Acquired and germline predisposition to bone marrow failure: Diagnostic features and clinical implications

Accepted Manuscript

Acquired and Germline Predisposition to Bone Marrow Failure: Diagnostic features and clinical implications Michael E. Kallen MD , Alina Dulau-Florea MD , Katherine R. Calvo MD PhD PII: DOI: Reference:

S0037-1963(18)30066-0 10.1053/j.seminhematol.2018.05.016 YSHEM 50979

To appear in:

Seminars in Hematology

Received date: Accepted date:

10 May 2018 29 May 2018

Please cite this article as: Michael E. Kallen MD , Alina Dulau-Florea MD , Katherine R. Calvo MD PhD , Acquired and Germline Predisposition to Bone Marrow Failure: Diagnostic features and clinical implications, Seminars in Hematology (2018), doi: 10.1053/j.seminhematol.2018.05.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Acquired and Germline Predisposition to Bone Marrow Failure: Diagnostic features and clinical implications

AUTHORS: Michael E. Kallen MD1, Alina Dulau-Florea MD2, Katherine R. Calvo MD PhD2 1

CR IP T

National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Hematology Section, Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD 20892. 2

M

AN US

CORRESPONDENCE: Katherine R. Calvo NIH/CC/DLM 10 Center Dr. Bldg 10, 2C306 BETHESDA, MD 20892-1508 [email protected] 301-594-9578

ED

DISCLOSURES: The authors have no conflicts of interest to disclose.

AC

CE

PT

KEY WORDS: Bone marrow failure; aplastic anemia; MDS; GATA2 deficiency; flow cytometry

1

ACCEPTED MANUSCRIPT

ABSTRACT: Bone marrow failure and related syndromes are rare disorders characterized by ineffective bone marrow hematopoiesis and peripheral cytopenias. Although many are

CR IP T

associated with characteristic clinical features, recent advances have shown a more complicated picture with a spectrum of broad and overlapping phenotypes and imperfect

genotype – phenotype correlations. Distinguishing acquired from inherited forms of marrow failure can be challenging, but is of crucial importance given differences in the risk of disease progression to myelodysplastic syndrome, acute myeloid leukemia, and other malignancies, as

AN US

well as the potential to genetically screen relatives and select the appropriate donor if

hematopoietic stem cell transplantation becomes necessary. Flow cytometry patterns in combination with morphology, cytogenetics, and history can help differentiate several diagnostic marrow failure/insufficiency entities and guide genetic testing. Herein we review several

M

overlapping acquired marrow failure entities including aplastic anemia, hypoplastic

ED

myelodysplasia, and large granular lymphocyte disorders; and several bone marrow disorders with germline predisposition, including GATA2 deficiency, CTLA4 haploinsufficiency,

PT

dyskeratosis congenita / telomeropathies, Fanconi anemia, Shwachman – Diamond syndrome, congenital amegakaryocytic thrombocytopenia, severe congenital neutropenia, and Diamond –

CE

Blackfan anemia with a focus on advances related to pathophysiology, diagnosis, and

AC

management.

1. INTRODUCTION The bone marrow failure (BMF) syndromes are rare disorders characterized by

ineffective bone marrow hematopoiesis and resultant peripheral cytopenias. They comprise acquired forms, of which aplastic anemia is the most well-known, and inherited forms. The inherited subtypes can present with or without numerous associated physical anomalies and a

2

ACCEPTED MANUSCRIPT

spectrum of pathologic findings in multiple different organ systems. Both atypical and cryptic presentations are possible. Inherited marrow failure associated with autosomal recessive inheritance (e.g. Fanconi anemia) typically has high penetrance and presents early in life. In contrast, marrow disease associated with autosomal dominant gene mutations (e.g. GATA2

CR IP T

deficiency or a subset of teleomeropathies) may have variable penetrance with adolescent or adult onset of disease. A positive family history may suggest constitutional nature, although de novo germline mutations are also possible. While classic marrow failure syndromes frequently present clinically in children, there is increased recognition of potential underdiagnosis in older

AN US

patients.

An important diagnostic issue is the distinction of inherited marrow failure syndromes from idiopathic aplastic anemia (AA) or de novo hypoplastic myelodysplastic syndrome (MDS). This can present a potentially challenging differential relying on multiple modalities, and

M

synthesis of clinical information and family history with morphology, flow cytometry, cytogenetic, and molecular testing results. The distinction is critical, as the etiology of most acquired AA is

ED

thought to be related to T-cell mediated autoimmune destruction of hematopoietic precursors and therefore treated with immunosuppressive therapy, whereas inherited BMF syndromes

PT

warrant more aggressive surveillance due to increased risk of progression to MDS and acute myeloid leukemia (AML), as well as an associated risk of solid malignancies. Accurate

CE

diagnosis of BMF syndromes provides an opportunity for genetic testing of potentially affected family members, as well as hematopoietic stem cell transplantation (HSCT) at an earlier time

AC

point.

In this review, we briefly discuss AA, hypoplastic MDS and several inherited BMF

syndromes, including Fanconi anemia, dyskeratosis congenita and telomeropathies, GATA2 deficiency, Shwachman – Diamond syndrome, congenital amegakaryocytic thrombocytopenia, severe congenital neutropenia, and Diamond – Blackfan anemia, as well as the emergent entity

3

ACCEPTED MANUSCRIPT

of CTLA4 haploinsufficiency. Recent work has advanced our pathophysiologic understanding of these rare disorders, allowing greater diagnostic clarity and improved management.

2. ACQUIRED MARROW FAILURE APLASTIC ANEMIA

CR IP T

2.1.

AA is the paradigm of human BMF, and is characterized by hypocellular bone marrow and peripheral cytopenias, with an incidence of 2 per million in the west and 4-6 per million in Asia 1. AA may either be inherited or acquired, and in most cases the pathogenic mechanism is

AN US

thought to involve immune-mediated destruction of hematopoietic progenitors. Most cases are idiopathic, although a variety of toxic insults have been recognized, including chemotherapy and radiation, certain antibiotics and anticonvulsant drugs, certain viral infections 2, and hepatitis 3. AA patients are clinically separated by the Camitta criteria into moderate and severe categories,

M

with severe AA defined as two of three peripheral cytopenias (absolute neutrophil count <500/mm3, platelet count <20,000/mm3, corrected reticulocyte count <1%) and marrow

ED

cellularity of <30% 4.

Bone marrow cellularity is best assessed on the bone marrow core biopsy. In AA, bone

PT

marrow biopsies demonstrate marked hypocellularity with trilineage hypoplasia including rare to absent megakaryocytes, in the absence of marrow fibrosis (Figure 1A). We and others

CE

occasionally observe that the cellularity in AA can be patchy 5, with predominantly acellular areas and intermittent cellular foci of hematopoiesis, and is not always congruent with peripheral

AC

blood counts. Hematopoiesis is often erythroid predominant, potentially due to common involvement by clonal proliferations of paroxysmal nocturnal hemoglobinuria (PNH) cells

6

(Figure 1B and 1C). Careful examination of marrow aspirate smears is mandatory for determining the blast count, assessment of dysplasia, and exclusion of hypocellular MDS and AML 7. Maturation can infrequently show features resembling dyserythropoiesis and dysmegakaryopoiesis, involving <10% of erythroid and megakaryocytic precursors8, and these

4

ACCEPTED MANUSCRIPT

findings are generally mild in degree, including rare erythroid nuclear budding or megaloblastic maturation, and rare small megakaryocytes, micromegakaryocytes or hypolobated forms. Involvement of >10% of the erythroid or megakaryocyte precursors, or the presence of dysmyelopoietic features including pelgeroid or hypogranular neutrophils, should raise concern

CR IP T

for myelodysplasia. According to the WHO 2017 diagnostic guidelines 8 at least 30 megakaryocytes should be evaluated in the assessment of significant megakaryocytic

dysplasia. However, most hypoplastic marrows do not have enough megakaryocytes present to satisfy this recommendation. CD34 quantification, which can be performed either by multicolor

AN US

flow cytometry of BM aspirates, or by immunohistochemistry on bone marrow core biopsies, can aid in the diagnosis of MDS, where an elevation, in conjunction with morphologic and/or cytogenetic abnormalities, would favor a diagnosis of hypocellular MDS (Figure 1G). In contrast, a significant decrease in marrow CD34-positive cell numbers is characteristic for AA9, as the 10, 11

. CD61

M

CD34+ hematopoietic progenitors are targets of autoimmune destruction

immunohistochemistry is helpful in identifying atypical megakaryocytic features including

ED

micromegakaryocytes, cluster formation, large forms with separated and peripheralized nuclear lobes, and abnormal paratrabecular localization.

PT

Bone marrow flow cytometry on adequate samples from AA patients in our experience reveals decreased CD34+ cells, as well as frequent PNH clones. Flow cytometric analysis can

CE

be helpful in differentiating AA from hypoplastic MDS (Figure 1I), T-cell large granular lymphocytosis (T-LGL) (Figure 2B), GATA2 deficiency (Figure 4) and CTLA4 haploinsufficiency

AC

(Figure 5B) and will be discussed in more detail below. Bone marrow cytogenetic studies are crucial in detection of clonal proliferations, particularly given the 15% overall rate of clonal evolution in AA 12, and the higher rates in many inherited BMF disorders. Recurring chromosomal abnormalities of particular significance in the diagnosis of hypocellular MDS include -7, -5, i(17q), del(11q), del(12p), del(9q), and idic(X)(q13). Of note, +8, del(20q), and -Y are not currently considered definitive evidence of MDS if encountered as sole abnormalities in

5

ACCEPTED MANUSCRIPT

the absence of morphologic evidence based on WHO criteria8. Trisomy 8 and monosomy 7 are the most common stereotypical cytogenetic abnormalities seen, and monosomy 7 confers a dire prognosis with refractory cytopenias and progression to AML mandating prompt HSCT 1. Targeted next generation sequencing panels to identify recurrent mutations associated

CR IP T

with myeloid disease have recently become available as clinical tests. Genetic variants are detected in up to 50% of patients with aplastic anemia, and while some variants are fairly unique to AA, such as those involving PIGA and BCOR/BCORL1 mutations, others involve mutations more commonly found in myeloid malignancies, including DNMT3A and ASXL113.

AN US

Understanding the significance of variants and mutations detected in AA is complicated by the finding that the same mutations can also be detected in the normal aging population and in patients with idiopathic cytopenia of undetermined significance, termed clonal hematopoiesis of undermined significance (CHIP) 14, 15. A minority of patients with AA have heterozygous

M

mutations in genes encoding proteins involved in the maintenance of telomere length, TERT and TERC, discussed further in section 3.3.

ED

AA is treated with immunosuppressive regimens of antithymocyte globulin (ATG) and cyclosporine, which induce tolerance by depleting activated T-cells and directly inhibiting T-cell

PT

proliferation and activation 1. The majority of patients experience blood count improvement on this therapy, although relapse and need for additional immunosuppression is common, due to

CE

incomplete eradication of pathogenic clonal T-cell populations. Clinical trials of the thrombopoietin mimetic eltrombopag show efficacy in newly diagnosed AA and a subset of

AC

severe refractory patients, and sustainable responses have been seen after drug discontinuation 16, 17. Allogeneic HSCT has shown improvement in survival rates, particularly for HLA-matched unrelated donor transplants, which is of importance given the risks of eventual immunosuppressive therapy failure and clonal evolution 18.

2.2.

HYPOPLASTIC MYELODYSPLASIA

6

ACCEPTED MANUSCRIPT

Hypoplastic myelodysplastic syndromes (h-MDS) are a subset of myeloid neoplasms defined based on bone marrow (BM) hypocellularity of less than 30% in adults of <60 years of age, or less than 20% in those older than 70 years 7, in addition to morphologic dysplasia and peripheral cytopenia(s) 8 (Figure 1D-I). Although h-MDS accounts for only 10-15% of all MDS 19,

CR IP T

they constitute the majority of childhood MDS, particularly of the low-grade MDS, classified as refractory cytopenia of childhood (RCC) 20. Pathogenesis of some h-MDS is also different from traditional (normocellular and hypercellular) MDS, implicating immune dysregulation, more akin to aplastic anemia 19. Studies have shown the existence of immune-mediated apoptosis of

AN US

normal hematopoietic progenitors mediated by increased cytotoxic T lymphocytes, IFN-gamma producing CD4+ lymphocytes and Th17 cells, defective T regulatory cells, and increased production of pro-inflammatory and apoptosis-inducing cytokines 19 21.

In RCC, the bone marrow is hypocellular and blasts are less than 5%. h-MDS can often

M

constitute a diagnostic challenge because of the resemblance to aplastic anemia (AA) due to the paucity of cells in both core biopsies and aspirate smears of bone marrows. In addition to

ED

dysplastic changes in two or more lineages (preferably granulocytes and megakaryocytes) the presence of marrow fibrosis, disorganized microarchitecture and clusters of blasts, suggest a

PT

MDS diagnosis 7 . Granulocytes may show atypical maturation with nuclear-to-cytoplasmic

CE

maturation asynchrony, hyposegmentation or pelgeroid neutrophils, and hypogranular forms. Megakaryocytes may show atypical features including separation of nuclear lobes, hypolobation

AC

or micromegakyocytic forms (Figure 1E-F). Dyserythropoiesis can manifest as nuclear budding, binucleation, or megaloblastic changes. Increased blasts may be suggested by immunohistochemistry for CD34 on the core biopsy (Figure 1G), or by morphologic evaluation of the aspirate smear (Figure 1H). Subtle morphologic changes can be overlooked by unexperienced pathologists or hematologists, and a high degree of suspicion and expertise are needed to synthesize all data necessary for diagnosis.

7

ACCEPTED MANUSCRIPT

Besides morphologic abnormalities, flow cytometric analysis of cells from BM aspirates, can be essential for distinguishing MDS from AA or reactive cytopenias. Several abnormalities, such as higher CD34-positive blasts compared to aplastic anemia (AA) cases; aberrant immunophenotype of the myeloblasts (“lineage infidelity”), as well as decreased SSC of

CR IP T

granulocytes and/or abnormal patterns of maturation of the granulocytes and monocytes, favor a diagnosis of MDS 22 23. For childhood MDS, studies showed that flow cytometric

immunophenotyping is valuable in distinguishing RCC from healthy controls, and also from severe aplastic anemia (SAA) and advanced MDS 24. RCC patients show a severe reduction of

AN US

myeloid cells in comparison with healthy controls, but less severe than in SAA. However, in contrast to adult MDS, reduced SSC of granulocytes and lineage infidelity markers on myeloblasts, were uncommon in RCC, whereas heterogeneous expression of CD71 and CD36 on erythrocytes, and aberrant expression of CD56 on monocytes, were relatively frequent in

M

RCC 24. Flow cytometric analysis of marrow may reveal dysplastic myeloblasts with absence of CD38 and/or aberrant expression of CD7, clues that are highly suggestive of MDS (Figure 1I).

ED

Two or more flow cytometric abnormalities, were seen in MDS with monosomy 7, which in RCC is known to have a higher risk of progression to acute myeloid leukemia. Cytogenetic

PT

abnormalities are present in approximately 50% of cases and, in contrast to transient abnormalities that can occur in AA, they are persistent and sometimes complex. Molecular

CE

studies developed more recently, in particular massive parallel sequencing, lead to the identification of mutations and copy number alterations in several candidate genes implicated in

AC

MDS pathogenesis. However, somatic mutations, in particular those with low allele frequencies, have been also found in aplastic anemia and other BMF syndromes (Fanconi anemia, Shwachman-Diamond syndrome and others). A large interest has been given to germline mutations predisposing to MDS, and GATA2 was found the most common germline defect predisposing to pediatric MDS 25, of which the majority of cases are h-MDS and AA mimics.

8

ACCEPTED MANUSCRIPT

h-MDS risk stratification is currently based on parameters that define the Revised International Prognostic Scoring System (IPSS-R) established for all MDS, such as number of lineages affected by dysplasia, degree of cytopenia, blast percentages and cytogenetic abnormalities 26. However, given the heterogeneity and particularities of this group of diseases, the development

2.3.

CR IP T

of better prognostic markers and therapeutic targets for these entities is imperative.

T-CELL LARGE GRANULAR LYMPHOCYTOSIS (T-LGL) IN MARROW FAILURE

AN US

T-LGL is a hematologic disease characterized by expansions of mature T cells presenting as an indolent disease that affects predominantly middle aged adults (median age 60 years) 27, with equal distribution among men and women. Clinically, T-LGL usually presents with cytopenias and a variety of autoimmune manifestations. Neutropenia is the most common

M

cytopenia, although anemia is also frequent, and can manifest as autoimmune hemolytic anemia or associated with pure red cell aplasia 28 (Figure 2A-B). Thrombocytopenia is

ED

infrequent and moderate, but not unseen and therefore, peripheral blood counts can be similar to those encountered in aplastic anemia, in which pancytopenia is the most common

PT

presentation. Morphologic examination of the peripheral blood in T-LGL typically reveals

CE

lymphocytosis with large lymphocytes with abundant cytoplasm containing azurophilic granules, although absolute lymphocytosis may not be apparent in a minority of cases. T-LGL leukemia is

AC

defined as a persistent clonal lymphocytosis over 6 months. Although the bone marrow is, in the majority of cases of T-LGL, normo- or hypercellular, some cases present with hypocellular marrows, with features that can overlap with aplastic anemia 29. The bone marrow commonly shows nodular lymphocytic aggregates, mostly non-paratrabecular; although some cases may show interstitial lymphocytic infiltration without a nodular component. Immunohistochemical stains show increased CD3-positive T cells with CD8 predominance in an interstitial and

9

ACCEPTED MANUSCRIPT

intrasinusoidal distribution, whereas CD4 cells constitute a minority of the T cells and are almost exclusively confined to the nodular lymphocytic aggregates, which also contain non-clonal B cells. CD8 T cells have a mature cytotoxic phenotype, co-expressing TIA-1 (Figure 2A) and granzyme B 30.

CR IP T

T cell clonality in T-LGL can be demonstrated by molecular studies using polymerase chain reaction (PCR) for T-cell receptor gene rearrangements. A more recent method used to assess T cell clonality is immunophenotyping by flow cytometry combined with monoclonal antibodies against the Vbeta repertoire demonstrating a restricted expression of one TCR-Vbeta family

31

AN US

in T-LGL, as presumptive evidence for clonality. Careful analysis is needed in assessing and interpreting T cell clonality, as oligoclonal T cell proliferations have been described in many patients with aplastic anemia, as suggested by skewing of the variable region of B-chain (VB) Tcell receptor (TCR) repertoire in these patients compared with healthy controls 32. To complicate

M

the issue even more, associations of T-LGL with BMF syndromes including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS), have

ED

been reported 29 33. Flow cytometric analysis of the marrow in bone marrow failure and patients being evaluated for AA, can be helpful in identifying immunophenotypically abnormal clonal T-

PT

LGL (Figure 2B) that may contribute to marrow failure or for identification of T-LGL cases

CE

presenting with features of AA.

The clonal expansion in T-LGL is likely due to chronic antigenic stimulation followed by

AC

dysregulation of apoptosis due to constitutive activation of several survival pathways such as Jak/STAT, MapK, phosphatidylinositol 3-kinase-AKT, Ras-Raf-1-MEK1, NF-kB 34 35. Somatic mutations in STAT3 and STAT5B genes are important players in T-LGL pathogenesis 34 36.

10

ACCEPTED MANUSCRIPT

3. GERMLINE PREDISPOSITION, INHERITED MARROW FAILURE AND OVERLAPPING ENTITIES 3.1.

GATA2 DEFICIENCY

GATA2 deficiency results from germline mutations in the gene encoding the transcription

CR IP T

factor GATA2, causing a spectrum of overlapping phenotypes originally reported by separate groups as monocytopenia with mycobacterial infections (monoMAC)

37, 38

, dendritic cell,

monocyte, B-cell and NK-cell lymphoid deficiency (DCML deficiency) 39, primary lymphedema and MDS (Emberger syndrome) 40, and familial MDS / AML 41. Other features described include

AN US

pulmonary alveolar proteinosis, warts, and sensorineural hearing loss 42. This syndrome has autosomal dominant inheritance, typically presents in late childhood, adolescence, or adulthood, and confers an increased risk of immunodeficiency, bone marrow failure, MDS and AML 42. Infections are often the presenting symptoms and are followed by or concurrent with bone

M

marrow insufficiency and a high rate of progression to MDS/AML or CMML, although patients may present with aplasia or MDS/AML and lack a history of overt immunodeficiency 41. A recent

ED

study of pediatric and adolescent MDS found germline mutations in GATA2 in 15% of advanced MDS cases, and in 72% of adolescents with MDS and monosomy 7 25.

PT

GATA2 is a zinc-finger transcription factor involved in hematopoietic progenitor cell development, and haploinsufficiency through a variety of mutations in coding and non-coding

CE

regions causes BMF. GATA2 is known to interact with GATA1 in hematopoietic development, and with GATA3 in trophoblastic development 42, 43. The genetic lesions are heterozygous and

AC

heterogeneous, but cluster into three groups: missense mutations and in-frame deletions involving the C-terminal zinc finger, mutations predicted to result in null alleles, and regulatory mutations within the enhancer region of an intron designated by some groups as intron 5 44 45, or intron 4 25, depending on the splice variant of GATA2 used as reference. While many GATA2 patients have family histories of MDS/AML and/or infections, a proportion of cases represent de novo germline mutations, without a family history of disease. De novo germline mutations in

11

ACCEPTED MANUSCRIPT

GATA2 were reported in a major proportion of pediatric and adolescent cases of MDS with germline GATA2 mutation 25. Bone marrow biopsies in GATA2 deficient patients typically demonstrate hypocellularity (Figure 3A-B), with atypical / dysplastic megakaryocytic features including hypolobation,

CR IP T

micromegakaryocytes, and megakaryocytes with separated / peripheralized nuclear lobes (Figure 3C-D, G), as well as erythroid dysplasia (Figure 3E), atypical myeloid maturation (Figure 3F) increased reticulin fibrosis 46, 47. In a subset of cases, the bone marrow cellularity is

markedly reduced precluding adequate assessment of dysplastic changes, and morphologically

AN US

overlapping with severe AA (Figure 3A) 48 49. Bone marrow flow cytometry analysis can be helpful in identifying patients with bone marrow hypoplasia who may harbor germline GATA2 mutations (Figure 4). In contrast to idiopathic AA or de novo hypocellular MDS, the GATA2 deficient bone marrow often shows a distinctive profile with disproportionally and markedly

M

reduced to absent monocytes, dendritic cells, B-cells, B-cell precursors, and NK-cells 46, 47. These flow cytometry features are not common in idiopathic AA which typically shows intact BM

ED

lymphocyte subsets. Relative T-cell lymphocytosis, and a proportion of cases with atypical Tcells, increased T-LGLs, and / or atypical plasma cells, are also frequently seen.

PT

Some GATA2 bone marrows are hypocellular with atypical megakaryocytes, but without overt morphologic evidence of dysplasia in other lineages. In the setting of a normal karyotype,

CE

these cases can be diagnosed as “bone marrow and immunodeficiency disorder with germline GATA2 mutation”. In our experience, these patients should be carefully monitored as disease

AC

progression to overt MDS/AML is not uncommon and may happen suddenly. Clonal cytogenetic abnormalities are identified in approximately 50-60% of GATA2

deficiency associated marrow disease, most commonly monosomy 7, trisomy 8, and trisomy 1q. In a large study of 57 patients with GATA2 deficiency, 84% of patients developed MDS, 14% developed AML, and 8% developed CMML, with other non-hematologic neoplasms noted including human papillomavirus related tumors in 35% and Epstein-Barr virus related tumors in

12

ACCEPTED MANUSCRIPT

4% 42. The exact mechanisms behind clonal evolution and predisposition to malignancy are under investigation and likely multifactorial 25, 41, 50. Hematopoietic stem cell transplantation is the only effective therapy for GATA2 deficiency, and results in hematopoietic reconstitution and reversal of the clinical phenotype 51 52. Penetrance, expressivity, and age of disease onset can

patient’s GATA2 mutation regardless of the donor’s phenotype.

CR IP T

be variable within families, necessitating that all potential related donors be screened for the

3.2. DYSKERATOSIS CONGENITA AND TELOMEROPATHY ASSOCIATED BONE

AN US

MARROW FAILURE

Telomeres are hexanucleotide repeats at the ends of chromosomes, which serve a protective function and solve the “end replication problem” of successive DNA loss after each round of cell division 53 54. Telomere diseases or telomeropathies are rare disorders of telomere

M

maintenance and repair, causing accelerated telomere attrition, senescence, apoptosis, and chromosome instability, and leading to decreased numbers and impaired regeneration of

ED

hematopoietic stem cells 54. Germline mutations in genes encoding components of telomere maintenance predispose to bone marrow failure and abnormalities involving other organ

PT

systems 53 55. Bone marrow failure associated with germline mutations in TERT and TERC, coding for telomerase and its RNA template respectively, affect both children and adults, and

CE

cause isolated occurrence or combinations of bone marrow failure, hepatic fibrosis / steatosis, and pulmonary fibrosis 55, and are associated with an autosomal dominant inheritance pattern

AC

with variable penetrance. Dyskeratosis congenita (DC), a subtype of telomeropathy, is a childhood syndrome

classically characterized by the triad of dystrophic nails, skin rashes, and leukoplakia

54

and

diagnosis at a median age of 15 years 56. Like telomeropathies in general, DC is a complex, multisystem disorder with a broad spectrum of possible abnormalities 56, 57. Though many mutations have recently been identified, it is most commonly caused by X-linked recessive 13

ACCEPTED MANUSCRIPT

mutations in DKC1, primarily affecting males, in which loss of dyskerin destabilizes the telomerase complex. Additionally, altered dyskerin directly impacts ribosomal RNA processing and hematopoietic stem cell differentiation, linking this mechanism to ribosomopathies including Diamond - Blackfan anemia 55. Phenotypically severe DC variants include Hoyeraal –

CR IP T

Hreidarsson syndrome, with cerebellar hypoplasia associated with DKC1 mutations or compound mutations in RTEL158 59; and Revesz syndrome, with bilateral exudative retinopathy associated with autosomal dominant mutations in TINF260. In addition to abnormal cytogenetic clones 56 and hematologic malignancies, DC patients are at risk for numerous carcinomas,

AN US

including squamous cell carcinomas of the tongue, and adenocarcinomas of the head, neck, and GI tract 61 55.

Morphologic examination of bone marrow biopsies in telomeropathy patients frequently demonstrates hypocellularity, with additional findings including intermittent cellular foci and

M

megakaryocyte atypia (Figure 5A-C). Diagnosis can be assisted by obtaining a careful family history, and clues such as early graying of the hair may prove helpful. Laboratory testing

ED

focuses on detection of short telomeres in peripheral blood leukocytes, by methods including flow cytometric measurement of fluorescence in situ hybridization to telomere repeats (flow-

PT

FISH), Southern blot measurement of terminal restriction fragment length, and quantitative polymerase chain reaction (qPCR) 55 62. Cautious interpretation is advised, as telomere length

CE

varies by ethnicity, test sensitivity and specificity are not well established outside of DC patients, and sensitivity may be lower in older patients 55.

AC

Telomere loss has been linked to increased risk of progression to MDS/AML and other

malignancies 63, 64, through complex underlying biologic mechanisms involving the activation of DNA damage response pathways and causation of chromosomal instability 54. Short telomeres may be a biomarker of hematopoietic stem cell damage, and have been seen in acquired AA as well as other inherited BMF disorders including FA, Shwachman – Diamond syndrome, and Diamond – Blackfan anemia 55, 65. A recent study demonstrated marked telomere attrition

14

ACCEPTED MANUSCRIPT

preceding monosomy 7 emergence in AA patients who evolved to MDS / AML, demonstrating a possible mechanism for development of aneuploidy 66. Another study demonstrated association between telomere length and hematopoietic relapse, clonal evolution, and survival in severe AA patients 64.

CR IP T

Androgens have been used to treat telomere diseases, and have been shown to upregulate TERT expression in lymphocytes and CD34+ hematopoietic cells 67. This effect is mediated through an estrogen responsive element in the TERT promoter 68. Recently, a two year trial of danazol therapy in telomeropathy patients showed telomere elongation in 11/12

3.3.

AN US

patients, and improved peripheral blood counts in 10/12 patients 68.

FANCONI ANEMIA

Fanconi anemia (FA) is reported to be the most frequent inherited cause of BMF 69, and

M

is a heterogeneous disease clinically characterized by a broad spectrum of congenital anomalies and a range of mild to severe phenotypes, a natural history of childhood bone

ED

marrow failure, and predisposition to both hematologic and solid tumors. The most common physical findings are short stature, café au lait skin spots, radial ray abnormalities,

PT

microcephaly, micropthalmia, renal abnormalities, and hypogonadism although asymptomatic patients with no physical findings have been incidentally diagnosed and the disease is

CE

underreported 70. The prevalence is estimated to be 1-5 per million, with a slight male predominance 70, carrier frequency of 0.3% to 1% 71, and initial hematologic complications at a

AC

median age of 7 years.

FA has largely autosomal recessive inheritance 71, with mutations identified in over 18

genes 69 involved in a coordinated process to maintain genomic stability and repair DNA interstrand crosslinks. Eight of the FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) form a complex that ubiquitinates FANCD2 and FANCI, which interacts with other downstream FA proteins to engage in chromatin repair 72. This process is

15

ACCEPTED MANUSCRIPT

additionally regulated by ATM and ATR, genes in which abnormalities are associated with ataxia-telangiectasia and Seckel’s syndrome, respectively 72; the pathway also intersects with NBS, mutated in Nijmegen breakage syndrome 73, BRCA1 and BRCA2 72, and the BLM helicase, lost in Bloom’s syndrome 74. The resultant DNA repair abnormality causes a

CR IP T

propensity for spontaneous chromosomal breaks and increased sensitivity to DNA crosslinking agents and radiation 71, and is thought to be present in the hematopoietic stem cell pool in utero, ultimately leading to spontaneous childhood BMF 75.

Laboratory diagnosis is based on hypersensitivity of FA cells to crosslinking agents such

AN US

as diepoxybutane and mitomycin C, causing increased numbers of chromosomal breaks when compared to cells from normal individuals 71. An additional step is determination of complementation groups by correction through retroviral transfection of known FA genes, followed by gene sequencing 70. The diagnosis of FA is critical, because patients do not

M

respond to immunosuppressive therapy as in AA, and will require alternate pre-transplant conditioning regimens and careful solid tumor surveillance 6. Additional laboratory findings

ED

include increased serum alfa fetoprotein (AFP) levels, and increased hemoglobin F concentrations by electrophoresis due to stress hematopoiesis 2.

PT

Bone marrow examination shares a similar morphologic picture overlapping with AA, featuring hypocellularity and patchy hematopoiesis (Figure 5D-E). A low level of dysplastic

CE

features is common, including mild dyserythropoiesis, which raises the challenge of distinction from progression to MDS, and necessitates frequent marrow examination and close follow-up.

AC

Features such as hypercellularity, marked dysplasia, acquired cytogenetic abnormalities, and rapid changes in peripheral blood counts should prompt concern for progression to MDS or AML 2, 69, 70

. The most frequent cytogenetic abnormalities are +1q, +3q, -7q, and RUNX1

abnormalities at 21q22, followed by -5q, -13q, and -20q; classical de novo translocations including t(8;21), t(15;17), and MLL translocations are virtually absent 69. A special circumstance seen in FA is somatic mosaicism, where genetic reversion of one mutated FANC

16

ACCEPTED MANUSCRIPT

allele causes functional correction and hematopoiesis, and this must be distinguished from clonal evolution 69. The incidence of MDS in FA patients ranges from 11% - 34%, and the cumulative incidence of AML ranges from 10% - 37% by age 50 69. HSCT is the only definitive treatment

CR IP T

for AML and MDS with excess blasts or with significant dysplasia and/or poor-prognosis cytogenetic abnormalities, with a 30% - 40% long-term overall survival rate 69. A recent expert review provisionally retained poor prognostic cytogenetic findings as indications for HSCT, including -7q, +3q, RUNX1 abnormalities, and complex karyotypes, while isolated -5q, -11q, and

AN US

-20q can be closely followed without prompt HSCT, depending on the patient context, bone marrow morphology, and cytopenias 69. Transfusion-dependency in FA patients (with severe BMF or severe isolated cytopenias) is also an indication for HCST 76. A recent study has shown 94% survival at 5 years in patients who received alternative donor HSCT without prior history of

M

opportunistic infections or transfusions 77. Post-transplant patients have a 28% cumulative incidence of solid malignancy by age 50, including a cumulative incidence of squamous cell

ED

carcinoma of 24% at 15 years to 34% at 20 years, and a 4.4-fold higher rate of squamous cell carcinoma compared to pretransplantation 69, 70, 78. As a result, close monitoring and avoidance

PT

of smoking and excessive alcohol are indicated. FA patients have excessive toxicity to DNAdamaging agents and therefore, lower doses of chemotherapeutic drugs are mandated if

SHWACHMAN-DIAMOND SYNDROME

AC

3.4.

CE

cytoreduction pre-HSCT is needed.

Shwachman – Diamond syndrome (SDS) is a rare autosomal recessive disorder

characterized by the triad of BMF, exocrine pancreatic insufficiency, and skeletal changes, but can also feature immunodeficiency, hepatic anomalies, dental dysplasia, and low IQ 2. SDS has a slight male predominance, an estimated incidence of 1 in 75,000, and is the second most common cause of childhood exocrine pancreatic insufficiency after cystic fibrosis 2, 70. Children

17

ACCEPTED MANUSCRIPT

typically present with neutropenia and steatorrhea, and a subset of approximately 20% develop severe AA at a median age of 3 70. Approximately 90% of patients harbor biallelic mutations of SBDS, which is located at the centromeric region of chromosome 7, localized to the nucleolus, and involved in ribosomal

CR IP T

subunit association and rRNA processing 70, 79, 80. Low levels of SBDS protein cause impaired ribosome subunit association in SDS 81. Increased Fas antigen expression by hematopoietic stem cells results in decreased colony formation, early neutrophil apoptosis, and ineffective erythropoiesis with a shift from adult to fetal hemoglobin 2. Aberrant mitotic spindle formation

AN US

may contribute to the genomic instability seen in SDS 70. Heterozygous SBDS mutations have been reported in a small number of AA patients who showed no evidence of pancreatic exocrine failure or skeletal abnormalities; interestingly, short telomeres were described in both heterozygous AA patients and affected biallelic SDS patients, potentially linking SBDS

M

heterozygosity and AA via shortened telomeres 82, and highlighting an area of overlap between ribosomopathies and telomeropathies.

ED

Bone marrow biopsies in SDS patients demonstrate a range in cellularity from hypocellular to normocellular, with left-shifted or hypoplastic myeloid maturation present in a

PT

minority of cases 2 (Figure 5F-G). More than 15% of cases demonstrate clonal cytogenetics, most commonly i(7q) though not in AML cases, as well as -7 and del(20q) 70. PNH clones have 83

CE

not been detected, indicating that BMF in SDS does not select for PNH progenitor cells

.

Progression to the acute erythroid leukemia subtype (AML-M6) can be seen in SDS,

AC

representing 30% of cases of leukemic progression 84, 85. A recent study reported that clonal hematopoiesis due to mutations in TP53 was present in 13/27 (48%) of the SDS patients studied, and was thought to represent an early event in transformation to MDS/AML 86. No definitive evidence is available regarding SDS risk for solid tumors 79. HSCT is indicated for severe cytopenias or clonal evolution, and has a reported two year survival of 58%, with a high incidence of transplant-related cardiac, hepatic, and pulmonary toxicity 70.

18

ACCEPTED MANUSCRIPT

3.5.

DIAMOND-BLACKFAN ANEMIA

Diamond – Blackfan anemia (DBA) is a heterogeneous group of disorders clinically characterized by normochromic, usually macrocytic anemia with reticulocytopenia 2, presenting

CR IP T

in the first year of life in the majority of cases, at a median age of 3 months 70. A minority of cases have congenital anomalies including thumb malformations, craniofacial abnormalities, and urogenital anomalies 2, with at least one birth defect present in 25% of cases but of milder degree than in FA or DKC 70. Affected individuals also have an increased cancer predisposition.

AN US

The differential diagnosis includes transient erythroblastopenia of childhood (TEC) and

parvovirus B19 infection. There is no definitive diagnostic laboratory test as in FA and TEL, and diagnostic criteria have been established 87 including the categories of classical, non-classical, and probable DBA 2.

M

In addition to macrocytic anemia, laboratory abnormalities include elevated erythrocyte adenosine deaminase (eADA) activity in greater than 85% of patients 70; although this is

ED

nonspecific for DBA, can also be seen in immune deficiencies and other acquired anemias, and can be used in screening transplant donors 2. Other laboratory findings include elevated

PT

Hemoglobin F, strong erythrocyte expression of i antigen, increased serum erythropoietin, decreased haptoglobin, delayed plasma iron clearance, and low red cell iron utilization 2. Bone

CE

marrow biopsies in DBA patients demonstrate normocellular marrow for age with profound erythroid hypoplasia, occasional dyserythropoietic features with or without ring sideroblasts, and

AC

preserved myeloid and megakaryocytic hematopoiesis with unremarkable morphology and no dysplastic features 2. In addition to being an inherited bone marrow failure and cancer predisposition

syndrome, DBA is a ribosomopathy, caused by heterozygous mutations in genes encoding the 40 S or 60 S ribosomal subunits and affecting rRNA processing

70

. These mutations may be of

autosomal dominant inheritance or arise spontaneously 79, with approximately 75% of cases

19

ACCEPTED MANUSCRIPT

being sporadic 2 and 50% lacking identifiable gene mutations 70. X-linked mutations in GATA1 have also been associated with DBA 79. Mutations result in haploinsufficiency or reduced protein expression, with incomplete understanding of downstream effects 79, though one potential pathway links dysregulation of ribosomal proteins with accumulation of free

CR IP T

unassembled subunits, which in turn bind MDM2 and cause abnormal release and stabilization of p53 70, 79. Also of interest is the identification of the RPS14 gene in 5q- MDS (with somatically acquired deletion of the long arm of chromosome 5), where haploinsufficiency of this ribosomal protein resulted in impaired erythroid differentiation 70, 79. Mutations in CERC1 (ADA2

AN US

deficiency) have recently been associated with pure red cell aplasia resembling DBA 88.

Therapy for DBA includes corticosteroids, transfusion and iron chelation, and HSCT as the only curative treatment 89. The prognosis is superior to that of FA and DKC, with a median overall survival of 40 years and an apparent spontaneous remission rate of around 25% 70.

M

DBA patients suffer an increased risk of cancer, with a crude rate of approximately 3-4%, and reported hematolymphoid malignancies including MDS, AML, and ALL, and solid tumors

ED

including osteosarcomas 70, 90. Clonal cytogenetic evolution is uncommon. No specific cancer surveillance guidelines are recommended at this time, though comprehensive management

GERMLINE MUTATIONS ASSOCIATED WITH THROMBOCYTOPENIA

CE

3.6.

PT

recommendations are available 89.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited BMF

AC

syndrome, characterized by severe thrombocytopenia occurring at birth (platelet count < 50,000/mL), due to failure of megakaryopoiesis. The main symptoms are related to severe thrombocytopenia and include purpura, petechiae, epistaxis, sporadic gastric intestinal bleeding, and rarely, intracranial hemorrhage. Morphologic assessment of the peripheral blood smear at presentation reveals reduced platelets of normal size and granularity, and normal red and white cell morphology and indices. The bone marrow shows reduced or absent megakaryocytes, and

20

ACCEPTED MANUSCRIPT

gradual progression to bone marrow failure with pancytopenia. The molecular cause is attributed to a mutation in the myeloproliferative ligand (c-MPL) gene on chromosome 1p34 91, 92

, which encodes the thrombopoietin receptor. It is inherited in an autosomal recessive

manner. Two phenotypes are recognized, one associated with additional anomalies including

CR IP T

cardiac defects and growth abnormalities, and one without. Treatment includes platelet transfusions initially, whereas red cell transfusions and infections prophylaxis with antibiotics may become necessary as the disease progresses to BMF. HSCT is the only curative

treatment, having the potential for full reconstitution of hematopoiesis. The MECOM-associated

AN US

syndrome with heterozygous mutations in MECOM (MDS1 and EVI1 complex locus), also

causes congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Both familial and sporadic cases have been described 93.

Germline mutations in several other genes have been linked to familial

M

thrombocytopenia with predisposition to the development of myeloid neoplasms 94. These genes include RUNX1 (Familial platelet disorder with predisposition to AML [FPD/AML]) 95,

ED

ANKRD26 (Familial thrombocytopenia 2 [THC2]) 96, and ETV6 (Familial Thrombocytopenia 5) 97, and are associated with an autosomal dominant inheritance pattern. Bone marrow often shows

PT

presence of megakaryocytes in the setting of isolated thrombocytopenia. Megakaryocytes may demonstrate atypical morphologic features consistent with dysmegakaryopoiesis including

CE

separation of nuclear lobes and small mononuclear forms98-100, and may be present in asymptomatic family members with the mutation. Caution must be used in bone marrow

AC

interpretation to avoid over-diagnosis of MDS98, 100, although these patients are at risk of developing MDS/AML. The development of MDS is often associated with onset of additional cytopenias, cytogenetic abnormalities, or multilineage dysplasia98. Germline mutations in ETV6 additionally predispose to the development of B lymphoblastic leukemia, melanoma and solid tumors.

21

ACCEPTED MANUSCRIPT

3.7.

SEVERE CONGENITAL NEUTROPENIA

Severe congenital neutropenia encompasses a group of inherited disorders characterized by neutrophil counts below 0.5 x 109 per liter. The disease, usually inherited in

CR IP T

autosomal recessive manner, presents early in childhood with invasive bacterial infections such as omphalitis, skin abscesses, pneumonia, or septicemia, and predisposition to invasive fungal infections. Although in most cases neutropenia and its related consequences is the single manifestation, other clinical features including neurological, endocrine and immunological

AN US

abnormalities may be present. Among these, decreased bone mineral density has been

described, leading to osteopenia or osteoporosis 101 and increased propensity to fractures. Many patients show qualitative neutrophil aberrations in addition to low neutrophil counts. Bone marrow biopsies and aspirates show severely decreased mature granulocytes due to marrow

M

maturation block at the promyelocyte stage. Morphologically, the neutrophils show prominent vacuolization and aberrant azurophilic granules. Functional abnormalities include defective

ED

migration and bacterial killing, which may be attributed to deficiency of granule proteins102, and increased propensity to undergo apoptosis103.

PT

Mutations in different genes causing SCN have been identified, including those in ELA2/ELANE on 19q13, GFI1 on 1p22, HAX-1 on 1q21, SLC37A4 (encoding glucose-6-

CE

phosphate translocase, G6PT), and WASP, as well as mutations in the granulocyte colony stimulating factor (G-CSF) 104 105. A less commonly mutated gene is GATA2, which has been

AC

found in up to 10% of individuals with congenital neutropenia, and may be associated with immunodeficiency. Other gene mutations that generate neutropenia and are associated with immunodeficiencies include COH (Cohen syndrome), CD40L (Hyper IgM syndrome) and CXCR4 (WHIM syndrome). The roles of these genes in neutrophil development are diverse and involve complex signaling systems. For example, deficiency of the mitochondrial proteins HAX1 and AK2

22

ACCEPTED MANUSCRIPT

causes apoptosis of myeloid progenitors and abnormalities of the mitochondrial transmembrane potential, while mutations in elastase (ELA2/ELANE) are associated with signs of increased endoplasmic reticulum stress 106. Other pathogenic mechanisms include lack of or decreased sensitivity to endogenous G-CSF, due to a dysfunctional extracellular portion of the G-CSF

CR IP T

receptor (G-CSF3R) or to defective mobilization of bone marrow neutrophils (WHIM syndrome) 105, 107

.

Regardless of the mutation, the majority of SCN patients are at increased risk of

progression to MDS and AML. Chromosomal abnormalities including monosomy 7 and gain of

AN US

chromosome 21, as well as sequential acquisition of additional gene mutations (in CSF3R and RUNX1) 108 are frequently detected before AML transformation. Treatment options depend on clinical manifestations. In addition to appropriate antibiotics, the use of G-CSF has been successful in treating life-threatening infections associated with neutropenia, but its prolonged

M

use is related to CSF3R mutations and leukemic transformation 109. HSCT is indicated in

3.8.

ED

refractory patients and in those with poor G-CSF response or transformation to MDS / AML.

CTLA4 HAPLOINSUFFICIENCY

PT

Germline heterozygous mutations in the cytotoxic T-lymphocyte antigen-4 gene (CTLA4) have been recently described and result in haploinsufficiency with predisposition to severe

CE

systemic immune dysregulation 110 associated with features of both autoimmunity and immunodeficiency. The inheritance pattern is autosomal dominant with variable penetrance.

AC

Many patients were originally diagnosed with common variable immune deficiency (CVID) with hypogammaglobulinemia and recurrent infections, but also developed severe autoimmune complications 110 111 beyond the extent of typical CVID. Complications include infiltrative lymphocytic lesions of lungs (e.g. lymphocytic pneumonitis), GI tract (e.g. lymphocytic colitis and intestinal enteropathy), and brain.

Autoimmune cytopenias (anemia, thrombocytopenia and

neutropenia) can manifest in moderate to severe pancytopenia. The bone marrow features can

23

ACCEPTED MANUSCRIPT

resemble those seen in T-LGL with hypercellularity and massive T-cell infiltrates; however, a subset of patients present with markedly hypocellular marrows with trilineage hypoplasia leading to a mistaken diagnosis of AA. Several striking features can be seen in the bone marrow of hypoplastic CTLA4 deficiency cases including prominent atypical T-cell aggregates (Figure 6A)

CR IP T

and a subtle diffuse interstitial T-cell infiltrate in a background of severe trilineage hypoplasia. Flow cytometric analysis of the marrow is helpful in discriminating CTLA4 haploinsufficiency from AA and other hypoplastic marrow disease (Figure 6B) in that CTLA4 marrows typically show a marked decreased to absence of B-cells and B-cell precursors (uncommon in AA), with

AN US

increased T-cell populations (CD4:CD8 ratios vary). Unlike GATA2 deficiency, NK-cell, monocyte, and dendritic populations are typically present in the marrows of CTLA4 haploinsufficiency.

M

CTLA4 is required for proper function of regulatory T-cells. Deficiency of CTLA4 in mice has been shown to result in massive lymphoproliferation with fatal autoimmunity 112. Recently

ED

autosomal recessive mutations in the gene LRBA have been reported that are associated with a similar phenotype to CTLA4 haploinsufficiency 113. LRBA deficiency results in secondary CTLA4

PT

deficiency 114. Bone marrow features of LRBA deficiency are not yet fully characterized. The

CE

prevalence of CTLA4 and LRBA deficiency is currently unknown.

AC

4. Expansion of Gene Mutations Associated with Marrow Hypoplasia and Bone Marrow Failure

New disease entities are being defined based on the identification of underlying germline

mutations identified during targeted sequencing, whole exome sequencing, or whole genome sequencing. Concurrently, new mutations are being discovered in patients presenting with features of BMF, who are not found to harbor classic known mutations, expanding the number

24

ACCEPTED MANUSCRIPT

of genes associated with BMF. Several newly identified genes associated with BMF include SAMD9, SAMD9L, CERC1 and SRP72. Germline mutations in SAMD9 or SAMD9L have been associated with aplasia and monosomy 7 115 and pediatric MDS with monosomy 7 116 117. CERC1 encodes adenosine deaminase 2 (ADA2) and germline mutations have been shown to

CR IP T

underlie a syndrome of vasculopathy presenting in childhood 118; however, mutations in CERC1 have also been identified in patients with pure red cell aplasia, aplastic anemia, bone marrow failure, and immunodeficiency 88, 119, 120. Autosomal dominant mutations in SRP72 have been identified in familial aplastic anemia and MDS 121. The recent WHO classification of hematologic

AN US

malignancies 8 contains a new diagnostic category for myeloid neoplasms with germline

predisposition. Special recognition was given to germline mutations in CEBPA, DDX41, RUNX1, ANKRD26, ETV6, GATA2, and genes associated with inherited BMF syndromes and telomeropathies. The number of gene mutations associated with BMF, marrow hypoplasia, and

M

MDS/AML is anticipated to increase as genomic sequencing becomes more widespread.

ED

5. Conclusion

The inherited and acquired BMF syndromes feature complex molecular pathophysiology

PT

resulting in ineffective hematopoiesis and increased risk of progression to MDS and AML. Clinically diverse presentations and overlapping features make separation from AA challenging,

CE

and rely heavily upon synthesis of clinical information with bone marrow morphology, flow cytometry, cytogenetics, molecular data, and additional laboratory techniques. Advances in our

AC

understanding of recently characterized disorders are introducing promising pharmacologic therapies and improving outcomes with HSCT. Improvements in detection will only increase our cohort sizes and further our understanding and management of these rare disorders.

Acknowledgements

25

ACCEPTED MANUSCRIPT

This work was supported by the Intramural Research Program of the NIH Clinical Center and the National Cancer Institute. The authors would like to thank Neal S. Young, Danielle M. Townsley, Steven M. Holland, Dennis D. Hickstein, Gulbu Uzel, Blanche Alter, Sharon Savage,

CR IP T

Raul Braylan, Irina Maric, Jay Lozier, and Roger Kurlander.

REFERENCES:

8. 9. 10. 11. 12. 13.

AC

14.

AN US

6. 7.

M

5.

ED

4.

PT

2. 3.

Young NS, Calado RT, Scheinberg P. Current concepts in the pathophysiology and treatment of aplastic anemia. Blood. 2006;108:2509-2519. Diagnostic Pediatric Hematopathology: Cambridge University Press; 2011. Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS. Hepatitis-associated aplastic anemia. N Engl J Med. 1997;336:1059-1064. Camitta BM, Storb R, Thomas ED. Aplastic anemia (second of two parts): pathogenesis, diagnosis, treatment, and prognosis. N Engl J Med. 1982;306:712-718. DeZern AE, Sekeres MA. The challenging world of cytopenias: distinguishing myelodysplastic syndromes from other disorders of marrow failure. Oncologist. 2014;19:735-745. Scheinberg P, Young NS. How I treat acquired aplastic anemia. Blood. 2012;120:1185-1196. Bennett JM, Orazi A. Diagnostic criteria to distinguish hypocellular acute myeloid leukemia from hypocellular myelodysplastic syndromes and aplastic anemia: recommendations for a standardized approach. Haematologica. 2009;94:264-268. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 2017. Maciejewski JP, Anderson S, Katevas P, Young NS. Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure. Br J Haematol. 1994;87:227-234. Brodsky RA, Jones RJ. Aplastic anaemia. The Lancet. 2005;365:1647-1656. Young NS, Maciejewski J. The pathophysiology of acquired aplastic anemia. N Engl J Med. 1997;336:1365-1372. Maciejewski JP, Risitano A, Sloand EM, Nunez O, Young NS. Distinct clinical outcomes for cytogenetic abnormalities evolving from aplastic anemia. Blood. 2002;99:3129-3135. Yoshizato T, Dumitriu B, Hosokawa K, et al. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia. N Engl J Med. 2015;373:35-47. Steensma DP, Bejar R, Jaiswal S, et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood. 2015;126:9-16. Kwok B, Hall JM, Witte JS, et al. MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance. Blood. 2015;126:2355-2361. Townsley DM, Scheinberg P, Winkler T, et al. Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia. N Engl J Med. 2017;376:1540-1550. Olnes MJ, Scheinberg P, Calvo KR, et al. Eltrombopag and improved hematopoiesis in refractory aplastic anemia. N Engl J Med. 2012;367:11-19. Georges GE, Storb R. Hematopoietic stem cell transplantation for acquired aplastic anemia. Curr Opin Hematol. 2016;23:495-500.

CE

1.

15. 16. 17. 18.

26

ACCEPTED MANUSCRIPT

25.

26. 27. 28. 29. 30.

31.

32. 33.

AC

34.

CR IP T

24.

AN US

23.

M

22.

ED

21.

PT

20.

Calado RT. Immunologic aspects of hypoplastic myelodysplastic syndrome. Semin Oncol. 2011;38:667-672. Niemeyer CM, Baumann I. Classification of childhood aplastic anemia and myelodysplastic syndrome. Hematology Am Soc Hematol Educ Program. 2011;2011:84-89. Kordasti SY, Afzali B, Lim Z, et al. IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome. Br J Haematol. 2009;145:64-72. Matsui WH, Brodsky RA, Smith BD, Borowitz MJ, Jones RJ. Quantitative analysis of bone marrow CD34 cells in aplastic anemia and hypoplastic myelodysplastic syndromes. Leukemia. 2006;20:458-462. Westers TM, Ireland R, Kern W, et al. Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group. Leukemia. 2012;26:1730-1741. Aalbers AM, van den Heuvel-Eibrink MM, Baumann I, et al. Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood. Haematologica. 2015;100:315-323. Wlodarski MW, Hirabayashi S, Pastor V, et al. Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents. Blood. 2016;127:1387-1397; quiz 1518. Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood. 2012;120:2454-2465. Lamy T, Loughran TP, Jr. Clinical features of large granular lymphocyte leukemia. Semin Hematol. 2003;40:185-195. Go RS, Li CY, Tefferi A, Phyliky RL. Acquired pure red cell aplasia associated with lymphoproliferative disease of granular T lymphocytes. Blood. 2001;98:483-485. Go RS, Tefferi A, Li CY, Lust JA, Phyliky RL. Lymphoproliferative disease of granular T lymphocytes presenting as aplastic anemia. Blood. 2000;96:3644-3646. Morice WG, Kurtin PJ, Tefferi A, Hanson CA. Distinct bone marrow findings in T-cell granular lymphocytic leukemia revealed by paraffin section immunoperoxidase stains for CD8, TIA-1, and granzyme B. Blood. 2002;99:268-274. Lima M, Almeida J, Santos AH, et al. Immunophenotypic analysis of the TCR-Vbeta repertoire in 98 persistent expansions of CD3(+)/TCR-alphabeta(+) large granular lymphocytes: utility in assessing clonality and insights into the pathogenesis of the disease. Am J Pathol. 2001;159:1861-1868. Kook H, Risitano AM, Zeng W, et al. Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens. Blood. 2002;99:3668-3675. Risitano AM, Maciejewski JP, Muranski P, et al. Large granular lymphocyte (LGL)-like clonal expansions in paroxysmal nocturnal hemoglobinuria (PNH) patients. Leukemia. 2005;19:217222. Koskela HL, Eldfors S, Ellonen P, et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med. 2012;366:1905-1913. Schade AE, Powers JJ, Wlodarski MW, Maciejewski JP. Phosphatidylinositol-3-phosphate kinase pathway activation protects leukemic large granular lymphocytes from undergoing homeostatic apoptosis. Blood. 2006;107:4834-4840. Andersson EI, Tanahashi T, Sekiguchi N, et al. High incidence of activating STAT5B mutations in CD4-positive T-cell large granular lymphocyte leukemia. Blood. 2016;128:2465-2468. Vinh DC, Patel SY, Uzel G, et al. Autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses, and myelodysplasia. Blood. 2010;115:1519-1529.

CE

19.

35.

36. 37.

27

ACCEPTED MANUSCRIPT

44.

45. 46.

47. 48.

. 49. 50.

51.

Chu VH, Curry JL, Elghetany MT, Curry CV. MonoMAC versus idiopathic CD4+lymphocytopenia. Comment to Haematologica. 2011;96(8):1221–5. Haematologica. 2012;97:e9-e11. West RR, Hsu AP, Holland SM, Cuellar-Rodriguez J, Hickstein DD. Acquired ASXL1 mutations are common in patients with inherited GATA2 mutations and correlate with myeloid transformation. Haematologica. 2014;99:276-281. Cuellar-Rodriguez J, Gea-Banacloche J, Freeman AF, et al. Successful allogeneic hematopoietic stem cell transplantation for GATA2 deficiency. Blood. 2011;118:3715-3720. Parta M, Shah NN, Baird K, et al. Allogeneic Hematopoietic Stem Cell Transplantation for GATA2 Deficiency Using a Busulfan-Based Regimen. Biol Blood Marrow Transplant. 2018. Armanios M, Blackburn EH. The telomere syndromes. Nat Rev Genet. 2012;13:693-704. Calado RT, Young NS. Telomere diseases. N Engl J Med. 2009;361:2353-2365. Townsley DM, Dumitriu B, Young NS. Bone marrow failure and the telomeropathies. Blood. 2014;124:2775-2783. Savage SA, Alter BP. Dyskeratosis congenita. Hematol Oncol Clin North Am. 2009;23:215-231. Dokal I. Dyskeratosis congenita in all its forms. Brit J Haematol. 2000;110:768-779. Ballew BJ, Yeager M, Jacobs K, et al. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita. Hum Genet. 2013;132:473-480.

AC

52.

CR IP T

43.

AN US

42.

M

41.

ED

40.

PT

39.

Hsu AP, Sampaio EP, Khan J, et al. Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (MonoMAC) syndrome. Blood. 2011;118:2653-2655. Dickinson RE, Griffin H, Bigley V, et al. Exome sequencing identifies GATA-2 mutation as the cause of dendritic cell, monocyte, B and NK lymphoid deficiency. Blood. 2011;118:2656-2658. Ostergaard P, Simpson MA, Connell FC, et al. Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome). Nat Genet. 2011;43:929-931. Hahn CN, Chong CE, Carmichael CL, et al. Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia. Nat Genet. 2011;43:1012-1017. Spinner MA, Sanchez LA, Hsu AP, et al. GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity. Blood. 2014;123:809-821. Bresnick EH, Katsumura KR, Lee HY, Johnson KD, Perkins AS. Master regulatory GATA transcription factors: mechanistic principles and emerging links to hematologic malignancies. Nucleic Acids Res. 2012;40:5819-5831. Hsu AP, Johnson KD, Falcone EL, et al. GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome. Blood. 2013;121:3830-3837, S38313837. Dickinson RE, Milne P, Jardine L, et al. The evolution of cellular deficiency in GATA2 mutation. Blood. 2014;123:863-874. Calvo KR, Vinh DC, Maric I, et al. Myelodysplasia in autosomal dominant and sporadic monocytopenia immunodeficiency syndrome: diagnostic features and clinical implications. Haematologica. 2011;96:1221-1225. Ganapathi KA, Townsley DM, Hsu AP, et al. GATA2 deficiency-associated bone marrow disorder differs from idiopathic aplastic anemia. Blood. 2015;125:56-70. Calvo KR, Hickstein DD, Holland SM. MonoMAC and GATA2 deficiency: overlapping clinical and pathological features with aplastic anemia and idiopathic CD4+ lymphocytopenia. Reply to Haematologica 2012;97(4):058669. Haematologica. 2012;97:e12-e13.

CE

38.

53. 54. 55. 56. 57. 58.

28

ACCEPTED MANUSCRIPT

65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75.

AC

76.

CR IP T

64.

AN US

63.

M

62.

ED

61.

PT

60.

Walne AJ, Vulliamy T, Kirwan M, Plagnol V, Dokal I. Constitutional mutations in RTEL1 cause severe dyskeratosis congenita. Am J Hum Genet. 2013;92:448-453. Savage SA, Giri N, Baerlocher GM, Orr N, Lansdorp PM, Alter BP. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita. Am J Hum Genet. 2008;82:501-509. Alter BP, Giri N, Savage SA, Rosenberg PS. Cancer in dyskeratosis congenita. Blood. 2009;113:6549-6557. Gutierrez-Rodrigues F, Santana-Lemos BA, Scheucher PS, Alves-Paiva RM, Calado RT. Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans. PLoS One. 2014;9:e113747. Willeit P, Willeit J, Mayr A, et al. Telomere length and risk of incident cancer and cancer mortality. JAMA. 2010;304:69-75. Scheinberg P, Cooper JN, Sloand EM, Wu CO, Calado RT, Young NS. Association of telomere length of peripheral blood leukocytes with hematopoietic relapse, malignant transformation, and survival in severe aplastic anemia. JAMA. 2010;304:1358-1364. Alter BP, Giri N, Savage SA, Rosenberg PS. Telomere length in inherited bone marrow failure syndromes. Haematologica. 2015;100:49-54. Dumitriu B, Feng X, Townsley DM, et al. Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia. Blood. 2015;125:706-709. Calado RT, Yewdell WT, Wilkerson KL, et al. Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells. Blood. 2009;114:2236-2243. Townsley DM, Dumitriu B, Liu D, et al. Danazol Treatment for Telomere Diseases. N Engl J Med. 2016;374:1922-1931. Peffault de Latour R, Soulier J. How I treat MDS and AML in Fanconi anemia. Blood. 2016;127:2971-2979. Shimamura A, Alter BP. Pathophysiology and management of inherited bone marrow failure syndromes. Blood Rev. 2010;24:101-122. Smith AR, Wagner JE. Current clinical management of Fanconi anemia. Expert Rev Hematol. 2012;5:513-522. D'Andrea AD. Susceptibility pathways in Fanconi's anemia and breast cancer. N Engl J Med. 2010;362:1909-1919. Nakanishi K, Taniguchi T, Ranganathan V, et al. Interaction of FANCD2 and NBS1 in the DNA damage response. Nat Cell Biol. 2002;4:913-920. Meetei AR, Sechi S, Wallisch M, et al. A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome. Mol Cell Biol. 2003;23:3417-3426. Garaycoechea JI, Patel KJ. Why does the bone marrow fail in Fanconi anemia? Blood. 2014;123:26-34. MacMillan ML, Wagner JE. Haematopoeitic cell transplantation for Fanconi anaemia - when and how? Br J Haematol. 2010;149:14-21. MacMillan ML, DeFor TE, Young JA, et al. Alternative donor hematopoietic cell transplantation for Fanconi anemia. Blood. 2015;125:3798-3804. Rosenberg PS, Socie G, Alter BP, Gluckman E. Risk of head and neck squamous cell cancer and death in patients with Fanconi anemia who did and did not receive transplants. Blood. 2005;105:67-73. Ruggero D, Shimamura A. Marrow failure: a window into ribosome biology. Blood. 2014;124:2784-2792. Ganapathi KA, Austin KM, Lee CS, et al. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA. Blood. 2007;110:1458-1465.

CE

59.

77. 78.

79. 80.

29

ACCEPTED MANUSCRIPT

88. 89. 90.

91. 92. 93.

94. 95. 96.

97.

AC

98.

CR IP T

87.

AN US

85. 86.

M

84.

ED

83.

PT

82.

Burwick N, Coats SA, Nakamura T, Shimamura A. Impaired ribosomal subunit association in Shwachman-Diamond syndrome. Blood. 2012;120:5143-5152. Calado RT, Graf SA, Wilkerson KL, et al. Mutations in the SBDS gene in acquired aplastic anemia. Blood. 2007;110:1141-1146. Keller P, Debaun MR, Rothbaum RJ, Bessler M. Bone marrow failure in Shwachman-Diamond syndrome does not select for clonal haematopoiesis of the paroxysmal nocturnal haemoglobinuria phenotype. Brit J Haematol. 2002;119:830-832. Dokal I, Rule S, Chen F, Potter M, Goldman J. Adult onset of acute myeloid leukaemia (M6) in patients with Shwachman-Diamond syndrome. Br J Haematol. 1997;99:171-173. Dror Y. Shwachman-Diamond syndrome. Pediatr Blood Cancer. 2005;45:892-901. Xia J, Miller CA, Baty J, et al. Somatic mutations and clonal hematopoiesis in congenital neutropenia. Blood. 2018;131:408-416. Vlachos A, Ball S, Dahl N, et al. Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference. Br J Haematol. 2008;142:859-876. Sasa GS, Elghetany MT, Bergstrom K, et al. Adenosine Deaminase 2 Deficiency As a Cause of Pure Red Cell Aplasia Mimicking Diamond Blackfan Anemia. Blood. 2015;126. Vlachos A, Muir E. How I treat Diamond-Blackfan anemia. Blood. 2010;116:3715-3723. Vlachos A, Rosenberg PS, Atsidaftos E, Alter BP, Lipton JM. Incidence of neoplasia in Diamond Blackfan anemia: a report from the Diamond Blackfan Anemia Registry. Blood. 2012;119:38153819. Ihara K, Ishii E, Eguchi M, et al. Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia. Proc Natl Acad Sci U S A. 1999;96:3132-3136. Ballmaier M, Germeshausen M, Schulze H, et al. c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia. Blood. 2001;97:139-146. Germeshausen M, Ancliff P, Estrada J, et al. MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia. Blood Adv. 2018;2:586-596. Brown AL, Churpek JE, Malcovati L, Dohner H, Godley LA. Recognition of familial myeloid neoplasia in adults. Semin Hematol. 2017;54:60-68. Schlegelberger B, Heller PG. RUNX1 deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM). Semin Hematol. 2017;54:75-80. Noris P, Perrotta S, Seri M, et al. Mutations in ANKRD26 are responsible for a frequent form of inherited thrombocytopenia: analysis of 78 patients from 21 families. Blood. 2011;117:66736680. Zhang MY, Churpek JE, Keel SB, et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nat Genet. 2015;47:180-185. Kanagal-Shamanna R, Loghavi S, DiNardo CD, et al. Bone marrow pathologic abnormalities in familial platelet disorder with propensity for myeloid malignancy and germline RUNX1 mutation. Haematologica. 2017;102:1661-1670. Noetzli L, Lo RW, Lee-Sherick AB, et al. Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nat Genet. 2015;47:535-538. Zaninetti C, Santini V, Tiniakou M, Barozzi S, Savoia A, Pecci A. Inherited thrombocytopenia caused by ANKRD26 mutations misdiagnosed and treated as myelodysplastic syndrome: report on two cases. J Thromb Haemost. 2017;15:2388-2392. Yakisan E, Schirg E, Zeidler C, et al. High incidence of significant bone loss in patients with severe congenital neutropenia (Kostmann's syndrome). J Pediatr. 1997;131:592-597.

CE

81.

99.

100.

101.

30

ACCEPTED MANUSCRIPT

108.

109. 110. 111. 112. 113.

114. 115. 116. 117. 118.

AC

119.

CR IP T

107.

AN US

106.

M

105.

ED

104.

PT

103.

Pütsep K, Carlsson G, Boman HG, Andersson M. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. The Lancet. 2002;360:1144-1149. Schaffer AA, Klein C. Genetic heterogeneity in severe congenital neutropenia: how many aberrant pathways can kill a neutrophil? Curr Opin Allergy Clin Immunol. 2007;7:481-494. Xia J, Bolyard AA, Rodger E, et al. Prevalence of mutations in ELANE, GFI1, HAX1, SBDS, WAS and G6PC3 in patients with severe congenital neutropenia. Br J Haematol. 2009;147:535-542. Triot A, Jarvinen PM, Arostegui JI, et al. Inherited biallelic CSF3R mutations in severe congenital neutropenia. Blood. 2014;123:3811-3817. Klein C. Genetic defects in severe congenital neutropenia: emerging insights into life and death of human neutrophil granulocytes. Annu Rev Immunol. 2011;29:399-413. Hernandez PA, Gorlin RJ, Lukens JN, et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat Genet. 2003;34:70-74. Skokowa J, Steinemann D, Katsman-Kuipers JE, et al. Cooperativity of RUNX1 and CSF3R mutations in severe congenital neutropenia: a unique pathway in myeloid leukemogenesis. Blood. 2014;123:2229-2237. Rosenberg PS, Zeidler C, Bolyard AA, et al. Stable long-term risk of leukaemia in patients with severe congenital neutropenia maintained on G-CSF therapy. Br J Haematol. 2010;150:196-199. Kuehn HS, Ouyang W, Lo B, et al. Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4. Science. 2014;345:1623-1627. Schubert D, Bode C, Kenefeck R, et al. Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nat Med. 2014;20:1410-1416. Waterhouse P, Penninger JM, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science. 1995;270:985-988. Lopez-Herrera G, Tampella G, Pan-Hammarstrom Q, et al. Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. Am J Hum Genet. 2012;90:986-1001. Lo B, Fritz JM, Su HC, Uzel G, Jordan MB, Lenardo MJ. CHAI and LATAIE: new genetic diseases of CTLA-4 checkpoint insufficiency. Blood. 2016;128:1037-1042. Bluteau O, Sebert M, Leblanc T, et al. A landscape of germline mutations in a cohort of inherited bone marrow failure patients. Blood. 2017. Pastor VB, Sahoo S, Boklan J, et al. Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7. Haematologica. 2017. Schwartz JR, Ma J, Lamprecht T, et al. The genomic landscape of pediatric myelodysplastic syndromes. Nat Commun. 2017;8:1557. Zhou Q, Yang D, Ombrello AK, et al. Early-onset stroke and vasculopathy associated with mutations in ADA2. N Engl J Med. 2014;370:911-920. Van Montfrans JM, Hartman EA, Braun KP, et al. Phenotypic variability in patients with ADA2 deficiency due to identical homozygous R169Q mutations. Rheumatology (Oxford). 2016;55:902910. Hsu AP, West RR, Calvo KR, et al. Adenosine deaminase type 2 deficiency masquerading as GATA2 deficiency: Successful hematopoietic stem cell transplantation. J Allergy Clin Immunol. 2016;138:628-630 e622. Kirwan M, Walne AJ, Plagnol V, et al. Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial aplasia and myelodysplasia. Am J Hum Genet. 2012;90:888892.

CE

102.

120.

121.

31

ACCEPTED MANUSCRIPT

FIGURE LEGENDS

Figure 1. Aplastic Anemia (AA), AA with PNH clone, and hypocellular MDS.

PT

ED

M

AN US

CR IP T

A. Bone marrow core biopsy from 22-year-old female with severe AA, showing markedly hypocellular marrow with trilineage hypoplasia (100x magnification, Hematolylin & Eosin [H&E]). B and C. Bone marrow core biopsy (100x, H&E) and aspirate (1000x, Wright Giemsa [WG]) from a 7 year-old-male with AA and a PNH clone involving 13% of peripheral blood granulocytes. The marrow is markedly hypocellular for age (B) with erythroid predominance and rare nuclear budding on aspirate smear (C, and in inset) in less than 10% of erythroid precursors. D-I. Bone marrow from an 8-year-old female with pancytopenia and hypocellular MDS EB-1. Bone marrow cytogenetic analysis showed 46,XX,der(5)t(1;5)(q11;q11.2) in 3 out of 20 metaphases. D. Hypocellular bone marrow core biopsy, (100x, H&E stain). E. Atypical/dysplastic monolobated megakaryocytes (500x, H&E). F. CD61 immunohistochemistry (IHC) stain highlighting dysplastic megakaryocytes (500x). G. CD34 IHC stain revealing increased mononuclear cells consistent with blasts and estimated to represent 5-6% of the cellular marrow (200x). H. Paucicellular aspirate with presence of blasts(1000x, WG). No overt morphologic dysplastic changes were seen in the few erythroid and myeloid precursors present. I. Flow cytometric analysis of marrow aspirate of same 8-year-old with MDS-EB-1, in comparison to healthy control marrow. Marrow from healthy control demonstrates that the CD34 positive blasts (red) show a normal spectrum of CD13 expression from dim to bright (top left panel). The control myeloblasts express CD38 (top middle panel) and are negative for CD7 (top right panel). In contrast, an abnormal immunophenotype of myelobasts is detected in the MDS sample with tighter clustering of blasts on CD13 vs CD34 (bottom left panel), that are negative for CD38 (abnormal)(bottom middle panel) and are positive for CD7 (abnormal) (bottom right panel) consistent with a dysplastic phenotype.

AC

CE

Figure 2. Pure red cell aplasia (PRCA) in the setting of T-Large Granular Leukemia (TLGL) A. Bone marrow from a 60 year old male with transfusion dependent anemia and neutropenia. H&E stain (top left, 200x) shows a mildly hypocellular marrow for age. Immunohistochemistry for CD71 (top middle, 200x) demonstrates the absence of erythroid precursors. Myeloid precursors are highlighted by myeloperoxidase (MPO) stain (top right, red, 200x). CD3 IHC stain (bottom left, red, 200x) shows a prominent T-cell interstitial infiltrate in the BM. The majority of T-cells are positive for CD8 (bottom middle, brown, 200x) and TIA-1 (bottom right, brown, 500x). B. Flow cytometry analysis of normal healthy control marrow and marrow from patient with T-LGL and PRCA. Erythroid precursors (left plots under heading entitled “Erythroid”) in the marrow of the normal control (upper left) show normal maturation based on expression of CD105 and CD117. As indicated by the arrows, the earliest erythroid precursors (very few in number in the control) are positive for CD117 and CD105; as erythroid precursors differentiate, they lose expression of CD117 and subsequently downregulate expression of CD105 during maturation. In contrast, the erythroid precursors in the T-LGL patient with PRCA (bottom left) show phenotypic evidence of maturation arrest with the majority

32

ACCEPTED MANUSCRIPT

of erythroid cells expressing CD105 and variable CD117 (immature), with absence of mature forms that are negative for CD117 and CD105. T-cells (right three plots under heading entitled “Lymphocytes”) in the normal control (upper plots) are positive for CD3 and predominantly positive for CD7, show a normal distribution of CD8+ and CD4+ T-cells, and are largely negative for CD57. In the patient with LGL, a large abnormal T-cell population (red) is identified that is negative for CD7, dim to negative for CD8, negative for CD4, and expresses a spectrum of CD57 ranging from bright to negative; normal T-cells are also present in lower numbers (blue).

ED

M

AN US

CR IP T

Figure 3. GATA2 deficiency. A. Markedly hypocellular marrow from a 37-year-old male with pancytopenia (100x, H&E). Of note, paucicellularity limited morphologic evaluation for dysplastic changes. Morphologically, the initial findings were compatible with AA. However, the cytogenetic analysis revealed an abnormal karyotype with trisomy 8 and trisomy 1q. Based on a family history of hematologic disorders (cytopenias, MDS), the patient was tested and found positive for a germline mutation in GATA2 and was diagnosed with Myelodysplasia with germline GATA2 mutation. B-F. 23-year-old male with prior diagnosis of severe AA and previous normal bone marrow karyotype. There was no prior recognized history of immunodeficiency or infections. A bone marrow biopsy was performed revealing: B. Hypocellular marrow (100x, H&E). C. Focal areas showing dysplastic megakaryocytes with separated nuclear lobes(500x, H&E). D. Aspirate showing characteristic large dysplastic megakaryocyte with separated and peripheralized nuclear lobes (1000x, WG). E. Binucleated erythroid precursor (1000x, WG, cropped). F. Pelgeroid hyposegmented neutrophil on peripheral smear (1000x, WG, cropped). B-cells were absent in bone marrow based on CD20 IHC (not shown). Cytogenetic analysis of the marrow revealed an abnormal karyotype with monosomy 7. Germline mutation in GATA2 was later identified. G. Dysplastic megakaryocytes in the hypocellular bone marrow of a neutropenic 17 year-old-male with a history of warts, severe respiratory infections, and skin infections (500x, WG). Bone marrow flow cytometry showed absence of monocytes, B-cell precursors, dendritic cells, and marked B-cell and NK-cell lymphopenia. Cytogenetic analysis showed a normal karyotype. DNA sequencing revealed a germline mutation in GATA2.

AC

CE

PT

Figure 4. Flow cytometry (FC) analysis of bone marrow in AA and GATA2 deficiency (GATA2). Despite hypocellularity of bone marrow in AA and GATA2, most cases have enough cells for detailed FC analysis. A. Monocytes. FC in AA (left panel) typically shows presence of monocyte populations based on expression of CD14 and CD64 (cells in red box, plot displays all BM cells); in contrast GATA2 marrow (right panel) shows absence of monocytes. B. B-cells and precursors. FC in AA (left panel) shows presence of B-cell precursors expressing CD10 [cells in red box], and mature B-cells expressing CD20 and lacking CD10 [cells in black box] (plot displays BM lymphocytes); in contrast, GATA2 marrow (right panel) shows absence of Bcell precursors and decreased mature B-cells. C. NK-cells. FC in AA (left panel) shows presence of NK-cells based on expression of CD56 and negativity for CD3 (cells in red box, plot displays BM lymphocytes); in contrast, GATA2 marrow (right panel) shows absence of NK-cells. D. Plasmacytoid dendritic cells. FC in AA (left panel) shows presence of plasmacytoid dendritic cells based on expression of CD123 and HLA-DR (cells in red box, plot displays all BM cells); in contrast, GATA2 marrow (right panel) shows absence of plasmacytoid dendritic cells. Figure 5. Telomeropathy, Fanconi Anemia and Shwachman Diamond Syndrome. A-C. 21-year-old male with pancytopenia and a history of portal hypertension, esophageal varices, and fingernail clubbing. A compound heterozygous germline mutation in TERT was identified. Cytogenetic analysis of the marrow showed a normal karyotype. A. Bone marrow core biopsy showing hypocellular marrow for age (40x, H&E). B. Focal atypical megakaryocytes clusters (200x, H&E). C. Aspirate with atypical megakaryocyte (1000x,

33

ACCEPTED MANUSCRIPT

WG). D-E. 10-year-old male with Fanconi anemia (germline mutation in FANCJ). D. Hypocellular bone marrow biopsy with focal atypical megakaryocyte clustering (200x, H&E). E. Marrow aspirate showing myeloid hypoplasia (1000x, WG). F-G. 21-year-old male with history of Schwachman Diamond syndrome and neutropenia with mild thrombocytopenia. F. Bone marrow is hypocellular for age (100x, H&E). G. Aspirate smear shows myeloid hypoplasia without overt dysplasia (1000x, WG).

AC

CE

PT

ED

M

AN US

CR IP T

Figure 6. CTLA4 deficiency. A. Bone marrow core biopsy from a 19-year-old male with a history of combined variable immune deficiency (CVID), molluscum contagiousum, shingles, and frequent pneumonias who developed progressive pancytopenia and was diagnosed with severe AA (40x, H&E). A germline mutation in CTLA4 was identified. The bone marrow biopsy shows multiple atypical lymphoid aggregates in a background of a markedly hypocellular marrow with trilineage hypoplasia (upper left). Immunohistochemistry for CD3 shows that the lymphoid aggregates are strongly positive for CD3 (upper right, 40x) and highlights an interstitial T-cell infiltrate throughout the hypocellular regions. The majority of T-cells, particularly in the interstitial regions, are positive for CD8 (lower left, 40x). CD79a reveals a paucity of B-lineage cells (lower right, 40x). B. Flow cytometric analysis of the marrow aspirate of the patient and normal control marrow for comparison. The CTLA4 marrow shows marked decrease in mature B-cells (black box, CD20 positive and CD10 negative, plot displays lymphocytes), and B-cell precursors (red box, CD10 positive cells), in comparison to the healthy control marrow.

34

AC

CE

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

35

AC

CE

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

36

AC

CE

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

37

AC

CE

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

38

AC

CE

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

39

AC

CE

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

p

40