia in >10% in one or more lineages •  No significant cytopenia •  chromosomal defects indicaIve of MDS may be detectable

•  ring sideroblasts someImes found (diagnosis of RARS is established as soon as diagnosIc cytopenia occurs…)

•  … but dysplasia seen in viral infec3ons (eg, HIV infec3on), alcohol abuse, exposure to cytotoxic agents (eg, azathioprine, methotrexate), or nutri3onal deficiencies (eg, copper, folate, cobalamin).

P. Valent et al. / Leukemia Research 36 (2012) 1– 5 3

Table 2Key features discriminating between MDS, ICUS, and IDUS.

Features/findings MDS ICUS IDUS

Peripheral blood:Diagnostic cytopeniaa + + −Transfusion dependence +/− −/+ −Pseudo-Pelger-Huët Cells +/− − −/+Circulating blast cells −/+ − −BFU-E markedly reduced +/− −/+ −/+Clonality by HUMARA + −/+ n.k.Bone marrow:Ring sideroblasts ≥15% +/− − +/−Marked bone marrow dysplasiab + − +Blast cell increase ≥5% +/− − −Marked bone marrow fibrosis −/+ − −MDS-related Karyotype +/− − −/+Flow cytometry abnormalities +/− n.k. n.k.

MDS, myelodysplastic syndromes; ICUS, idiopathic cytopenia of undetermined sig-nificance; IDUS, idiopathic dysplasia of uncertain significance; BFU-E, burst formingunit erythroid progenitor cells; HUMARA, human androgen receptor X-chromosomeinactivation assay; n.k., not known.

a Diagnostic cytopenia: neutrophils <1.0 × 109/L or/and hemoglobin <110 g/Lor/and platelets <100 × 109/L.

b Erythroid dysplasia >10% or/and granulocyte dysplasia >10% or/and megakary-ocyte dysplasia >10%. +, seen in all patients; +/−, seen in a majority of patients; −/+,seen in a minority of patients; −, not seen.

are not suffering from persistent cytopenia [17]. It is importantto note that the ‘IDUS-state’ can be stable for many years or evendecades [19,20]. Use of the term MDS for these patients is inap-propriate as some of them subsequently develop another myeloidneoplasm rather than MDS, and the type of neoplasm that subse-quently occurs is unpredictable [19]. Moreover, some IDUS patientsshow a constant course and remain in an ‘IDUS-state’ for decades[19,20]. Finally, for neither ICUS nor IDUS is there proof that everypatient actually has or will develop a neoplastic process, whichis important in terms of patient management and therapeuticconsiderations.

2. Idiopathic cytopenia of undetermined significance—ICUS

The definition of ICUS is based on unexplained persistentcytopenia, and the failure to establish a diagnosis of MDS byaccepted criteria [4,6–8]. In each patient, it is important to studythe peripheral blood and bone marrow cytology and histology, andto exclude dysplasia (10% cut-off) in any of the 3 major lineages(Table 3). Based on the lineage(s) involved we propose to separatefour different variants of ICUS: idiopathic anemia of undeterminedsignificance (ICUS-A), idiopathic neutropenia of undetermined sig-nificance (ICUS-N), idiopathic thrombocytopenia of undeterminedsignificance (ICUS-T), and idiopathic bi/pancytopenia of undeter-mined significance (ICUS-BI/PAN) (Table 3). An important pointis that, as for MDS, cytopenia must be substantial and must berecorded over a time period of at least 6 months to count as ‘diag-nostic’ for ICUS [6–8]. Another important point is that a number ofinvestigations, including a thorough analysis of bone marrow sec-tions and aspirate material by standard cytological, cytochemical,histologic, and immunohistochemical techniques need to be per-formed before one can arrive at the diagnosis of ICUS [6–8]. Finally,the diagnosis of ICUS should lead to a hematologic follow up sim-ilar to that for low risk MDS. Notably, in some instances, ICUS willprogress to MDS or another myeloid disorder over time, with avariable latency period [6–8,14].

So far little is known about the biochemical defects andmechanisms underlying ICUS. Sometimes, clonal hematopoiesisis detectable by molecular studies or FISH [14,16]. These clonesare often small at diagnosis or are only transiently detectable. Inother cases, no karyotypic abnormality and no gene defects are

Table 3Proposed classification of idiopathic cytopenia of uncertain significance (ICUS).

Proposed term Suggestedabbreviation

Definition (ICUScriteria already meta)

Idiopathic anemia of uncertainsignificance

ICUS-A Hb <110 g/L

PLT ≥100 × 109/LANC ≥1.0 × 109/L

Idiopathic neutropenia ofuncertain significance

ICUS-N Hb ≥110 g/L

PLT ≥100 × 109/LANC <1.0 × 109/L

Idiopathic thrombocytopeniaof uncertain significance

ICUS-T Hb ≥110 g/L

PLT <100 × 109/LANC ≥1.0 × 109/L

Idiopathic bi/pancytopenia ofuncertain significance

ICUS-BI/PAN Hb <110 g/L and/orb

PLT <100 × 109/L and/orb

ANC <1.0 × 109/L

a ICUS criteria have already been fulfilled: the patient has constant cytopenia forat least 6 months, MDS criteria are not met, and no other reason/underlying diseasethat could explain cytopenia has been found.

b Two or three cytopenias required.

found even if an extensive search is performed [14–16]. In somepatients with ICUS, flow cytometry reveals an aberrant pheno-type of myeloid cells suggesting clonality [17]. Currently, genomics,deep sequencing studies, and other approaches are being appliedin investigational studies in order to learn more about molecularfeatures and lesions that may lead to ICUS. One important addi-tional factor may be the microenvironment, including stromal cells,immune cells, and membrane bound or soluble cytokines. Espe-cially the inadequate production of cytokines has been discussedas a potential mechanisms contributing to ICUS. Likewise, in casesof ICUS with anemia (ICUS-A), a suboptimal response in erythro-poietin (EPO) production by the kidney (even if renal function isotherwise normal) has been described and has been discussed as apotential reason for the development of anemia in these patients[18].

3. Idiopathic dysplasia of uncertain significance—IDUS

Idiopathic dysplasia is a term that was created to describepatients who have clonal myelopoiesis without diagnosticcytopenia (Table 1) [13,15,19]. Sometimes, chromosomal defectsindicative of MDS are detectable [19,20]. In other patients, ringsideroblasts are found and the diagnosis of RARS is establishedas soon as diagnostic cytopenia (Table 1) develops [19]. However,patients with IDUS may also progress to AML, a myeloproliferativeneoplasm (MPN), or an MDS/MPN overlap disease [19] (Table 4).The latency period between IDUS and MDS is variable. Sometimes,stable clonal myelopoiesis is recorded over many years or evendecades [19,20]. In other cases, a relatively fast evolution to MDSand AML is seen. Here, it is extremely important to note that IDUScan only be diagnosed when blood counts remain stable (abovediagnostic cut off levels) over at least 6 months. An important ques-tion is how blood cell production can remain normal (or almostnormal) in IDUS even if normal myelopoiesis in the bone marrowhas been completely replaced by the IDUS clone.

This phenomenon has three potential explanations: one is thatresidual normal stem cells are capable of keeping blood countswithin the normal or near-normal range. The second explana-tion is that clonal cells retain their ability to differentiate intomature hematopoietic cells, and retain (a certain) responsivenessto differentiation-inducing cytokines [18,20]. In both hypotheses, itis important that the cytokine-network is also acting and respond-ing (to imminent cytopenia) in an appropriate manner. Likewise, it

•  144 paIents with unexplained cytopenias. 17% MDS, 15% ICUS and some evidence of dysplasia, and 69% with ICUS and no dysplasia

•  SomaIc mutaIons : 71% of MDS , 62% ICUS and some dysplasia, 20% of ICUS paIents and no dysplasia.

•  35% of ICUS paIents carried a somaIc mutaIon or chromosomal abnormality

online October 1, 2015 originally publisheddoi:10.1182/blood-2015-08-667063

2015 126: 2355-2361

Matthew J. McGinniss, Shareef A. Nahas, Julie Kines and Rafael BejarBashar Dabbas, Aine Yung, Jenan Al-Hafidh, Emily Balmert, Christine Vaupel, Carlos El Hader, Brian Kwok, Jeff M. Hall, John S. Witte, Yin Xu, Prashanti Reddy, Keming Lin, Rachel Flamholz, common in idiopathic cytopenias of undetermined significanceMDS-associated somatic mutations and clonal hematopoiesis are

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A total of 144 enrolled patients with at least 1 unexplained, clinicallymeaningful cytopenia (defined as a hemoglobin level ,11 g/dL, a neu-trophil count of,18003109/L, or a platelet count,100 0003109/L)had diagnostic blood and bone marrow samples sent to GenoptixMedical Laboratory (supplemental Table 1). Routine diagnostic pro-cedures were performed (morphologic analysis, metaphase karyo-typing, FISH, and flow cytometry) and patients were assigned to1 of 3 groups based on expert hematopathology review (Figure 1):24 patients were found to meet the diagnostic criteria for MDS andwere assigned to group A, 21 patients had evidence of dysplasia thatdid not meet diagnostic criteria for MDS and were assigned to groupB, and 99 patients were found to have no evidence of dysplasia or anMDS defining karyotype abnormality and were assigned to group C.The 120 patients in groups B andC can be described as having ICUS,suggesting that among cytopenic patients suspected of havingMDS,the occurrence of ICUS is many times higher than the diagnosis ofMDS. To corroborate this finding, we examined similar cases sent toGenoptix during 2014. Among several thousand diagnostic samplesfrom cytopenic patients suspected of having MDS, 30% had an alter-native diagnosis, 62%did notmeet any diagnostic criteria, and only8%were diagnosed with MDS.

DNA isolated from the bone marrow of trial participants under-went next generation sequencing of 22 MDS-associated genes (sup-plemental Table 2). One or more somatic mutations were identifiedin 71% of patients with MDS (group A), 62% of patients with onlyrare dysplastic morphology (group B), and 20% of patients with noevidence of dysplasia (group C) (P , .0001). Combining groups Band C, 33 of 120 of these ICUS patients (28%) carried at least 1somatic mutation indicative of clonal hematopoiesis (Figure 1).Another 9 ICUS patients without a detected somaticmutation harboreda clonal chromosomal abnormality. Therefore,we identified42patientswith CCUS compared with 24 with a diagnosis of MDS in this pro-spectively defined cohort.

Characteristics of somatic mutations in cytopenic patients

GroupApatientswith clear evidence ofMDSweremore likely than thegroup B or C patients to have 2 or more somatic mutations, and had ahigher mean number of mutations per patient with at least 1mutation(2.5 vs 1.5 and 1.3 in groups B and C, respectively; P5 .004). Thegenes mutated in each group were largely, but not completely,overlapping (Figure 2). SF3B1 mutations and TET2mutations werethe most common in group A and B patients, consistent with priorstudies of somatic mutations in MDS. In contrast, group C patients

with no evidence of dysplasiamost frequently hadmutations ofTP53followed byTET2 andDNMT3A, but lackedmutations of SF3B1 andother splicing factors (supplemental Figure 1A).

Figure 1. Prospective trial schema and diagnostic groups. (A) Schema showing how patients were screened and selected for entry. Patients were placed into 1 of 3 groups based ontheir bone marrow findings. (B) Proportions of patients with and without mutations in each diagnostic group are shown after DNA sequencing 22 genes associated with myeloid neoplasms.

Figure 2. Mutations and VAFs in prospective study groups. (A) The frequency ofmutations in each gene listed on the x-axis is shown for prospective cohort patients withMDS (red bars in lower half) and CCUS, separated by whether dysplasia was seen in thebone marrow or not (blue and purple bars, respectively, in upper half). (B) Variant allelefractions by diagnostic group. The VAF for each mutation identified in the prospectiveMDS and CCUS groups is shown. Colored dots represent the highest VAF found in apatient whereas black dots represent mutations with VAFs less than the maximum for agiven patient. The colored dotted lines represent the mean of the highest VAFs (coloreddots) whereas solid black lines represent the mean of all mutations (colored and black dots).

BLOOD, 19 NOVEMBER 2015 x VOLUME 126, NUMBER 21 SOMATIC MUTATIONS ARE COMMON IN ICUS 2357


•  spectrum of mutated genes similar between the 3 groups except SF3B1 rarely mutated in paIents without dysplasia

•  Variant allele fracIons, mean age and blood counts. comparable between clonal ICUS and MDS

•  Clonal ICUS (CCUS) is a more frequent diagnosis than MDS in cytopenic paIents.

online October 1, 2015 originally publisheddoi:10.1182/blood-2015-08-667063

2015 126: 2355-2361

Matthew J. McGinniss, Shareef A. Nahas, Julie Kines and Rafael BejarBashar Dabbas, Aine Yung, Jenan Al-Hafidh, Emily Balmert, Christine Vaupel, Carlos El Hader, Brian Kwok, Jeff M. Hall, John S. Witte, Yin Xu, Prashanti Reddy, Keming Lin, Rachel Flamholz, common in idiopathic cytopenias of undetermined significanceMDS-associated somatic mutations and clonal hematopoiesis are


(483 articles)Plenary Papers (1540 articles)Myeloid Neoplasia

(4047 articles)Free Research Articles (4392 articles)Clinical Trials and Observations

Articles on similar topics can be found in the following Blood collections






cytopenias. In theprospective study,patientswere assignedadiagnosticgroupprior to genetic sequencing. In the retrospective group, sequentialcases of ICUS that had undergone genetic sequencing were comparedwith 91 cases of lower-risk MDS diagnosed in the same period. Bothcohorts demonstrated that somatic mutations indicative of clonalhematopoiesis are common in patients with ICUS, particularly if theydemonstrate some degree of dysplasia.

The frequency of ICUS in cytopenic patients suspected of havingMDS could be estimated from our prospective cohort. In this group,ICUS was diagnosed 5 times as often as MDS, with the majority ofICUS patients showing no evidence of dysplasia in their bone marrow.There is little data on the incidence of ICUS, but a reviewof cases sent toGenoptix for evaluation of cytopenias is consistent with our findings.However, an accurate measure of the incidence of ICUS will requirelarger, carefully controlled prospective studies. Our retrospective studyincluded a higher proportion of ICUS patients with some degree of dys-plasia vs none.However, it is likely that ICUSpatientswithout dysplasiawere underrepresented in this group because they were less likely tohave a genetic sequencing test ordered by the reviewing pathologist.

Although the proportion of patients with evidence of clonal he-matopoiesis was lower in ICUS patients, the absolute number withCCUS was substantially larger than the number diagnosed withMDS. The actual fraction of patients with CCUS may actually behigher because somepatientsmay have hadmutations not detected inour panel. Our findings conservatively suggest that the incidence ofCCUS is as great, or greater, than the incidence of MDS. This hasimplications for the diagnostic utility of somaticmutations in cytopenicpatients. Outcomes for CCUS patients are not known. It is unlikely thatthey all will progress to frank MDS as some may never develop dys-plasia or excess blasts and others might develop AML without an in-tervening diagnosis of MDS.30 Recognition of CCUS will allow thesepatients to be studied so that the incidence and the clinical significanceof their somatic mutations can be determined.

Mutations of different genes may have distinct prognostic sig-nificance in CCUS as they do inMDS. Our data demonstrate that the

spectrum of mutation in CCUS and MDS is fairly similar. Evenmutated genes associated with poor outcomes in MDS, like TP53,ASXL1, RUNX1, and DNMT3A, were identified in patients withCCUS at comparable frequencies. The only mutated gene overre-presented in MDS patients was SF3B1. This is likely because of thestrong association between SF3B1 mutations and the presence of ringsideroblasts, a readily identifiable feature associated with MDS.31,32

In fact, without consideration of ring sideroblasts, all 37 cases classifiedas having refractory anemia with ring sideroblasts (RARS) in theretrospective studywould not havemet the diagnostic criteria forMDS.Furthermore, pathologists who see ring sideroblasts even in the absenceof significant dysplasia may be more inclined to order geneticsequencing in these patients. Knowledge of an SF3B1 mutation (oranother mutation) could influence the quantification of ring sideroblastsand other forms of dysplasia in favor of meeting the diagnostic criteriafor MDS. However, in this study, all diagnoses were made prior to thereceipt of sequencing results and were not altered by this information.

The second major impediment to the use of somatic mutations asdiagnostic biomarkers comes from the high rate of age-dependentsomaticmutations in personswithout a knownhematologic disorder.These individuals are said to have clonal hematopoiesis of indeter-minate potential (CHIP).21-23 The prevalence of CHIP increasesgreatly with age and approaches 10% among 70- to 80-year olds, thetypical age range forMDSpatients. Because the incidence ofMDS inthis age group is orders of magnitude lower than 10%, most personswith somatic mutations indicative of clonal hematopoiesis neverdevelop MDS (or any other hematologic malignancy).33 Therefore,the presence of a somatic mutation in the blood of a patient withunexplained cytopenias may be an incidental finding unrelated totheir blood counts and cannot currently be relied upon to establish adiagnosis of MDS absent traditional criteria.10,26 It is certainly pos-sible that mutations found in our cohorts were unrelated to theirblood counts. However, more than one-third of ICUS patientshad CCUS as evidenced by somatic mutations or chromosomalabnormalities. Even in patients with no evidence of dysplasia, 27%had CCUS, which is over twofold greater than the rate of CHIP in asimilarly aged “normal” population. Furthermore, CHIP is a signif-icant risk factor for developing a hematologic malignancy, partic-ularly when allele frequency of a somatic variant is .10%. In ourstudies, the majority of CCUS patients had mutations with VAFs.10% and their distribution of VAFs showed little difference whencompared with lower-risk MDS patients. This alone would indicatethat CCUS patients have a substantially increased risk, comparedwith an age-matched population, of developing a frank hematologicmalignancy, albeit not necessarily MDS.21,30,34,35 Whether the pres-ence of a clinically significant cytopenia further increases that riskremains unknown and the clinical utility of gene sequencing in ICUSpatients has not been proven.

Table 2. Proposed criteria for CCUS

Peripheral blood findings Bone marrow findings Genetic findings

1 or more of the following: None of the following: 1 or more of the following:

Hemoglobin, ,11 g/dL $10% dysplasia in the granulocytic, erythroid, or

megakaryocytic lineage

An acquired chromosomal abnormality not diagnostic

of a heme malignancy

ANC ,1500/mL, 1.5 3 109/L Myeloblasts comprise $5% of total cellularity Presence of a somatic mutation with a VAF $2% in a

heme malignancy–associated gene in the peripheral

blood or bone marrow

Platelet count ,100 000/mL, 100 3 109/L An acquired chromosomal abnormality specific for

MDS/AML

Additional criteria: No other likely cause of cytopenias or evidence of another hematologic disorder.

Table 1. Clinical measures by mutation status for both cohorts

MDS CCUSNonclonal

ICUS P

Group size, N 115 136 233

Male:female ratio 1.0 1.65 0.68 .0003

Age, mean (SD), y 74.3 (10.4) 75.1 (12.5) 66.6 (13.6) ,.0001*

Hgb, mean (SD), g/dL 9.8 (1.8) 10.7 (1.8) 11.1 (2.0) ,.0001†

ANC, mean (SD), 3109/L 3.2 (2.1) 3.5 (2.3) 3.4 (2.4) .71

Platelets, mean (SD),

3109/L

184 (110) 164 (104) 179 (101) .27

ANC, absolute neutrophil count; SD, standard deviation.*No difference between MDS and CCUS (P 5 .87).†No difference between CCUS and ICUS (P 5 .21).

BLOOD, 19 NOVEMBER 2015 x VOLUME 126, NUMBER 21 SOMATIC MUTATIONS ARE COMMON IN ICUS 2359


Clonal ICUS (CCUS)

Significance of myelodysplastic syndrome-associated somatic variants in the evaluationof patients with pancytopenia and idiopathiccytopenias of undetermined significanceSebastian Fernandez-Pol, Lisa Ma, Robert S Ohgami and Daniel A Arber

Department of Pathology, Stanford University, Stanford, CA, USA

In this study, we set out to evaluate the frequency of mutations in 20 myelodysplastic syndrome-associatedgenes in 53 individuals with pancytopenia in which bone marrow evaluation failed to meet standard criteria for adiagnosis of myelodysplastic syndrome. These idiopathic pancytopenia cases were associated with no specificcause for their pancytopenia (n=28), aplastic anemia (n=13), pancytopenia attributable to liver disease (n=4),pancytopenia associated with autoimmune disease (n= 4), and pancytopenia attributed to drug effect (n=4). Wealso selected 38 bone marrow aspirates from patients presenting with pancytopenia and meeting criteria for adiagnosis of myelodysplastic syndrome (n=21) or acute myeloid leukemia (n=17) as malignant comparisoncases. Targeted sequencing of the 20 genes was performed on all cases. The idiopathic pancytopenia group hada lower average age (46 vs 66 years, Po0.0001) and a lower number of mutations per case that were statisticallysignificant (0.81 vs 1.18, P =0.045). The frequency of cases with at least one mutation was higher for cases with adiagnosable myeloid neoplasm (68 vs 38%, P= 0.012). Except for mutations in U2AF1, which was mutated in 5 ofthe 38 malignant cases (13.2%) and in none of the idiopathic pancytopenia cases (P=0.011), the frequency ofmutations in the genes evaluated was not significantly different between idiopathic pancytopenia and malignantcases. Median and mean clinical follow-up for the idiopathic pancytopenia group was available for 444 and739 days, respectively. Over this time frame, none of the idiopathic pancytopenia patients was diagnosed with amyelodysplastic syndrome or an acute myeloid leukemia. These findings provide further evidence thatidentification of mutations in several genes associated with myelodysplastic syndromes should not be usedalone to support a diagnosis of a myelodysplastic syndrome.Modern Pathology (2016) 29, 996–1003; doi:10.1038/modpathol.2016.100; published online 3 June 2016

The molecular characterization of myelodysplasticsyndromes has dramatically improved stratificationof this heterogeneous group of disorders intoclinically relevant subgroups. Although identifica-tion of chromosomal abnormalities has demon-strated utility in the diagnosis and prognosticstratification of myelodysplastic syndromes, thesignificance of somatic mutations in the diagnosisof these disorders is not as well established.1–5 It hasbeen estimated that approximately 80% of myelo-dysplastic syndrome cases have at least one muta-tion in one of approximately 40 genes.6–18 However,

it is essential to note that numerous studies havedemonstrated that many of the same mutationsassociated with myelodysplastic syndromes arefound in 5–10% of individuals over 70 years of ageand without evidence of overt neoplasia or anyclinical evidence of hematologic abnormality,a condition termed clonal hematopoiesis of indeter-minate potential.19–21 Individuals with clonal hema-topoiesis of indeterminate potential appear to havean increased risk of developing a hematologicmalignancy, a risk that increases with increasingvariant allele frequency. Clonal hematopoiesis ofindeterminate potential thus appears to represent abenign precursor condition that increases the risk ofsubsequent malignancy, similar to the relationshipbetween monoclonal gammopathy of undeterminedsignificance and plasma cell myeloma and smallmonoclonal B-cell lymphocytosis and chroniclymphocytic leukemia. However, the absolute risk

Correspondence: Dr S Fernandez-Pol MD, PhD, Department ofPathology, Stanford University, 300 Pasteur Drive, Lane 235,Stanford, CA 94305-5324, USA.E-mail: [email protected] 23 February 2016; revised 22 April 2016; accepted 23April 2016; published online 3 June 2016

Modern Pathology (2016) 29, 996–1003

996 © 2016 All rights reserved 0893-3952/16 $32.00

www.modernpathology.org

variant alleles were 1895 and 1019, respectively. Themean variant allele frequency of all of the variantalleles from all 91 cases was 0.31. Boxplot figuresof the read depths and variant allele frequencies forall genes and cases are shown in SupplementaryFigures 1 and 2. The distribution of the differenttypes of mutations (eg, transitions, transversions,insertions, and deletions) of the variants selectedfor further analysis is shown in SupplementaryFigure 4.

Somatic Variant Annotation

After sequencing, we used several criteria to identifysomatic variants. Somatic variants were called ifthey were (i) predicted to cause a change in amino-acid sequence (ie, substitutions, deletions, non-sense, and frame-shift mutations) and (ii) hada variant allele fraction that did not fall withinthe range of 45–55% to exclude cases that mightbe heterozygous variants. In addition, variantswere excluded if the allele frequency in theExome Aggregation Consortium (ExAC) databasewas greater than 1% in order to exclude potentialsingle-nucleotide polymorphisms. We also searchedCbioportal (http://www.cbioportal.org/) and the Cat-alogue of Somatic Mutations in Cancer (COSMIC,http://cancer.sanger.ac.uk/cosmic) to see whethervariants had been identified as somatic variants inother malignancies. All variants that passed theabove inclusion and exclusion criteria are shown inSupplementary Table 1 together with patient clinicalcharacteristics.

Statistics

All statistics reported were performed using R orExcel software.

Results

Cohort Description

The 53 patients with idiopathic pancytopeniaincluded 28 cases (53%) with no identifiable asso-ciated reason for their pancytopenia (idiopathic cyto-penia of undetermined significance group), 13 (25%)with aplastic anemia, 4 (8%) with pancytopeniaattributable to liver disease, 4 (8%) with pancytopeniaassociated with autoimmune disease, and 4 (8%) withpancytopenia suspected to be due to drug effect. Theassociation of these patients’ pancytopenia withparticular conditions was made based on the lack ofevidence of hematologic malignancy after bone mar-row examination and review of the clinical notes. Wegrouped the cases of myelodysplastic syndrome,myelodysplastic syndrome/myeloproliferative neo-plasm, and acute myeloid leukemia into a malignantpancytopenia group. The idiopathic pancytopeniagroup had a lower average age (46 vs 66 years,Po0.0001, Wilcoxon rank-sum test) and a lowernumber of mutations per case that were statisticallysignificant (idiopathic pancytopenia=0.81, range 0–5,vs malignant=1.18, range 0–5, P=0.041). The malig-nant group was more likely to have at least onemutation in any gene than the idiopathic pancytopeniagroup (68 vs 38%, P=0.012 chi-square test, Figure 1a).There was no difference between the groups in termsof the frequency of cases with two or more mutations.The distribution of the number of mutations in the twogroups is shown in Figure 1b. When looking withinage decades, among patients between 51 and 60 yearsof age, the malignant cases were more likely to have atleast one gene mutation than idiopathic pancytopeniacases (Po0.05, Fisher’s exact test, Figure 1c).

For the idiopathic pancytopenia group, we had amean follow-up time of 739 days (median= 444 days,range 0–7.7 years). For the idiopathic pancytopeniacases with at least one mutation (n=20), the mean

Table 1 Cohort Characteristics

Case subclassificationNumber of cases

(% of total) Average age (s.d.)

Number of cases with atleast one mutation (% of

subgroup)Number ofdeaths (%)

Mean follow-up in days

Median follow-up in days

Idiopathic pancytopeniaICUS 28 (31%) 50 (25) 12 (43%) 7 (13%) 830 430Aplastic anemia 13 (14%) 35 (15) 3 (23%) 0 730 461Liver disease 4 (4%) 59 (12) 2 (50%) 1 (2%) 433 455Autoimmune disease 4 (4%) 27 (7) 2 (50%) 0 730 794Drug effect 4 (4%) 54 (12) 1 (25%) 0 264 180Non-neoplastic total 53 (58%) 46 (22)a Med=49 20 (38%) 8 (15%) 739 444

MalignantMDS and MDS/MPN 21 (23%) 69 (30) 15 (71%)AML 17 (19%) 62 (25) 11 (65%)Malignant total 38 (42%) 66 (16)a Med=70 26 (68%)

Abbreviations: AML, acute myeloid leukemia; ICUS, idiopathic cytopenia of undetermined significance; MDS, myelodysplastic syndrome; MPN,myeloproliferative neoplasm.aIndicates Po0.0001 for Wilcoxon rank-sum test.


ICUS and MDS-associated mutations

998 S Fernandez-Pol et al

follow-up was 797 days, median was 397, range0–2799 (7.7 years). Eight of the individuals withidiopathic pancytopenia died in the follow-up periodavailable. Seven of the patients who died hadco-morbidities with high mortality rate, includingmalignancy (1 glioblastoma multiforme, 1 metastatichepatocellular carcinoma), cirrhosis (4 patients), andnecrotizing pancreatitis (1 patient). One patient diedin a motor vehicle accident. Of the eight idiopathicpancytopenia cases who died during the follow-upperiod, 5 cases had no mutation identified, 2 caseshad 1 mutation, and 1 case had 3 mutations. Of the 53individuals in the idiopathic pancytopenia group,none progressed to a myelodysplastic syndrome or anacute myeloid leukemia in the follow-up time avail-able (Table 1).

Somatic Mutations in U2AF1 are More FrequentlyFound in Acute Myeloid Leukemia and MyelodysplasticSyndromes than in Idiopathic Pancytopenias

In all, 11 of the 20 genes evaluated were morefrequently mutated in malignant cases than in

idiopathic pancytopenia cases, 7 were more fre-quently mutated in idiopathic pancytopenia cases,and no mutations were identified in 2 genes (IDH1and NRAS; Figure 2). The frequency of somaticmutations between the two groups was statisticallysignificant for U2AF1, which was mutated in 5 of the38 malignant cases (13%) and in none of theidiopathic pancytopenia cases (P-value = 0.0108,Fisher’s exact test, Figure 2). A U2AF1 mutationwas found in 2 cases of refractory cytopenia withmultilineage dysplasia, 1 case of refractory anemiawith excess blasts-2, 1 case of mixed myelodysplas-tic syndrome/myeloproliferative neoplasm with17% circulating blasts, and 1 case of acute myeloidleukemia. Four of the U2AF1 mutations identified inour study result in Q157P substitutions and the othermutation results in a Q157R substitution. Ourfindings are consistent with several reports thatidentify heterozygous Q157 substitutions in U2AF1(also known as U2AF35) mutations in cases ofmyelodysplastic syndrome and acute myeloidleukemia.27,28 Although the frequency of mutationsin the other genes was not different between thegroups at the Po0.05 significance level, TP53,

Figure 1 Frequency of mutations in 20 genes in 91 cases of pancytopenia. (a) Percent of cases with at least one mutation (P=0.012 chi-squaretest). (b) Distribution of the frequency of cases with 0, 1, 2, 3, 4, or 5 mutations per case. (c) Percent of cases with at least one mutation indifferent age decades. The asterisk indicates that Po0.05, Fisher’s exact test. IP, idiopathic pancytopenia cases; M, malignant cases.



S Fernandez-Pol et al 999

SETBP1, and RUNX1 were more frequently mutatedin malignant cases (Figure 2).

Discussion

Our findings are remarkably similar to those pub-lished in previous work that finds a significantnumber of somatic mutations in myelodysplasticsyndrome-associated genes in patients with idio-pathic cytopenias. In addition, in our cohort,individuals with idiopathic pancytopenia did notprogress to overt myelodysplastic syndrome or acutemyeloid leukemia within the clinical follow-upavailable for our study, even when a mutation isdetected. In aggregate, the findings of our work andthose of others raise additional questions regardingthe utility of testing for myelodysplastic syndromegenes in patients with cytopenias who do not meetconventional diagnostic criteria for myelodysplasticsyndrome.

It is interesting to compare our findings with thoseof Kwok et al, who also evaluated the frequency ofsomatic mutations in myelodysplastic syndrome-associated genes in cases of idiopathic cytopenia ofundetermined significance. The genes studied byKwok et al included the same 20 genes evaluated inour study except that they did not evaluate CEBPAand they additionally examined ETV6, PHF6, andZRSR2 in their study. Somatic mutations in the fourgenes that differ between our study and the priorstudy represent a small fraction of the variants foundin either study. In our idiopathic pancytopeniagroup, which includes patients with idiopathiccytopenia of undetermined significance as well as

patients with co-morbidities who are often asso-ciated with cytopenias, we find evidence of clonalhematopoiesis in 38% of cases. It is likely that this isa significant underestimate of the true frequency ofclonal hematopoiesis in these patients as we onlylooked at selected exons in 20 genes. In the study byKwok et al, it is not entirely clear whether theidiopathic cytopenia of undetermined significancecases selected included cases with the co-morbidities whom we include in our idiopathicpancytopenia group. It will be interesting to seewhether other investigators find evidence of muta-tions in myelodysplastic syndrome-associated genesin patients with cytopenias that are attributed topatient co-morbidities.

Although our study cohort differs from that ofKwok et al in several ways, our findings largelycorroborate the prior ones. In both studies, thefrequency of cases with at least one somatic variantin a myelodysplastic syndrome-associated gene issimilar (38% in our idiopathic pancytopenia groupand 35% overall in their idiopathic cytopenia ofundetermined significance group). TET2, DNMT3A,and ASXL1 were three most frequently mutatedgenes in both our idiopathic pancytopenia group andtheir idiopathic cytopenia of undetermined signifi-cance group. Importantly, the frequency of mutationsin these genes and most of the others evaluated didnot significantly differ between the idiopathic pan-cytopenia/idiopathic cytopenia of undeterminedsignificance groups and the myelodysplastic syn-drome/acute myeloid leukemia cohorts, suggestingthat variants in these genes are not useful indeciphering between myelodysplastic syndromeand several other causes of cytopenia. In both

Figure 2 Frequency of mutations in 20 genes evaluated in idiopathic pancytopenia vs malignant cases. The asterisk indicatesP-value =0.0108, Fisher’s exact test.



1000 S Fernandez-Pol et al

Significance of myelodysplastic syndrome-associated somatic variants in the evaluationof patients with pancytopenia and idiopathiccytopenias of undetermined significanceSebastian Fernandez-Pol, Lisa Ma, Robert S Ohgami and Daniel A Arber

Department of Pathology, Stanford University, Stanford, CA, USA

In this study, we set out to evaluate the frequency of mutations in 20 myelodysplastic syndrome-associatedgenes in 53 individuals with pancytopenia in which bone marrow evaluation failed to meet standard criteria for adiagnosis of myelodysplastic syndrome. These idiopathic pancytopenia cases were associated with no specificcause for their pancytopenia (n=28), aplastic anemia (n=13), pancytopenia attributable to liver disease (n=4),pancytopenia associated with autoimmune disease (n= 4), and pancytopenia attributed to drug effect (n=4). Wealso selected 38 bone marrow aspirates from patients presenting with pancytopenia and meeting criteria for adiagnosis of myelodysplastic syndrome (n=21) or acute myeloid leukemia (n=17) as malignant comparisoncases. Targeted sequencing of the 20 genes was performed on all cases. The idiopathic pancytopenia group hada lower average age (46 vs 66 years, Po0.0001) and a lower number of mutations per case that were statisticallysignificant (0.81 vs 1.18, P =0.045). The frequency of cases with at least one mutation was higher for cases with adiagnosable myeloid neoplasm (68 vs 38%, P= 0.012). Except for mutations in U2AF1, which was mutated in 5 ofthe 38 malignant cases (13.2%) and in none of the idiopathic pancytopenia cases (P=0.011), the frequency ofmutations in the genes evaluated was not significantly different between idiopathic pancytopenia and malignantcases. Median and mean clinical follow-up for the idiopathic pancytopenia group was available for 444 and739 days, respectively. Over this time frame, none of the idiopathic pancytopenia patients was diagnosed with amyelodysplastic syndrome or an acute myeloid leukemia. These findings provide further evidence thatidentification of mutations in several genes associated with myelodysplastic syndromes should not be usedalone to support a diagnosis of a myelodysplastic syndrome.Modern Pathology (2016) 29, 996–1003; doi:10.1038/modpathol.2016.100; published online 3 June 2016

The molecular characterization of myelodysplasticsyndromes has dramatically improved stratificationof this heterogeneous group of disorders intoclinically relevant subgroups. Although identifica-tion of chromosomal abnormalities has demon-strated utility in the diagnosis and prognosticstratification of myelodysplastic syndromes, thesignificance of somatic mutations in the diagnosisof these disorders is not as well established.1–5 It hasbeen estimated that approximately 80% of myelo-dysplastic syndrome cases have at least one muta-tion in one of approximately 40 genes.6–18 However,

it is essential to note that numerous studies havedemonstrated that many of the same mutationsassociated with myelodysplastic syndromes arefound in 5–10% of individuals over 70 years of ageand without evidence of overt neoplasia or anyclinical evidence of hematologic abnormality,a condition termed clonal hematopoiesis of indeter-minate potential.19–21 Individuals with clonal hema-topoiesis of indeterminate potential appear to havean increased risk of developing a hematologicmalignancy, a risk that increases with increasingvariant allele frequency. Clonal hematopoiesis ofindeterminate potential thus appears to represent abenign precursor condition that increases the risk ofsubsequent malignancy, similar to the relationshipbetween monoclonal gammopathy of undeterminedsignificance and plasma cell myeloma and smallmonoclonal B-cell lymphocytosis and chroniclymphocytic leukemia. However, the absolute risk

Correspondence: Dr S Fernandez-Pol MD, PhD, Department ofPathology, Stanford University, 300 Pasteur Drive, Lane 235,Stanford, CA 94305-5324, USA.E-mail: [email protected] 23 February 2016; revised 22 April 2016; accepted 23April 2016; published online 3 June 2016


996 © 2016 All rights reserved 0893-3952/16 $32.00

www.modernpathology.org

« Median and mean clinical follow-‐up for the idiopathic pancytopenia group available for 444 and 739 days, respecIvely. Over this Ime frame, none of the idiopathic pancytopenia paIents was diagnosed with MDS or AML »

MDS or not MDS ?




1472 VOLUME 20 | NUMBER 12 | DECEMBER 2014 NATURE MEDICINE

A N A LY S I S

Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5–6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.

Blood cells are continuously regenerated by hematopoietic stem/progenitor cells (HSPCs). Human HSPCs divide only rarely (estimated at once a month) but have self-renewal properties that sus-tain survival for decades. As HSPCs divide, they accumulate rare ran-dom mutations that generally do not affect function1. However, some mutations confer advantages in self-renewal, proliferation or both,

resulting in clonal expansion of the affected cells. Although these ‘initiating’ mutations do not lead directly to disease, they can cooper-ate with subsequent mutations to cause hematopoietic malignancies. For example, BCR-ABL and BCL2 oncogenic translocations have been found in blood cells of individuals without overt hematological malig-nancies2–4. The frequency of such events appears to increase with age, with a similar trend being found for somatic structural changes in the nuclear genomes of blood cells1,5. Single nucleotide polymorphism array analysis from large genome-wide association study cohorts showed ~2–3% of normal individuals of advanced age (70s and 80s) harbor leukemia-associated copy number changes that include genes such as DNMT3A (encoding DNA (cytosine-5-)-methyltransferase 3 ) and TET2 (encoding tet methylcytosine dioxygenase 2)6,7. More recently, somatic recurrent TET2 mutations were detected in the blood of elderly women without overt hematological malignancies8, and DNMT3A mutations were reported in nonleukemic cells9.

These findings have collectively led to the hypothesis that certain genetic mutations may confer advantages to affected HSPCs, result-ing in enhanced cell renewal, clonal expansion or both. However, it is unclear whether the effect involves only a small number of genes or many more genes related to leukemia and lymphoma and whether their participation in promoting clonal expansion necessarily leads to clones resembling cancer cells. Here, we address the former question by analyzing variations in 2,728 blood samples in The Cancer Genome Atlas (TCGA). We observed many individuals with age-related hematopoietic clonal mosaicism and concurrent presence of over 60 mutations in 19 leukemia- and/or lymphoma-associated genes. Our study identified not only genes but also specific mutations, associated with the clonal expansion process. Additional statistical analysis identified low-level (2–10% variant allele fractions (VAFs)) recurrent leukemic mutations in a substantial number of cases, pos-sibly in the early stages of clonal expansion. Moreover, our analysis suggests that DNMT3A, TET2, JAK2 (encoding Janus kinase 2), ASXL1 (encoding additional sex combs–like transcriptional regulator 1), SF3B1 (encoding splicing factor 3b, subunit 1) and TP53 (encoding tumor protein p53) have distinct and overlapping roles in the develop-ment of myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL) and/or acute myelogenous leukemia (AML). Finally, these results also incidentally highlight the need for caution when using blood as a reference for a surrogate ‘germline’ genome, especially in older individuals.

1The Genome Institute, Washington University in St. Louis, St. Louis, Missouri, USA. 2Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, USA. 3Brown School Master of Public Health Program, Washington University in St. Louis, St. Louis, Missouri, USA. 4Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, USA. 5Department of Mathematics, Washington University in St. Louis, St. Louis, Missouri, USA. 6Siteman Cancer Center, Washington University in St. Louis, St. Louis, Missouri, USA. 7These authors contributed equally to this work. Correspondence should be addressed to L.D. ([email protected]).

Received 29 May; accepted 21 September; published online 19 October 2014; doi:10.1038/nm.3733

Age-related mutations associated with clonal hematopoietic expansion and malignanciesMingchao Xie1,2,7, Charles Lu1,7, Jiayin Wang1,2,7, Michael D McLellan1, Kimberly J Johnson3, Michael C Wendl1,4,5, Joshua F McMichael1, Heather K Schmidt1, Venkata Yellapantula1,2, Christopher A Miller1, Bradley A Ozenberger1,2, John S Welch2,6, Daniel C Link2,6, Matthew J Walter2,6, Elaine R Mardis1,2,4,6, John F Dipersio2,6, Feng Chen2,6, Richard K Wilson1,2,4,6, Timothy J Ley1,2,4,6 & Li Ding1,2,4,6

Nature Medecine, 2014

•  N=2718 •  blood cells of more than 2% of individuals (5–6% of people

older than 70 years) show a somaIc mutaIon

A N A LY S I S


present in 1000 Genomes (see Online Methods) and having greater than 0.1% MAF as reported in the current Exome Variant Server data release (ESP6500SI-V2, http://evs.gs.washington.edu/EVS/) from 6,503 samples drawn from multiple NHLBI Exome Sequencing Project (ESP) cohorts. We focused on those mutations found in blood normal samples but not present or present only at very low levels in either the tumor samples or tumor-adjacent normal samples, as this pattern is highly suggestive of somatic mutation in HSPCs introduced by the clonal expansion process. Inflammatory lymphocytes, macrophages and neutrophils will infiltrate different tumors to different extents. Therefore, the hematopoietic mutations do not have to be completely absent in the tumor sample.

Our analysis of RTVs and KHVs in 556 selected cancer-associated genes identified 70 blood-specific mutations in 58 individuals. We fur-ther performed comparative analysis of blood versus tumor samples for these 58 individuals with the goal of detecting all blood-specific nonsynonymous mutations in the 556 cancer-associated genes; this analysis identified seven additional events (those that were probably loss of heterozygosity related to copy number alterations in the tumor were not included), yielding a final list of 77 blood-specific mutations in 58 cases (Table 1 and Supplementary Table 8). For 5 of those 58 cases that also had adjacent normal tissue analyzed, blood variants were absent in the adjacent normal tissue. Notably, among the 31 genes harboring these events, 19 have already been linked to hema-tological malignancies (Fig. 1a). In fact, 64 out of the total 77 events (83%) were in these 19 genes, examples as follows: DNMT3A (ref. 16; 18 cases), TET2 (ref. 17; nine cases), JAK2 (ref. 18; eight cases), ASXL1 (refs. 19,20; six cases), TP53 (ref. 21; four cases), SF3B1 (ref. 22;

two cases), BCORL1 (ref. 23; two cases), ASXL2 (ref. 24; one case) and SH2B3 (ref. 25; one case) (Table 1 and Fig. 1a). The overall frequency of blood-specific mutations increased with age (logistic regression analysis, P = 2.38 × 10−8); for example, 0.9% of the cases were in their 40s, 1.0% in their 50s, 1.8% in their 60s, 5.3% in their 70s and 6.1% in their 80s (Fig. 1b). The blood-specific mutations were found in all 11 cancer types (Fig. 1b). Frequencies of individual TET2, DNMT3A, ASXL1 and SF3B1 mutations also show association with age (all false discovery rate (FDR) values <0.034, logistic regression). TET2, ASXL1 and SF3B1 mutations were predominantly found in the oldest age groups (70s and 80s) and DNMT3A mutations in 60s to 80s. In con-trast, JAK2 mutations, which did not achieve significance, trended in both younger (40s and 50s) and older age groups (70s) (Fig. 1c).

The average age of the 54 individuals with blood-specific mutations in the 19 leukemia- and/or lymphoma-associated genes was 70.0 9.9 years, significantly higher than that of the larger TCGA cohort used in this study (difference of means test, P = 3.4 × 10−10) (Fig. 2a). Notably, the 6 individuals having 2 blood-specific mutations in the 9 recurrently mutated genes are relatively older, with ages of 64 years (DNMT3A: R882H, 36% VAF; TET2: H863fs, 12% VAF), 72 years

c

0

1

2

3

4

5

6

% o

f affe

cted

TC

GA

cas

es in

eac

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<40(n = 214)

40–49(n = 341)

50–59(n = 707)

60–69(n = 767)

70–79(n = 475)

80–89(n = 132)

Age (years)

GenesASXL1

DNMT3A

GNAS

JAK2

SF3B1

TET2

Total

a

0

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0

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Age (years)

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ASXL1

TP53

GNAS

PPM1D

BCORL1

JAK2

TET2

SF1ASXL2

SH2B3

CDKN2A

AXL

ZRSR2

IDH2

CREBBP

SETBP1

MYLK

CBL

Samples with two mutationsAge data missing

*ATM, DIDO1, GUCY1A2, HDAC4,MBD1, MECOM, NOTCH3, PRKDC,RICTOR, SNX25, SOS1

Can

cer-a

ssocia

ted genes not known

to b

e inv

olved in

hematological malignancy*

DNMT3A

SF3B

1

PRAD

LGG UCEC

OVLUSC

KIRC

GBMHNSC

STAD

BRCA

LUAD

b100

Age (years)

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0

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70–79 (5.26%)

n = 475

60–6

9 (1

.83%

)n

= 76

7

50–59 (0.99%)

n = 707

40–49 (0.88%)n = 341

80–89 (6.06%)

n = 132

Figure 1 Blood-specific mutations identified in 58 out of 2,728 TCGA cases from 11 cancer types. (a) Rose chart illustrating the distribution of blood-specific nonsynonymous mutations in 31 genes. The VAFs of the 77 mutations are indicated in the center. (b) Rose chart illustrating the age distribution of samples with blood-specific mutations. The cancer type distribution is shown in the center. (c) Distribution of blood-specific mutations in DNMT3A, TET2, JAK2, ASXL1, SF3B1 and GNAS in different age groups. Total includes all blood-specific mutations in 556 cancer-associated genes identified in each age group.

T h e n e w e ngl a nd j o u r na l o f m e dic i n e

n engl j med nejm.org 1

original article

Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes

Siddhartha Jaiswal, M.D., Ph.D., Pierre Fontanillas, Ph.D., Jason Flannick, Ph.D., Alisa Manning, Ph.D., Peter V. Grauman, B.A., Brenton G. Mar, M.D., Ph.D.,

R. Coleman Lindsley, M.D., Ph.D., Craig H. Mermel, M.D., Ph.D., Noel Burtt, B.S., Alejandro Chavez, M.D., Ph.D., John M. Higgins, M.D., Vladislav Moltchanov, Ph.D., Frank C. Kuo, M.D., Ph.D., Michael J. Kluk, M.D., Ph.D., Brian Henderson, M.D., Leena Kinnunen M.Sc., Heikki A. Koistinen, M.D., Ph.D., Claes Ladenvall, Ph.D.,

Gad Getz, Ph.D., Adolfo Correa, M.D., Ph.D., Benjamin F. Banahan, Ph.D., Stacey Gabriel, Ph.D., Sekar Kathiresan, M.D., Heather M. Stringham, Ph.D.,

Mark I. McCarthy, M.D.,* Michael Boehnke, Ph.D.,* Jaakko Tuomilehto, M.D., Ph.D., Christopher Haiman, Sc.D., Leif Groop, M.D., Ph.D., Gil Atzmon, Ph.D.,

James G. Wilson, M.D., Donna Neuberg, Sc.D., David Altshuler, M.D., Ph.D.,* and Benjamin L. Ebert, M.D., Ph.D.†

The authors’ affiliations are listed in the Appendix. Address reprint requests to Dr. Ebert at the Department of Medicine, Division of Hematology, Brigham and Women’s Hospital, Harvard Medical School, 1 Blackfan Cir., Karp 5.210, Boston, MA 02115, or at [email protected].

* Dr. McCarthy is listed on behalf of the Type 2 Diabetes (T2D) Genetic Explora-tion by Next-Generation Sequencing in Multi-Ethnic Samples (T2D-GENES) study investigators; Dr. Boehnke, on be-half of the Genetics of Type 2 Diabetes (GoT2D) study investigators; and Dr. Altshuler, on behalf of the SIGMA T2D study investigators.

† A complete list of investigators in the T2D-GENES, GoT2D, and SIGMA T2D studies is provided in the Supplemen-tary Appendix, available at NEJM.org.

This article was published on November 26, 2014, at NEJM.org.

DOI: 10.1056/NEJMoa1408617Copyright © 2014 Massachusetts Medical Society.

A BS TR AC T

BackgroundThe incidence of hematologic cancers increases with age. These cancers are associ-ated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders.

MethodsWe analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide vari-ants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events.

ResultsDetectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respec-tively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8).

ConclusionsAge-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the lat-ter possibly due to an increased risk of cardiovascular disease. (Funded by the Na-tional Institutes of Health and others.)

The New England Journal of Medicine Downloaded from nejm.org by PIERRE FENAUX on November 26, 2014. For personal use only. No other uses without permission.

Copyright © 2014 Massachusetts Medical Society. All rights reserved.

17,182 individuals Muta3ons, predominantly of DNMT3A, TET2, and ASXL1 genes in normal subject •  70 to 79 years of age, 9.5% •  80 to 89 years : 11.7% •  90 to 108 years :18.4%

somaIc mutaIon associated with increased risk of developing hematologic malignancy (HR 11), increased all-‐cause mortality (HR 1.4), and increased risk of incident coronary heart disease (HR 2.0 ) and ischemic stroke (HR 2.6).

Age-Related Clonal Hematopoiesis and Adverse Outcomes

n engl j med nejm.org 3

The identification of somatic driver mutations in cancer has come largely from studies that have compared differences in DNA sequence between tumor and normal tissue obtained from the same person. Once mutations are identified, investiga-tors may genotype samples for these somatic vari-ants without relying on matched normal tissue. Because we had DNA from only one source (blood), we limited our examination to variants that had been described previously in the litera-ture in 160 recurrently mutated candidate genes in myeloid and lymphoid cancers (Table S2 in the Supplementary Appendix). We removed potential false positive variants by using variant-calling algorithms that had filters for known artifacts such as strand-bias and clustered reads, as well as by performing additional filtering for rare error modes using a “panel of normals” (sequence data from a panel of normal persons).25 The lower limit of detection for variants depended on the depth of coverage. The median average sequenc-ing depth for exons from the 160 genes was 84 reads (range, 13 to 144). At a sequencing depth of 84 reads, the limit of detection for SNVs was at a variant allele fraction of 0.035; the limit of detection for indels was 0.070.

With this approach, we identified a total of 805 candidate somatic variants (hereafter referred to as mutations) in 73 genes from 746 persons (Table S3 in the Supplementary Appendix). As a negative control, we searched for previously de-scribed, nonhematologic cancer-associated vari-ants in 40 genes (Table S4 in the Supplementary Appendix)27 and found only 10 such variants in these genes (Table S5 in the Supplementary Ap-pendix), indicating that the rate of false discovery due to technical artifacts was low. We also verified a subset of the variants using amplicon-based, targeted sequencing; 18 of 18 variants were con-firmed, with a correlation coefficient of 0.97 for the variant allele fraction between the two meth-ods (Fig. S1 in the Supplementary Appendix).

Increase in the Frequency of Clonal Somatic Mutations with Age

Hematologic cancers, as well as other cancers and premalignant states, increase in frequency with age. Mutations were very rare in samples obtained from patients younger than 40 years of age but rose in frequency with each decade of life thereafter (Fig. 1). Mutations in genes implicated in hematologic cancers were found in 5.6% (95%

confidence interval [CI], 5.0 to 6.3) of persons 60 to 69 years of age, 9.5% (95% CI, 8.4 to 10.8) of persons 70 to 79 years of age (219 of 2300 per-sons), 11.7% (95% CI, 8.6 to 15.7) of persons 80 to 89 years of age (37 of 317), and 18.4% (95% CI, 12.1 to 27.0) of persons 90 years of age or older (19 of 103). These rates greatly exceed the inci-dence of clinically diagnosed hematologic cancer in the general population.28

Though we searched for mutations in genes implicated in many different hematologic can-cers, we primarily identified genes that were most frequently mutated in AML and the myelodys-plastic syndrome. The most commonly mutated gene was DNMT3A (403 variants) (Fig. 2A, and Fig. S2 in the Supplementary Appendix), fol-lowed by TET2 (72 variants) and ASXL1 (62 vari-ants). In TET2, only exon 3 was obtained by exon capture (corresponding to approximately 50% of the coding region), and the portion of exon 12 of ASXL1 that accounts for approximately 50% of the mutations in this gene had poor coverage depth. Thus, mutations in TET2 and ASXL1 are probably underrepresented in this study. Other frequently mutated genes included TP53 (33 vari-ants), JAK2 (31 variants), and SF3B1 (27 variants).

In sequencing studies of the myelodysplastic syndrome and AML, most patients have mutations in two or more driver genes (the median number of recurrently mutated genes in patients with de

Freq

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y

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No. with MutationTotal

0240

517

1486

37317

2192300

2825002

1385441

502894

1855

Figure 1. Prevalence of Somatic Mutations, According to Age.

Colored bands, in increasingly lighter shades, represent the 50th, 75th, and 95th percentiles.




n engl j med nejm.org4

novo AML is five17). In this study, we found that 693 of 746 persons with a detectable mutation had only one mutation in the set of genes we examined (Fig. 2B, and Fig. S2 in the Supplemen-tary Appendix), a finding that was consistent with the hypothesis that these persons had clones har-boring only an initiating lesion.

The most common base-pair change in the somatic variants was a cytosine-to-thymine (C→T) transition (Fig. 2C), which is considered to be a somatic mutational signature of aging.16,29 The

median variant allele fraction for the identified mutations was 0.09 (Fig. 2D), suggesting that the variants are present in only a subset of blood cells and supporting their somatic rather than germ-line origin.

Persistence of Somatic Mutations over TimeBlood-cell DNA obtained 4 to 8 years after the initial DNA collection was available for targeted sequencing in 13 persons with 17 somatic muta-tions (4 persons had 2 mutations). In all cases,

No.

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Allele Fraction

Nonsense Variants Indel Variants

Splice-Site Variants Missense Variants

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A403

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693

492 2

No.

of P

atie

nts

800

600

400

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01 2 3 4

Mutations

B

Mean AF=0.12Median AF=0.09




Figure 2. Characteristics of Candidate Somatic Variants.

Panel A shows the 10 most frequently mutated genes implicated in hematologic cancers. Panel B shows the number of persons with 1, 2, 3, or 4 candidate variants. Panel C shows the distribution of the types of single-nucleotide base-pair changes seen in the candidate variants. Panel D shows the allele fractions (AFs) of candidate somatic variants. The allele fraction was calculated as the number of vari-ant reads divided by the number of variant-plus-reference reads. For variants on the X chromosome in men, this number was divided by 2. Indel denotes insertions and deletions.




n engl j med 371;26 nejm.org december 25, 2014 2477

original article

Clonal Hematopoiesis and Blood-Cancer Risk Inferred from Blood DNA SequenceGiulio Genovese, Ph.D., Anna K. Kähler, Ph.D., Robert E. Handsaker, B.S.,

Johan Lindberg, Ph.D., Samuel A. Rose, B.S., Samuel F. Bakhoum, M.D., Ph.D., Kimberly Chambert, M.S., Eran Mick, B.S., Benjamin M. Neale, Ph.D.,

Menachem Fromer, Ph.D., Shaun M. Purcell, Ph.D., Oscar Svantesson, M.S., Mikael Landén, Ph.D., Martin Höglund, M.D., Ph.D., Sören Lehmann, M.D., Ph.D.,

Stacey B. Gabriel, Ph.D., Jennifer L. Moran, Ph.D., Eric S. Lander, Ph.D., Patrick F. Sullivan, M.D., Pamela Sklar, M.D., Ph.D., Henrik Grönberg, M.D., Ph.D.,

Christina M. Hultman, Ph.D., and Steven A. McCarroll, Ph.D.

The authors’ affiliations are listed in the Appendix. Address reprint requests to Dr. McCarroll at the Department of Genetics, Harvard Medical School, 77 Ave. Louis Pas-teur, Boston, MA 02115, or at [email protected]; or to Dr. Geno-vese at the Broad Institute of MIT and Har-vard, 75 Ames St., Cambridge, MA 02142, or at [email protected].

This article was published on November 26, 2014, at NEJM.org.

N Engl J Med 2014;371:2477-87.DOI: 10.1056/NEJMoa1409405Copyright © 2014 Massachusetts Medical Society.

A BS TR AC T

BackgroundCancers arise from multiple acquired mutations, which presumably occur over many years. Early stages in cancer development might be present years before cancers be-come clinically apparent.

MethodsWe analyzed data from whole-exome sequencing of DNA in peripheral-blood cells from 12,380 persons, unselected for cancer or hematologic phenotypes. We identified somatic mutations on the basis of unusual allelic fractions. We used data from Swed-ish national patient registers to follow health outcomes for 2 to 7 years after DNA sampling.

ResultsClonal hematopoiesis with somatic mutations was observed in 10% of persons older than 65 years of age but in only 1% of those younger than 50 years of age. Detect-able clonal expansions most frequently involved somatic mutations in three genes (DNMT3A, ASXL1, and TET2) that have previously been implicated in hematologic cancers. Clonal hematopoiesis was a strong risk factor for subsequent hematologic cancer (hazard ratio, 12.9; 95% confidence interval, 5.8 to 28.7). Approximately 42% of hematologic cancers in this cohort arose in persons who had clonality at the time of DNA sampling, more than 6 months before a first diagnosis of cancer. Analysis of bone marrow–biopsy specimens obtained from two patients at the time of diag-nosis of acute myeloid leukemia revealed that their cancers arose from the earlier clones.

ConclusionsClonal hematopoiesis with somatic mutations is readily detected by means of DNA sequencing, is increasingly common as people age, and is associated with increased risks of hematologic cancer and death. A subset of the genes that are mutated in patients with myeloid cancers is frequently mutated in apparently healthy persons; these mutations may represent characteristic early events in the development of hematologic cancers. (Funded by the National Human Genome Research Institute and others.)

The New England Journal of Medicine Downloaded from nejm.org on September 19, 2016. For personal use only. No other uses without permission.


•  12,380 persons •  10% persons > 65 years of age but in only 1% < 50 years . •  most frequently involved somaIc mutaIons in three genes (DNMT3A,

ASXL1, and TET2) •  Clonal hematopoiesis strong risk factor for subsequent hematologic

cancer (hazard raIo, 12.9).


n engl j med 371;26 nejm.org december 25, 20142480

tions might also promote clonal hematopoiesis. We therefore considered 208 specific variants reported as recurring (found in ≥7 patients) in hematopoietic and lymphoid cancers, as listed in the Catalogue of Somatic Mutations in Cancer30

(see the Supplementary Appendix for details). We found 98 instances of these recurring muta-tions, including JAK2 mutation p.V617F31 in 24 participants, DNMT3A mutation p.R882H32 in

15 participants, and SF3B1 mutation p.K700E33

in 9 participants.Disruptive mutations in DNMT3A, TET2, ASXL1,

and PPM1D, missense mutations in DNMT3A, and the recurring cancer-associated mutations com-prised a set of 327 candidate driver somatic mutations for clonal hematopoiesis in 308 par-ticipants (Fig. 2B, and Table S3 in the Supple-mentary Appendix), with 18 participants carry-

11

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71315

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35190DNMT3A

ASXL1

JAK2TET2

PPM1DSF3B1SRSF2

TP53CBL

MYD88U2AF1STAT3IDH2ATM

0 20 40 180 200

No. of Mutations

C

A B

Gene

All genes — % of mutations

Specific genes — no. of mutationsDNMT3A TET2 ASXL1 PPM1D

DisruptiveMutation

MissenseMutation

SynonymousMutation P Value

4.4×10−26

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6.0×10−13

7.0×10−9

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D

Clonal hematopoiesis withcandidate drivers

Clonal hematopoiesis withunknown drivers

Clonal hematopoiesis

DNMT3AASXL1

JAK2TET2

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TP53CBL

U2AF1IDH2

Participants with Multiple Candidate Drivers

One mutation Two mutations

6

33 20 15 10

64

47 2 4 0

31

1200

Figure 2. Candidate Driver Somatic Mutations.

Panel A shows all genes identified as carrying a significant excess of disruptive (nonsense, frame-shift, and splice-site) somatic mutations among 11,845 participants with sequencing data of sufficient quality for the detection of somatic mutations. To control for potential systematic sequencing artifacts due to sequence context, somatic mu-tations observed in multiple participants were counted only once. Panel B shows the contribution of individual genes to the total number of candidate driver somatic mutations that were observed. Panel C shows a comutation plot for participants with multiple candidate driver somatic mutations. Panel D shows estimates for participants with clonal hematopoiesis with candidate drivers (carrying at least one candidate driver mutation), those with clon-al hematopoiesis with unknown drivers (carrying three or more detectable somatic mutations and no candidate drivers), and those with clonal hematopoiesis and candidate or unknown drivers. The shaded bands represent 95% confidence intervals.





tions might also promote clonal hematopoiesis. We therefore considered 208 specific variants reported as recurring (found in ≥7 patients) in hematopoietic and lymphoid cancers, as listed in the Catalogue of Somatic Mutations in Cancer30

(see the Supplementary Appendix for details). We found 98 instances of these recurring muta-tions, including JAK2 mutation p.V617F31 in 24 participants, DNMT3A mutation p.R882H32 in

15 participants, and SF3B1 mutation p.K700E33

in 9 participants.Disruptive mutations in DNMT3A, TET2, ASXL1,

and PPM1D, missense mutations in DNMT3A, and the recurring cancer-associated mutations com-prised a set of 327 candidate driver somatic mutations for clonal hematopoiesis in 308 par-ticipants (Fig. 2B, and Table S3 in the Supple-mentary Appendix), with 18 participants carry-

11

11

134

71315

2431

35190DNMT3A

ASXL1

JAK2TET2

PPM1DSF3B1SRSF2

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MYD88U2AF1STAT3IDH2ATM

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No. of Mutations

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A B

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All genes — % of mutations

Specific genes — no. of mutationsDNMT3A TET2 ASXL1 PPM1D

DisruptiveMutation

MissenseMutation

SynonymousMutation P Value

4.4×10−26

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7.0×10−9

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Clonal hematopoiesis withunknown drivers

Clonal hematopoiesis

DNMT3AASXL1

JAK2TET2

PPM1DSF3B1SRSF2

TP53CBL

U2AF1IDH2

Participants with Multiple Candidate Drivers

One mutation Two mutations

6

33 20 15 10

64

47 2 4 0

31

1200

Figure 2. Candidate Driver Somatic Mutations.

Panel A shows all genes identified as carrying a significant excess of disruptive (nonsense, frame-shift, and splice-site) somatic mutations among 11,845 participants with sequencing data of sufficient quality for the detection of somatic mutations. To control for potential systematic sequencing artifacts due to sequence context, somatic mu-tations observed in multiple participants were counted only once. Panel B shows the contribution of individual genes to the total number of candidate driver somatic mutations that were observed. Panel C shows a comutation plot for participants with multiple candidate driver somatic mutations. Panel D shows estimates for participants with clonal hematopoiesis with candidate drivers (carrying at least one candidate driver mutation), those with clon-al hematopoiesis with unknown drivers (carrying three or more detectable somatic mutations and no candidate drivers), and those with clonal hematopoiesis and candidate or unknown drivers. The shaded bands represent 95% confidence intervals.





DNA sampling (Table S6 in the Supplementary Appendix) than participants without any detect-able putative somatic mutations (hazard ratio, 12.9; 95% confidence interval [CI], 5.8 to 28.7; P<0.001 by Cox proportional-hazards analysis of time to a diagnosis of hematologic cancer, with adjustment for age and sex) (Fig. 3A). Participants with candidate drivers and those with unknown drivers had similarly elevated risks (Fig. 3B). Of the 31 participants who received a diagnosis of hematologic cancer more than 6 months after

DNA sampling, 13 participants (42%) had shown clonal hematopoiesis in their initial DNA sample (Fig. 3C). Participants with clonal hematopoiesis were also more likely than those without clonal hematopoiesis to have a history of hematologic cancer, though this did not contribute to the above result (Table S7 in the Supplementary Appendix).

Participants with clonal hematopoiesis had reduced overall survival (hazard ratio for death, 1.4; 95% CI, 1.0 to 1.8; P = 0.03 by Cox propor-tional-hazards model, with adjustment for age

Prop

ortio

n w

ithou

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atol

ogic

Can

cer

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0.99

0.97

0.96

0.94

0.95

0.000 20 40 60 80

Months

D

C

E

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Hazard ratio, 12.9 (95% CI, 5.8–28.7)P<0.001

Prop

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ing

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0.95

0.90

0.85

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0.000 20 40 60 80

Months

Hazard ratio, 1.4 (95% CI, 1.0–1.8)P=0.03

0.50 5.00 50.00

No mutationsOne mutationTwo mutationsCH-UDCH-CDCH

Hazard Ratio for Hematologic Cancer (95% CI)No. of

PatientsCategory

0.47 (0.06–3.56)

13.73 (5.74–32.83)11.34 (3.44–37.41)1.99 (0.26–15.27)

1.00 (1.00–1.00)

12.89 (5.78–28.72)

0.05

87831122237170269439

1.00 2.00

No mutationsOne mutationTwo mutationsCH-UDCH-CDCH

Hazard Ratio for Death (95% CI)No. of

PatientsCategory

0.98 (0.75–1.29)

1.53 (1.07–2.19)1.16 (0.73–1.83)1.30 (0.84–2.01)

1.00 (1.00–1.00)

1.38 (1.03–1.84)

0.50

88241128240175280455

No clonalhematopoiesis

(N=8783)

No clonalhematopoiesis

(N=8824)

Clonalhematopoiesis

(N=439)

Clonalhematopoiesis

(N=455)

Persons in Whom HematologicCancer Was Later Diagnosed

Age-Matched Personswithout Cancer

CH-CDCH-UD

CH-UD4

CH-CD9

No CH18

No CH

Figure 3. Risk of Hematologic Cancer for Participants with Clonal Hematopoiesis.

Panels A and D show Kaplan–Meier plots of the proportions of participants who did not receive a diagnosis of hematologic cancer (Panel A) and for surviving participants (Panel D). Panels B and E show hazard ratios for hematologic cancer (Panel B) and death (Panel E) for participants with exactly one putative somatic mutation and no candidate drivers (one mutation), those with exactly two putative somatic mutations and no candidate drivers (two mutations), those with clonal hematopoiesis and unknown drivers (CH-UD), those with clonal hematopoiesis and candidate drivers (CH-CD), and those having clonal hematopoiesis with candidate or unknown drivers (CH), all compared with participants with no candidate drivers and no putative somatic mutations (no mutations). Panel C shows the proportions of participants who had clonal hematopoiesis with candidate or unknown drivers among 31 participants in whom hematologic cancers were diagnosed at least 6 months after DNA sampling, as compared with the proportions in a group of age-matched persons without hematologic cancer over a similar follow-up period.



Proposed criteria for CHIP

Wepropose that CHIPbe used to describe patientswhohave detectablesomatic clonal mutations in genes recurrently mutated in hematologicmalignancies (Figure 2A) but who lack a known hematologic malig-nancy or other clonal disorder. Under this definition, CHIP wouldencompass cytopenic patients with concurrent cancer-associatedmutations who do not meet diagnostic criteria for MDS, as well asthose with normal peripheral blood counts. CHIP would not includeclearly described clonal conditions such as paroxysmal nocturnalhemoglobinuria,MBL, orMGUS.We also propose that as aworkingdefinition, the mutant allele fraction must be$2% in the peripheral

blood, because with deep enough sequencing, a mutation can befound in every individual, and current outcomes data are based ona minimum variant allele fraction of.2% in peripheral blood.7,68 Itis currently unknown if variants present below this threshold alsocarry increased risk of adverse outcomes, and this threshold may needto be revised. A copy number variant resulting from a chromosomalrearrangement involving a chromosomal region where hematologicneoplasia-associated genes are encoded is also consistent with CHIP.

CHIP is distinct fromMDSbecauseCHIP is associatedwith amuchlonger survival, normal blood counts in most cases, and low rate ofprogression to AML. Individuals with CHIP have an increased risk ofdisease progression to hematologic neoplasia compared with individ-uals without detectable mutations, and this risk appears to be propor-tional to the size of the somatic clone; however, the rate of progressionappears to be only 0.5% to 1% per year, similar to MBL and MGUS.Although the annual rate of progression of CHIP,MBL, andMGUS toovert neoplasia is comparable, MBL andMGUS represent expansionsof lineage-committed cells,whereasCHIP involveshematopoietic stemcells or less mature progenitor cells, and thus CHIP is a precursor statefor a broader range of hematologic neoplasms (Figure 1B).

Although a growing body of evidence confirms that individualswith a first “hit” leading to CHIP are at increased risk of developingovert malignancy and that CHIP is also associated with increasedmortality from nonneoplastic causes, the optimal clinical managementof these individuals is uncertain. In the absence of any available inter-vention to prevent progression, surveillance similar towhat is currentlyrecommended for MGUS could be considered.4,8,69 In the near term,most individuals who will be screened for somatic mutations will bethose with hematologic abnormalities that do not meet criteria forcurrent diagnostic entities. Future studies will be needed to delineatewhether certain subgroupswithCHIP have a higher risk of progressionto malignancy or other adverse outcomes. Such subgroups couldinclude those with an unexplained cytopenia (see below), lymphocy-tosis, leukocytosis, persistent eosinophilia, or persistent monocytosisthat do not meet criteria for recognized entities. The specific geneticmutations, number of mutations, and mutant allele fraction may alsoinfluence the risk of progression and could further refine diagnosticcriteria. It is likely that a substantial number of people without he-matologic abnormalities who have genetic sequencing performed forpersonal, research, or medical reasons (eg, on a blood-contaminatedsolid tumor biopsy) will incidentally be found to have CHIP. In certainsettings, such as following cytotoxic therapy for a nonhematologicdisorder, CHIP may have more ominous implications.70

Proposed diagnostic criteria for MDS

To diagnose MDS, it is important to rule out other disorders that canmimic MDS. Testing algorithms such as those advocated by theNational Comprehensive Cancer Network and the European Leuke-miaNet are helpful for this purpose.71,72 Given the large percentage ofindividuals with CHIP, a somaticmutation is not sufficient to diagnoseMDS, even in the context of prior therapy for another malignancy withradiation or cytotoxic drugs or a history of another marrow failuredisorder such as aplastic anemia.73 Like dysplasia or MDS-associatedcytogenetic abnormalities, somaticmutationsmust have a known asso-ciation with MDS and be accompanied by clinically meaningful cyto-penias to consider an MDS diagnosis.

Patients with cytopenias who lack MDS-defining features but inwhom no other cause for cytopenias is evident are said to have ICUS.The ICUS label includes a highly heterogeneous population, and ICUS

Figure 2. Definition of CHIP and its distinction from MDS and non-clonal cyto-penic states. (A) A proposed definition of CHIP. A mutation that is commonly associatedwith clonal expansion of hematopoietic cells in older persons should be present, whereascriteria for other diagnoses should not be met. Evidence of mildly disordered erythro-poiesis such as an elevated red cell distribution width or mean corpuscular volume canbe compatible with CHIP rather than MDS, and occasional dysplastic cells might beseen, as is common in the general population with careful scrutiny of blood and marrow.CHIP is associated with an increased risk of all-causemortality and subsequent diagnosisof hematological malignancy. The 19 genes most commonly mutated in healthy olderadults in sequencing studies to date are listed. The roster of CHIP-associated mutationswill likely change in the future, with some genes being removed and others being added.As a working definition, we propose a variant allele frequency of 2% in order to beconsidered CHIP (since extremely deep sequencing will detect mutations in almost everyperson), but this may need to be revised with further population analyses. PNH, par-oxysmal nocturnal hemoglobinuria. (B) The spectrum of clonal hematopoiesis, ICUS,and MDS. ICUS is a broad category that includes a heterogeneous group of individuals,some of whom have benign (nonclonal) hematopoiesis. Other patients with ICUS mayhave CHIP, differing only from lower risk MDS by their lack of dysplasia and, currently, anundetermined disease risk. CHIP can also include patients with clonal hematopoiesisand nonmalignant causes of cytopenias (eg, immune cytopenias, liver disease, or nutri-tional deficiencies) that would not be considered to have ICUS because of the presenceof a clone, but may have a distinct natural history. Obs, observation; BM, bone marrow;CCUS, clonal cytopenias of undetermined significance; HMA, hypomethylating agent(eg, azacitidine); GF, hematopoietic growth factor (eg, epoetin); IMiD, immunomodula-tory drug (eg, lenalidomide); IST, immunosuppressive therapy; BSC, best supportivecare; HSCT, hematopoietic stem cell transplant.

BLOOD, 2 JULY 2015 x VOLUME 126, NUMBER 1 CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL 13

For personal use only.on September 18, 2016. by guest www.bloodjournal.orgFrom online April 30, 2015

originally publisheddoi:10.1182/blood-2015-03-6317472015 126: 9-16

Robert P. Hasserjian and Benjamin L. EbertDavid P. Steensma, Rafael Bejar, Siddhartha Jaiswal, R. Coleman Lindsley, Mikkael A. Sekeres, myelodysplastic syndromesClonal hematopoiesis of indeterminate potential and its distinction from


(188 articles)Perspectives (1540 articles)Myeloid Neoplasia

(4051 articles)Free Research Articles Articles on similar topics can be found in the following Blood collections






disease (HR, 2.0) and ischemic stroke (HR, 2.6). Increased mortal-ity was synergistic with elevated red cell distribution width, whichmight be a marker of disordered erythropoiesis because of the ex-panded clone.

Current understanding of the multistep pathogenesis of cancersuggests that individuals with clonal mutations may already bepartway along the path to evolution of frank malignancy. However,although a higher rate of subsequent cancer diagnosis in patientswith

Figure 1. CHIP as a precursor state for hematological neoplasms. (A) A model for evolution from normal hematopoiesis to CHIP and then, in some cases, to MDS or AML.(B) Comparison of evolution patterns of MGUS, MBL, and CHIP. Hematopoietic progenitor or stem cells commonly acquire mutations throughout the human lifespan; most ofthese are passenger mutations that have no consequence for hematopoiesis. Certain mutations, however, confer a survival advantage to the mutated cell and its progeny andallow clonal expansion. Serial acquisition of mutations in an expanded clone can lead to a disease phenotype and ultimately morbidity and mortality. Although this article primarilydiscusses CHIP in the context of its distinction from MDS, CHIP can also directly progress to AML without an intervening MDS stage, and CHIP can progress to other conditionssuch as myeloproliferative neoplasms or lymphoid neoplasms. Just as with MGUS and MBL, the majority of patients with CHIP will never develop an overt neoplasm, andpatients will eventually die of unrelated causes.



online April 30, 2015 originally publisheddoi:10.1182/blood-2015-03-631747

2015 126: 9-16










Proposed criteria for CHIP

Wepropose that CHIPbe used to describe patientswhohave detectablesomatic clonal mutations in genes recurrently mutated in hematologicmalignancies (Figure 2A) but who lack a known hematologic malig-nancy or other clonal disorder. Under this definition, CHIP wouldencompass cytopenic patients with concurrent cancer-associatedmutations who do not meet diagnostic criteria for MDS, as well asthose with normal peripheral blood counts. CHIP would not includeclearly described clonal conditions such as paroxysmal nocturnalhemoglobinuria,MBL, orMGUS.We also propose that as aworkingdefinition, the mutant allele fraction must be$2% in the peripheral

blood, because with deep enough sequencing, a mutation can befound in every individual, and current outcomes data are based ona minimum variant allele fraction of.2% in peripheral blood.7,68 Itis currently unknown if variants present below this threshold alsocarry increased risk of adverse outcomes, and this threshold may needto be revised. A copy number variant resulting from a chromosomalrearrangement involving a chromosomal region where hematologicneoplasia-associated genes are encoded is also consistent with CHIP.

CHIP is distinct fromMDSbecauseCHIP is associatedwith amuchlonger survival, normal blood counts in most cases, and low rate ofprogression to AML. Individuals with CHIP have an increased risk ofdisease progression to hematologic neoplasia compared with individ-uals without detectable mutations, and this risk appears to be propor-tional to the size of the somatic clone; however, the rate of progressionappears to be only 0.5% to 1% per year, similar to MBL and MGUS.Although the annual rate of progression of CHIP,MBL, andMGUS toovert neoplasia is comparable, MBL andMGUS represent expansionsof lineage-committed cells,whereasCHIP involveshematopoietic stemcells or less mature progenitor cells, and thus CHIP is a precursor statefor a broader range of hematologic neoplasms (Figure 1B).

Although a growing body of evidence confirms that individualswith a first “hit” leading to CHIP are at increased risk of developingovert malignancy and that CHIP is also associated with increasedmortality from nonneoplastic causes, the optimal clinical managementof these individuals is uncertain. In the absence of any available inter-vention to prevent progression, surveillance similar towhat is currentlyrecommended for MGUS could be considered.4,8,69 In the near term,most individuals who will be screened for somatic mutations will bethose with hematologic abnormalities that do not meet criteria forcurrent diagnostic entities. Future studies will be needed to delineatewhether certain subgroupswithCHIP have a higher risk of progressionto malignancy or other adverse outcomes. Such subgroups couldinclude those with an unexplained cytopenia (see below), lymphocy-tosis, leukocytosis, persistent eosinophilia, or persistent monocytosisthat do not meet criteria for recognized entities. The specific geneticmutations, number of mutations, and mutant allele fraction may alsoinfluence the risk of progression and could further refine diagnosticcriteria. It is likely that a substantial number of people without he-matologic abnormalities who have genetic sequencing performed forpersonal, research, or medical reasons (eg, on a blood-contaminatedsolid tumor biopsy) will incidentally be found to have CHIP. In certainsettings, such as following cytotoxic therapy for a nonhematologicdisorder, CHIP may have more ominous implications.70

Proposed diagnostic criteria for MDS

To diagnose MDS, it is important to rule out other disorders that canmimic MDS. Testing algorithms such as those advocated by theNational Comprehensive Cancer Network and the European Leuke-miaNet are helpful for this purpose.71,72 Given the large percentage ofindividuals with CHIP, a somaticmutation is not sufficient to diagnoseMDS, even in the context of prior therapy for another malignancy withradiation or cytotoxic drugs or a history of another marrow failuredisorder such as aplastic anemia.73 Like dysplasia or MDS-associatedcytogenetic abnormalities, somaticmutationsmust have a known asso-ciation with MDS and be accompanied by clinically meaningful cyto-penias to consider an MDS diagnosis.

Patients with cytopenias who lack MDS-defining features but inwhom no other cause for cytopenias is evident are said to have ICUS.The ICUS label includes a highly heterogeneous population, and ICUS

Figure 2. Definition of CHIP and its distinction from MDS and non-clonal cyto-penic states. (A) A proposed definition of CHIP. A mutation that is commonly associatedwith clonal expansion of hematopoietic cells in older persons should be present, whereascriteria for other diagnoses should not be met. Evidence of mildly disordered erythro-poiesis such as an elevated red cell distribution width or mean corpuscular volume canbe compatible with CHIP rather than MDS, and occasional dysplastic cells might beseen, as is common in the general population with careful scrutiny of blood and marrow.CHIP is associated with an increased risk of all-causemortality and subsequent diagnosisof hematological malignancy. The 19 genes most commonly mutated in healthy olderadults in sequencing studies to date are listed. The roster of CHIP-associated mutationswill likely change in the future, with some genes being removed and others being added.As a working definition, we propose a variant allele frequency of 2% in order to beconsidered CHIP (since extremely deep sequencing will detect mutations in almost everyperson), but this may need to be revised with further population analyses. PNH, par-oxysmal nocturnal hemoglobinuria. (B) The spectrum of clonal hematopoiesis, ICUS,and MDS. ICUS is a broad category that includes a heterogeneous group of individuals,some of whom have benign (nonclonal) hematopoiesis. Other patients with ICUS mayhave CHIP, differing only from lower risk MDS by their lack of dysplasia and, currently, anundetermined disease risk. CHIP can also include patients with clonal hematopoiesisand nonmalignant causes of cytopenias (eg, immune cytopenias, liver disease, or nutri-tional deficiencies) that would not be considered to have ICUS because of the presenceof a clone, but may have a distinct natural history. Obs, observation; BM, bone marrow;CCUS, clonal cytopenias of undetermined significance; HMA, hypomethylating agent(eg, azacitidine); GF, hematopoietic growth factor (eg, epoetin); IMiD, immunomodula-tory drug (eg, lenalidomide); IST, immunosuppressive therapy; BSC, best supportivecare; HSCT, hematopoietic stem cell transplant.



disease (HR, 2.0) and ischemic stroke (HR, 2.6). Increased mortal-ity was synergistic with elevated red cell distribution width, whichmight be a marker of disordered erythropoiesis because of the ex-panded clone.

Current understanding of the multistep pathogenesis of cancersuggests that individuals with clonal mutations may already bepartway along the path to evolution of frank malignancy. However,although a higher rate of subsequent cancer diagnosis in patientswith

Figure 1. CHIP as a precursor state for hematological neoplasms. (A) A model for evolution from normal hematopoiesis to CHIP and then, in some cases, to MDS or AML.(B) Comparison of evolution patterns of MGUS, MBL, and CHIP. Hematopoietic progenitor or stem cells commonly acquire mutations throughout the human lifespan; most ofthese are passenger mutations that have no consequence for hematopoiesis. Certain mutations, however, confer a survival advantage to the mutated cell and its progeny andallow clonal expansion. Serial acquisition of mutations in an expanded clone can lead to a disease phenotype and ultimately morbidity and mortality. Although this article primarilydiscusses CHIP in the context of its distinction from MDS, CHIP can also directly progress to AML without an intervening MDS stage, and CHIP can progress to other conditionssuch as myeloproliferative neoplasms or lymphoid neoplasms. Just as with MGUS and MBL, the majority of patients with CHIP will never develop an overt neoplasm, andpatients will eventually die of unrelated causes.



online April 30, 2015 originally publisheddoi:10.1182/blood-2015-03-631747

2015 126: 9-16










A N A LY S I S

NATURE MEDICINE VOLUME 20 | NUMBER 12 | DECEMBER 2014 1477

individuals. First, there is an obvious risk of blood-specific variants in individuals without overt hematological malignancy being mis-taken as germline variants. Second, germline alleles in cancer samples could be mistaken as tumor-specific variants when comparing to blood samples. Lastly, connections were made between mosaic PPM1D mutations in lymphocytes and the predisposition to breast and ovarian cancers31. Although the influence of the immune system on tumorigenesis is well known, it is also possible that the blood-specific mutations are independent, unrelated events that are simply associated with the clonal expansion of HSPCs.

Our unbiased mutational analysis using sequencing data of rela-tively high depth (average of 107.5× coverage, Supplementary

Table 13) allowed us to detect hotspot mutations down to 2–3% VAFs; we discovered that 5–6% cases with advanced age (over 70 years) carry blood-specific mutations known to be involved in hematological malignancies. Some of these individuals may be under-going hematopoietic clonal expansion (Fig. 5), but most probably do not progress to overt disease, as the incidence of myeloid malignancies in the elderly is less than 0.1% (ref. 37). Participants providing specimens to TCGA are de-identified and de-coded, so it is not feasible to determine whether a participant with a leukemia-associated mutation actually progressed to a malignant hematologic disease (Supplementary Fig. 2). Using single nucleotide polymorphism array data from GENEVA study cohorts (melanoma, lung health, prostate cancer, etc.), Laurie et al.7 showed that roughly 2–3% of elderly individuals (over 70 years) have chromosomal anomalies in blood samples. We therefore suggest that our estimate of frequency may be conservative, as other types of alterations (such as gene fusions and copy number alterations) were not included in our study. Regardless, these data clearly show that the elderly often acquire clonal skewing in their hematopoietic compartments and that this may represent a contributing factor to the development of hematologic malignancies.

METHODSMethods and any associated references are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKNOWLEDGMENTSThis work was supported by US National Cancer Institute grants R01CA180006 to L.D. and PO1CA101937 to T.J.L. and US National Human Genome Research Institute grants U54HG003079 to R.K.W. and U01HG006517 to L.D. M.J.W. is supported by a Leukemia and Lymphoma Society Scholar Award (1230-14), and J.S.W. is supported by R00HL103975. We thank M. Wyczalkowski for suggestions on figures and C. Kandoth on somatic mutation analysis. We also acknowledge The Cancer Genome Atlas as the source of primary data.

AUTHOR CONTRIBUTIONSL.D. designed and supervised research. M.X., C.L., J.W., M.D.M., M.C.W., H.K.S., V.Y., C.A.M., J.S.W., D.C.L., M.J.W., T.J.L., F.C. K.J.J. and L.D. analyzed the data. T.J.L., J.S.W., D.C.L., M.J.W. and J.F.D. provided disease-specific analysis. M.C.W. performed statistical analysis. E.R.M. and R.K.W. directed sequencing experiments. M.X., M.D.M., C.L., M.C.W. and J.F.M. prepared figures and tables. L.D. and F.C. wrote the manuscript. L.D., T.J.L., B.A.O. and M.C.W. revised the manuscript.

Early initiationmutations inDNMT3ATET2JAK2ASXL1GNASPPM1DSF3B1SH2B3

Subsequent cooperatingmutations inFLT3NPM1WT1IDH1RUNX1NRASCEBPAU2AF1PHF6STAG2

Backgroundmutation

Figure 5 Clonal expansion model. Proposed roles of a set of genes including DNMT3A, ASXL1, TET2, GNAS, JAK2, PPM1D, IDH1, NRAS, NPM1 and FLT3 in the initiation of hematopoietic clonal expansion. Proposed early initiation mutations are in green circles and proposed subsequent cooperating mutations are in red triangles. The genes were ordered on the basis of their mutation frequencies in this study as well as in ref. 21. ‘Background mutation’ refers to mutations that arise stochastically in a genome, which are random and do not have roles in disease.

Figure 4 Comparison of mutation frequencies in blood samples from 58 TCGA cases with mutations in cancer-associated genes in 151 MPN, 150 MDS, 160 CLL and 200 AML cases. (a) Mutation frequencies of major genes involved in hematological malignancies. (b) The average number of nonsynonymous mutations found in TCGA blood normal cases, MPN, MDS and AML patients across 556 cancer-associated genes.

75TCGA blood samples with mutations in cancer-associated genes (n = 58)Myeloproliferative neoplasm (MPN) (n = 151)Myelodysplastic syndrome (MDS) (n = 150)Chronic lymphocytic leukemia (CLL) (n = 160)Acute myeloid leukemia (AML) (n = 200)Genes not analyzed in MDS*

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atio

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ncy

(%)

Ave

rage

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utat

ions

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DNMT3AJAK2TET2

ASXL1

GNAS

SF3B1TP53

PPM1D*

SH2B3*

ASXL2*PHF6

NRAS

STAG2

RUNX1

U2AF1

RAD21*

SMC3

SMC1A*

KRAS KI

T

PTPN11WT1

CEBPAIDH1IDH2

NPM1FLT3

TCGAMPN

CLLAML

a b


A N A LY S I S

Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5–6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.

Blood cells are continuously regenerated by hematopoietic stem/progenitor cells (HSPCs). Human HSPCs divide only rarely (estimated at once a month) but have self-renewal properties that sus-tain survival for decades. As HSPCs divide, they accumulate rare ran-dom mutations that generally do not affect function1. However, some mutations confer advantages in self-renewal, proliferation or both,

resulting in clonal expansion of the affected cells. Although these ‘initiating’ mutations do not lead directly to disease, they can cooper-ate with subsequent mutations to cause hematopoietic malignancies. For example, BCR-ABL and BCL2 oncogenic translocations have been found in blood cells of individuals without overt hematological malig-nancies2–4. The frequency of such events appears to increase with age, with a similar trend being found for somatic structural changes in the nuclear genomes of blood cells1,5. Single nucleotide polymorphism array analysis from large genome-wide association study cohorts showed ~2–3% of normal individuals of advanced age (70s and 80s) harbor leukemia-associated copy number changes that include genes such as DNMT3A (encoding DNA (cytosine-5-)-methyltransferase 3 ) and TET2 (encoding tet methylcytosine dioxygenase 2)6,7. More recently, somatic recurrent TET2 mutations were detected in the blood of elderly women without overt hematological malignancies8, and DNMT3A mutations were reported in nonleukemic cells9.

These findings have collectively led to the hypothesis that certain genetic mutations may confer advantages to affected HSPCs, result-ing in enhanced cell renewal, clonal expansion or both. However, it is unclear whether the effect involves only a small number of genes or many more genes related to leukemia and lymphoma and whether their participation in promoting clonal expansion necessarily leads to clones resembling cancer cells. Here, we address the former question by analyzing variations in 2,728 blood samples in The Cancer Genome Atlas (TCGA). We observed many individuals with age-related hematopoietic clonal mosaicism and concurrent presence of over 60 mutations in 19 leukemia- and/or lymphoma-associated genes. Our study identified not only genes but also specific mutations, associated with the clonal expansion process. Additional statistical analysis identified low-level (2–10% variant allele fractions (VAFs)) recurrent leukemic mutations in a substantial number of cases, pos-sibly in the early stages of clonal expansion. Moreover, our analysis suggests that DNMT3A, TET2, JAK2 (encoding Janus kinase 2), ASXL1 (encoding additional sex combs–like transcriptional regulator 1), SF3B1 (encoding splicing factor 3b, subunit 1) and TP53 (encoding tumor protein p53) have distinct and overlapping roles in the develop-ment of myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL) and/or acute myelogenous leukemia (AML). Finally, these results also incidentally highlight the need for caution when using blood as a reference for a surrogate ‘germline’ genome, especially in older individuals.

1The Genome Institute, Washington University in St. Louis, St. Louis, Missouri, USA. 2Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, USA. 3Brown School Master of Public Health Program, Washington University in St. Louis, St. Louis, Missouri, USA. 4Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, USA. 5Department of Mathematics, Washington University in St. Louis, St. Louis, Missouri, USA. 6Siteman Cancer Center, Washington University in St. Louis, St. Louis, Missouri, USA. 7These authors contributed equally to this work. Correspondence should be addressed to L.D. ([email protected]).

Received 29 May; accepted 21 September; published online 19 October 2014; doi:10.1038/nm.3733

Age-related mutations associated with clonal hematopoietic expansion and malignanciesMingchao Xie1,2,7, Charles Lu1,7, Jiayin Wang1,2,7, Michael D McLellan1, Kimberly J Johnson3, Michael C Wendl1,4,5, Joshua F McMichael1, Heather K Schmidt1, Venkata Yellapantula1,2, Christopher A Miller1, Bradley A Ozenberger1,2, John S Welch2,6, Daniel C Link2,6, Matthew J Walter2,6, Elaine R Mardis1,2,4,6, John F Dipersio2,6, Feng Chen2,6, Richard K Wilson1,2,4,6, Timothy J Ley1,2,4,6 & Li Ding1,2,4,6

»  nature medicine 2014

Bone progenitor dysfuncIon induces myelodysplasia and secondary leukaemia.

Raaijmakers MH Nature 2010

Primary stromal dysfunc3on can result in secondary neoplas3c disease, suppor3ng the concept of niche-‐induced oncogenesis.

•  deleIon of Dicer 1 in mouse osteoprogenitors results in MDS having intact Dicer1.

•  reduced expression of Sbds ( Schwachman-‐Bodian-‐Diamond syndrome) protein in osteoprogenitors

LETTERdoi:10.1038/nature12883

Leukaemogenesis inducedbyanactivating b-cateninmutation in osteoblastsAruna Kode1, John S. Manavalan1, Ioanna Mosialou1, Govind Bhagat2, Chozha V. Rathinam3, Na Luo1, Hossein Khiabanian4,Albert Lee4, Vundavalli V. Murty5, Richard Friedman6, Andrea Brum1,7, David Park8, Naomi Galili9, Siddhartha Mukherjee10,Julie Teruya-Feldstein8, Azra Raza9, Raul Rabadan4, Ellin Berman11 & Stavroula Kousteni1,12

Cells of the osteoblast lineage affect the homing1,2 and the numberof long-term repopulating haematopoietic stem cells3,4, haemato-poietic stem cell mobilization and lineage determination and B celllymphopoiesis5–7.Osteoblastswere recently implicated inpre-leukaemicconditions inmice8,9.However, a single genetic change inosteoblaststhat can induce leukaemogenesis has not been shown.Herewe showthat an activatingmutation of b-catenin inmouse osteoblasts altersthe differentiation potential of myeloid and lymphoid progenitorsleading to development of acute myeloid leukaemia with commonchromosomal aberrations andcell autonomousprogression.Activatedb-catenin stimulates expression of theNotch ligand jagged 1 in osteo-blasts. Subsequent activation of Notch signalling in haematopoieticstemcell progenitors induces themalignant changes.Genetic orphar-macological inhibitionofNotch signallingameliorates acutemyeloidleukaemia and demonstrates the pathogenic role of the Notch path-way. In 38% of patients with myelodysplastic syndromes or acutemyeloid leukaemia, increasedb-catenin signalling andnuclear accumu-lationwas identified inosteoblasts andthesepatients showed increasedNotch signalling in haematopoietic cells. These findings demonstratethat genetic alterations in osteoblasts can induce acute myeloid leuk-aemia, identifymolecular signals leading to this transformationandsuggest a potential novel pharmacotherapeutic approach to acutemyeloid leukaemia.Mice expressing a constitutively activeb-catenin allele in osteoblasts,

referred to here as Ctnnb1CAosb (CA, constitutively active; osb, osteo-blast specific constitutive activity) are osteopetrotic10, and die before6weeks of age (Fig. 1a) for unknown reasons. Upon further examina-tion,Ctnnb1CAosb mice were anaemic at 2weeks of age with peripheralbloodmonocytosis, neutrophilia, lymphocytopenia and thrombocyto-penia (Extended Data Fig. 1a). Erythroid cells were decreased in themarrow and extramedullary haematopoiesis was observed in the liver(Fig. 1c and Extended Data Fig. 1b, l, m). Although the number ofmyeloid (CD11b1/Gr11) cells decreased due to osteopetrosis, theirrelative percentage increased, indicating a shift in the differentiationof HSCs to the myeloid lineage (Fig. 1d and Extended Data Fig. 1c, d).The haematopoietic stem and progenitor cell (HSPC) populationin the bone marrow (Lin2Sca1c-Kit1, LSK) cells decreased twofoldin Ctnnb1CAosb mice, but their percentage was twofold greater than inwild-type littermates (Fig. 1e and ExtendedData Fig. 1e, f). The long-termrepopulating HSC progenitors (LT-HSCs) increased in numbers andpercentage, whereas the lymphoid-biased multipotential progenitors,LSK1/FLT31, and thegranulocyte/monocyteprogenitors (GMP)(ExtendedData Fig. 1g–j) decreased. The GMP percentage increased (Fig. 1f).Identical abnormalities were observed in the spleen of Ctnnb1CAosb

mice (Extended Data Fig. 1n–p). The mutation was introduced inosteoblasts but not in any cells of the haematopoietic compartment(Extended Data Fig. 1q–t) of Ctnnb1CAosb mice.Blasts (12–90%) and dysplastic neutrophils (13–81%) were noted in

the blood and therewas dense anddiffuse infiltrationwithmyeloid andmonocytic cells, blasts (30–53% for n5 12 mice) and dysplastic neu-trophils in the marrow and spleen of Ctnnb1CAosb mice (Fig. 1g–k,Extended Data Fig. 2a–c). In the liver, clusters of immature cells withatypical nuclear appearancewere seen (Fig. 1l). The increase in immaturemyeloid cellswas confirmedby stainingwithmyeloidmarkers in bones,spleen and liver (ExtendedData Fig. 2d–h). ReducedB-cell lymphopoi-esis without changes inT-cell populationswas observed inCtnnb1CAosb

mice (Extended Data Fig. 2i–t). Differentiation blockade was demon-strated by the presence of immaturemyeloid progenitors inCtnnb1CAosb

marrow and differentiation cultures (Fig. 1m, n and Extended DataFig. 2u–x). These cellular abnormalities fulfil the criteria of AML dia-gnosis in mice11 with principle features of human AML12,13.A clonal abnormality involving aRobertsonian translocationRb(1;19)

was identified in myeloid cells of the spleen of a Ctnnb1CAosb mouse(Extended Data Fig. 2y). Recurrent numerical and structural chromo-somal alterations were also detected inmyeloid cells of the spleen of allmutant mice examined (Fig. 2a and Extended Data Table 1). Frequentabnormalities were detected in chromosome 5, the mouse orthologueof human chromosome 7q associatedwith common cytogenetic abnor-malities in patients with myelodysplastic syndromes (MDS) or acutemyeloid leukaemia (AML)14. Whole-exome sequencing identified 4non-silent somatic mutations in myeloid cells from 3 Ctnnb1CAosb

mice (Fig. 2b and Extended Data Fig. 2z), including a recurrent onein Tnfrsf21 and a single somatic mutation in Crb1 previously reportedin human AML15, but sample size has insufficient statistical power todetermine if it is a driver or passenger mutation. Hence, constitutiveactivation of b-catenin in osteoblasts facilitates clonal progression andis associated with somatic mutations in myeloid progenitors.Transplantation of bone marrow cells from Ctnnb1CAosb leukaemic

mice into lethally irradiatedwild-type recipients induced all features ofhaematopoietic dysfunction and AML observed in Ctnnb1CAosb miceincluding blasts (15–80%) and dysplastic neutrophils (15–75%) in theblood and blasts (30–40%) and abnormal megakaryocytes in the mar-row and early lethality (Extended Data Fig. 3a–i). Transplantation ofwild-type bone marrow cells to lethally irradiated Ctnnb1CAosb micealso resulted in AML with early lethality (Extended Data Fig. 3j–r).Transplantation of LT-HSCs, but not other haematopoietic popula-tions, from Ctnnb1CAosb mice to sub-lethally irradiated wild-type reci-pients resulted in AML with early lethality (Fig. 2c, d and Extended

1Department of Medicine, Division of Endocrinology, College of Physicians & Surgeons, Columbia University, New York, New York 10032, USA. 2Department of Pathology and Cell Biology, College ofPhysicians & Surgeons, Columbia University, New York, New York 10032, USA. 3Department of Genetics and Development College of Physicians & Surgeons, Columbia University, New York, New York10032,USA. 4Department of Biomedical Informatics andCenter for Computational Biology andBioinformatics, ColumbiaUniversity, NewYork, NewYork10032, USA. 5Department of Pathology& Institutefor Cancer Genetics Irving Cancer Research Center, Columbia University, New York, New York 10032, USA. 6Biomedical Informatics Shared Resource, Herbert Irving Comprehensive Cancer Center andDepartment of Biomedical Informatics, College of Physicians&Surgeons, ColumbiaUniversity, NewYork, NewYork 10032, USA. 7Department of InternalMedicine, ErasmusMC, Dr.Molewaterplein50, NL-3015GERotterdam,TheNetherlands. 8DepartmentofPathology,Memorial Sloan-KetteringCancerCenter,NewYork,NewYork10021,USA. 9MyelodysplasticSyndromesCenter, ColumbiaUniversityNewYork, New York 10032, USA. 10Departments of Medicine Hematology & Oncology Columbia University New York, New York 10032, USA. 11Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. 12Department of Physiology & Cellular Biophysics, College of Physicians & Surgeons, Columbia University, New York, New York 10032, USA.

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Here we show that an acIvaIng mutaIon of b-‐catenin in mouse osteoblasts alters the differenIaIon potenIal of myeloid and lymphoid progenitors leading to development of acute myeloid leukaemia with common chromosomal aberraIons and cell autonomous progression

Research article

The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 11 November 2013 4595

Induction of myelodysplasia by myeloid-derived suppressor cells

Xianghong Chen,1 Erika A. Eksioglu,1 Junmin Zhou,1 Ling Zhang,1 Julie Djeu,1 Nicole Fortenbery,1 Pearlie Epling-Burnette,1 Sandra Van Bijnen,2 Harry Dolstra,2

John Cannon,3 Je-in Youn,1 Sarah S. Donatelli,1 Dahui Qin,1 Theo De Witte,2 Jianguo Tao,1 Huaquan Wang,4 Pingyan Cheng,1 Dmitry I. Gabrilovich,1 Alan List,1 and Sheng Wei1,4

1H. Lee Moffitt Cancer Center, Tampa, Florida, USA. 2Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. 3Department of Pediatrics, Children’s Research Institute, University of South Florida, Tampa, Florida, USA. 4Tianjin Medical University Cancer Hospital, Tianjin, China.

Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited compo-nents to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppres-sive cytokines IL-10 and TGF- by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interrup-tion of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone mar-row expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

IntroductionUnderstanding the selective pressures and mechanisms involved in the initiation of stem cell malignancies is critical to development of effective strategies for prevention and treatment. Inflammatory molecules have long been implicated as regulatory cues driving the proliferation and apoptotic death of hematopoietic progenitors in myelodysplastic syndromes (MDS) (1–3). Chronic immune stimu-lation, coupled with senescence dependent changes in both hema-topoietic stem/progenitor cells (HSPC) and the BM microenviron-ment, are believed to be critical to the pathogenesis of the disease (4). Increasing evidence implicates the activation of innate immune signaling in both hematopoietic senescence and the pathobiology of MDS (5). TLR4, for instance, is overexpressed in MDS BM HSPC and is implicated in progenitor apoptosis and subsequent develop-ment of cytopenias (6). Furthermore, gene expression of the TLR adaptor E3 ubiquitin ligase, TNF receptor-associated factor-6 (TRAF6), is markedly upregulated in MDS CD34+ cells (7) accom-panied in some cases by amplification of genomic regions encod-ing TRAF6 or the Toll IL-1 receptor domain-containing adap-tor protein (TIRAP), key intermediates in TLR4 signaling (8, 9). Recent investigations showed that TLR signaling is constitu-tively activated in MDS with chromosome 5q deletion [del(5q)] as a result of allelic deletion of microRNA 145 (miRNA-145) and

miRNA-146a and consequent upregulation of their respective targets, TIRAP and TRAF6 (10). Moreover, knockdown of these specific miRNAs or overexpression of Traf6 in murine HSPC reca-pitulates the hematologic features of del(5q) MDS in a transplant model (10), providing convincing evidence that sustained TLR activation is a critical factor driving the malignant phenotype. More recent findings indicate that TRAF6 is essential for survival and proliferation of MDS HSPC (11) and sustained TLR activa-tion skews their commitment toward the myeloid lineage while suppressing osteoblast differentiation (12, 13), analogous to the senescence-dependent changes observed with normal aging (14).

Immature myeloid-derived suppressor cells (MDSC), known to accumulate in tumor-bearing mice and cancer patients, are site-specific inflammatory and T cell immunosuppressive effector cells that contribute to cancer progression (15, 16). Their suppres-sive activity is in part driven by inflammation-associated signal-ing molecules, such as the danger-associated molecular pattern (DAMP) heterodimer S100A8/S100A9 (also known as myeloid-related protein 8 [MRP-8] and MRP-14, respectively), which inter-act with several innate immune receptors that are involved in the biology of MDSC activation (17–20). Murine CD11b+Gr1+ MDSC form the basis of the vast majority of the mechanistic studies; how-ever, much less has been reported on their human counterparts. Human MDSC lack most markers of mature immune cells (LIN–, HLA-DR–) but possess CD33, the prototypical member of sialic acid–binding Ig-like super-family of lectins (Siglec) (15, 21–23). Importantly, while its precise action is unknown, CD33 possesses an immunoreceptor tyrosine-based inhibition motif (ITIM) that is associated with immune suppression (23).

Authorship note: Xianghong Chen, Erika A. Eksioglu, and Junmin Zhou contrib-uted equally to this work. Dmitry I. Gabrilovich, Alan List, and Sheng Wei Share are co–senior authors.

Conflict of interest: The authors have declared that no conflict of interest exists.

Citation for this article: J Clin Invest. 2013;123(11):4595–4611. doi:10.1172/JCI67580.

-‐primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS -‐

Somatic Mutations Identified by Means of Targeted Sequencing.

Yoshizato T et al. N Engl J Med 2015;373:35-47


n engl j med 373;1 nejm.org July 2, 2015 35

The authors’ full names, academic degrees, and affiliations are listed in the Appendix. Address reprint requests to Dr. Ogawa at the Dept. of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto Uni-versity, Yoshida-Konoe-cho, Kyoto-shi Sakyo-ku, Kyoto 606-8501, Japan, or at sogawa-tky@ umin . ac . jp; or to Dr. Young at the Hematology Branch, National Heart, Lung, and Blood Institute, 10 Center Dr., Bldg. 10/CRC, Rm. 3E-5140, Bethesda, MD 20892-1202, or at youngns@ mail . nih . gov.

Drs. Yoshizato and Dumitriu and Drs. Young and Ogawa contributed equally to this article.

N Engl J Med 2015;373:35-47.DOI: 10.1056/NEJMoa1414799Copyright © 2015 Massachusetts Medical Society.

BACKGROUNDIn patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immuno-suppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the di-agnosis of aplastic anemia.

METHODSWe performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients.

RESULTSSomatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as ac-quired mutations. The prevalence of the mutations increased with age, and muta-tions had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients.

CONCLUSIONSClonal hematopoiesis was prevalent in aplastic anemia. Some mutations were re-lated to clinical outcomes. A highly biased set of mutations is evidence of Darwin-ian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.)

A BS TR AC T

Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia

T. Yoshizato, B. Dumitriu, K. Hosokawa, H. Makishima, K. Yoshida, D. Townsley, A. Sato-Otsubo, Y. Sato, D. Liu, H. Suzuki, C.O. Wu, Y. Shiraishi, M.J. Clemente, K. Kataoka, Y. Shiozawa, Y. Okuno, K. Chiba, H. Tanaka, Y. Nagata, T. Katagiri,

A. Kon, M. Sanada, P. Scheinberg, S. Miyano, J.P. Maciejewski, S. Nakao, N.S. Young, and S. Ogawa

Original Article

The New England Journal of Medicine Downloaded from nejm.org at INSERM DISC DOC on September 20, 2016. For personal use only. No other uses without permission.


N= 82 Clonal hematopoiesis in 47% paIents, DNMT3A-‐mutated and ASXL1-‐mutated clones increased over Ime; BCOR-‐ and BCORL1-‐mutated and PIGA-‐mutated clones decreased or remained stable .MutaIons in PIGA and BCOR and BCORL1 correlated with a beoer response to immunosuppressive therapy mutaIons in DNMT3A and ASXL1 were associated with worse outcomes.

Clinical Correlations with Somatic Mutations.

Yoshizato T et al. N Engl J Med 2015;373:35-47

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« clinical » conclusions

•  Presence of somaIc mutaIons is not diagnosIc of MDS in the absence of unexplained cytopenias

•  Is CCUS MDS ? •  Is AA with MDS type mutaIons MDS ? •  Should CCUS be treated like MDS ? … just treat cytopenias

« clinical » conclusion

While discovery of somaIc mutaIons may help in the diagnosis and prognosis of definite MDS or cytopenias, somaIc mutaIons per se currently rarely have an impact on MDS treatment:

–  SF3B1 mutaIon without ringed sideroblasts (luspatercept) ?

–  TP53 mutaIon in lower risk MDS with del 5q? MDS without significant cytopenias rarely (never?) requires treatment

tMDS/AML or not ?

•  MDS post AML with NPM1 mutaIon

•  MDS /AML post APL

C o r r e s p o n d e n c e


n engl j med 375;6 nejm.org August 11, 2016 e9(1)

Assessment of Minimal Residual Disease in Standard-Risk AML

To the Editor: Ivey and colleagues (Feb. 4 issue)1 tested the prognostic value of the presence of a mutation in the gene encoding nucleophosmin (NPM1) in patients with acute myeloid leukemia (AML) who were in complete hematologic remis-sion after a second cycle of chemotherapy,2 al-though the recovery of cell counts in peripheral blood was not required for a determination of such remission. Some of the patients were in re-mission after the first cycle of chemotherapy and some were not. Moreover, some of the patients in remission had normal blood counts and some did not. Other studies have shown the prognostic importance of obtaining normal blood counts over shorter durations of time and with fewer cycles required to achieve remission. Thus, we wonder whether the patients who did not have minimal residual disease were more likely to have a remission with normal cell counts and more likely to be in remission after the first cycle of chemotherapy than were patients who were found to have minimal residual disease.

Michele Baccarani, M.D.University of Bologna Bologna, Italy michele . baccarani@ unibo . it

No potential conflict of interest relevant to this letter was re-ported.

1. Ivey A, Hills RK, Simpson MA, et al. Assessment of minimal residual disease in standard-risk AML. N Engl J Med 2016; 374: 422-33.2. Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recom-mendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol 2003; 21: 4642-9.

DOI: 10.1056/NEJMc1603847

To the Editor: Ivey et al. describe the persis-tence of mutations in genes other than NPM1 in

the blood and marrow of patients with AML who were in complete hematologic remission. The authors also state that patients with persis-tent NPM1 transcripts (the presence of which defined minimal residual disease) had a worse prognosis than those who did not carry this marker and that virtually all patients who had a relapse carried the NPM1 mutation. At our center, in 32 consecutive patients (median age, 55 years) with cytogenetically normal AML carrying a de novo mutation in NPM1 without internal tan-dem duplications in the gene encoding Fms-like tyrosine kinase 3 (FLT3-ITD) who had a com-plete remission after intensive chemotherapy, 14 (44%) had a relapse (all with the same NPM1 mutation as the one observed at diagnosis), 12 (38%) remained in complete remission, and 6 (19%) (all of whom were >55 years of age) had disease that developed into either the myelodysplastic syndrome (in 5 patients) or primary myelofibro-sis (in 1 patient) (Fig. 1A). In each of these 6 pa-tients, a preleukemic clone with at least one mutation in TET2, JAK2, ASXL1, IDH2, or a gene encoding a spliceosome protein was found at the time of the AML diagnosis and was present after the development of either the myelodys-plastic syndrome or primary myelofibrosis (Fig. 1B). However, the NPM1 mutation was absent. Thus, although we agree that relapse does not arise from preleukemic clones of cells with wild-type NPM1, such clones may promote the development of the myelodysplastic syndrome or primary myelofibrosis, especially in older pa-tients. Did Ivey et al. obtain data that support this conclusion and, if so, did they observe an age-specific association?

Sarah Morin-Zorman, M.D., Ph.D.Hôpital Saint Louis Paris, France sarah . morin@ aphp . fr

The New England Journal of Medicine Downloaded from nejm.org at INSERM DISC DOC on September 20, 2016. For personal use only. No other uses without permission.


Morin S el al

•  32 AML (median age, 55 years) with NPM1 without FLT3-‐ITD in CR aqer chemotherapy

•  44% relapse (with NPM1 mutaIon), 38% remained in

CR, 6 (19%) MDS •  In the 6 paIents, a preleukemic clone with at least one

mutaIon in TET2, JAK2, ASXL1, IDH2, or spliceosome gene was found at AML diagnosis and MDS diagnosis

Molecular analysis of MDS and AML occurring a@er treatment for APL Ph Aras, A Renneville (ASH 2016)

•  10 cases (1.2%) of MDS/AML during follow up of APL

•  MutaIons at APL diagnosis: 4 cases of FLT3 ITD or mutaIon

•  Muta3ons at MDS/AML:TP 53 (4 pts) ASXL1 (1/10 pts), CBL (1/10 pts), DNMT3A (1/10 pts), EZH2 (1/10 pts), GATA2 (1/10 pts), KRAS (1/10 pts), PTPN11 (1/10 pts), RUNX1 (1/10 pts), and TET2 (1/10 pts).

Groupe Francophone des Myélodysplasies

•  Activates clinical trials in MDS (35 centers in France and Belgium + Switzerland, Tunisia)

•  Website: www. gfmgroup.org •  Online registry of French MDS cases •  Close cooperation with: - a patient support group - the International MDS Foundation - the European Leukemia Net

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