Distinguishing constitutional from acquired bone marrow failure in the hematology clinic

Abstract

Distinguishing constitutional from immune bone marrow failure (BMF) has important clinical implications. However, the diagnosis is not always straightforward, and immune aplastic anemia, the commonest BMF, is a diagnosis of exclusion. In this review, we discuss a general approach to the evaluation of BMF, focusing on clinical presentations particular to immune and various constitutional disorders as well as the interpretation of bone marrow histology, flow cytometry, and karyotyping. Additionally, we examine the role of specialized testing in both immune and inherited BMF, and discuss genetic testing, both its role in patient evaluation and interpretation of results.

Introduction:

Bone marrow failure (BMF) poses a diagnostic challenge in the hematology clinic due to the rarity of syndromes and its wide clinical spectrum. Characterized by decreased production of one or more hematopoietic lineages and resulting in peripheral cytopenias, BMF is broadly classified as either constitutional or acquired. Hypoproliferative myelodysplastic syndrome (hMDS) and immune aplastic anemia (AA) are the commonest forms of marrow failure, discounting iatrogenic chemotherapy or radiotherapy. Immune AA in most cases presents acutely with marked cytopenias, termed severe AA (SAA). Absent specific testing, SAA is ultimately a diagnosis of exclusion in a patient meeting Camitta criteria: marrow cellularity <30% plus at least 2 of 3 cytopenias: absolute neutrophil count (ANC) <500 ×10⁹/L, absolute reticulocyte count (ARC) <60×10⁹/L and platelet count <20 ×10⁹/L). MDS is diagnosed and classified using the well-defined World Health Organization (WHO) 2016 classification of myeloid neoplasms(1) by the presence of cytopenias, morphological dysplasia, cytogenetic abnormalities, and certain somatic gene mutations. hMDS, however, can be difficult to distinguish from AA due to paucity of marrow cells and the overlap in cytogenetic findings with AA. Almost all MDS is acquired and occurs in older adults, when found in children and young adults there is a higher risk of an underlying germline predisposition syndrome. Many predisposition syndromes are also associated with marrow failure(2).

Constitutional BMF syndromes are less frequent than acquired, and in their classical forms they usually manifest in early childhood, have a characteristic clinical phenotype with physical abnormalities, and appear in conjunction with a pertinent family history. In recent years, advances in genetic sequencing have resulted in the identification of previously unrecognized etiologies, many of which can first appear in adulthood and with a less obvious clinical phenotype. The correct distinction between inherited and acquired syndromes is critical to providing proper care. Constitutional marrow failure does not typically respond to immunosuppression (IST), or other therapies used for immune BMF. Rather, patients with constitutional marrow failure are often referred for hematopoietic stem cell transplant (HSCT), disease specific medications, or investigational therapies. Additionally, identification of constitutional BMF prior to HSCT is important to allow for modifications to HSCT conditioning as well as exclusion of family donors harboring the same inherited gene defect. Patients with constitutional BMF often have multi-organ involvement, requiring care from multiple sub-specialists, as well as an increased cancer risk necessitating long term surveillance(3). We offer a practical diagnostic approach to BMF including common clinical presentations, useful laboratory tests, and genetic testing.

Clinical characteristics and basic laboratory evaluation

In the evaluation of a patient with bone marrow failure, a complete history paying particular attention to concurrent medical diagnoses, family history and potential exposures, as well as thorough physical examination are required. Patients with constitutional BMF may have a distinct syndromic pattern of multi-organ disease absent in immune AA (table 1). Cytopenias are the defining feature of either constitutional or acquired BMF, accompanied by the presence of marrow hypocellularity and reticulocytopenia (either absolute or relative), with some disease-dependent patterns(4). Before considering a diagnosis of BMF, other common causes of cytopenia should first be excluded: vitamin deficiencies (in particular B12 and folate), direct toxicity from alcohol use, viral infections (classically parvovirus B19 but also HIV, hepatitis C, EBV, CMV, and dengue), medication induced cytopenias, liver cirrhosis, hematologic malignancy, and common autoimmune diseases (such as rheumatoid arthritis and systemic lupus erythematosus). Results of the history, physical examination, and laboratory testing should be combined to guide further investigation. Classical clinical phenotypes and inheritance provide diagnostic clues(5). However, constitutional BMF can present atypically, and genetic factors underlying some constitutional marrow failure disorders are likely still undiscovered. In general, a BMF patient with multi-organ involvement, younger age, long standing or moderate cytopenias, or a family history of BMF, hematologic malignancy, or increased solid cancers should prompt consideration for constitutional BMF(Figure 1).

Table 1.

Inheritance and common clinical findings of constitutional BMF

	Fanconi anemia	Telomere disease	Schwachman diamond syndrome	Diamond-blackfan anemia	GATA2	SAMD9/L	Platelet disorders
Genes	FANC genes, BRCA2	DKC1, TERT, TERC, PARN, RTEL1, TINF2, CTC + others	SBDS	RP genes and TSR2	GATA2	SAMD9/L	c-MPL (CAMT) RBM8A (TAR) RUNX1, ETV6, ANKRD26
Inheritance	AR except for FANCB (XLR) and FANCR (AD)	AD: TERT, TERC, TINF2 AR: CTC, RTEL1 XR: DKC1	AR	AD or sporadic	AD	AD	AR: (CAMT, TAR) AD: RUNX1, ETV6, ANKRD26
Common non hematologic clinical findings	Limb abnormalities (absent radii / short thumbs) Short stature Renal anatomical defects Cafe au lait spots Microcephaly / microphthalmia	DC triad: oral leukoplakia, dyskeratotic nails, reticulated skin Pulmonary fibrosis Liver disease (fibrosis, fatty liver) AVM Early grey hair Immunodeficiency Osteoporosis	Failure to thrive/poor feeding Steatorrhea Recurrent infections Skeletal abnormalities Hepatomegaly Intellectual disability Congenital cardiac defects Endocrinopathy	Short stature/IUGR Limb abnormalities (triphalangeal thumb) Cardiac defects (VSD, ASD) Cephalic malformation (microcephaly) Developmental delay	Immunodeficiency (atypical mycobacteria, recurrent warts from HPV) Lymphedema Thrombosis Pulmonary alveolar proteinosis (dyspnea and cough)	MIRAGE: myelodysplasia, infection, growth restriction, adrenal hypoplasia, genital problems, enteropathy) Ataxia Pancytopenia: cerebellar symptoms and pancytopenia SAAD: nodular neutrophilic panniculitis, ILD, basal ganglia calcifications, cytopenia	CAMT: some neurological associations, possibly related to ICH TAR: skeletal defects (absent radii), cow’s milk intolerance, renal tract abnormalities, cardiac defects) RUNX1: platelet function defect
Malignancy risk	MDS/AML SCC of skin, head/neck, anogenital	MDS/AML SCC skin, head/neck, anogenital BCC skin	MDS/AML	MDS/AML Colon cancer Osteogenic sarcoma	MDS/AML SCC skin, anogenital BCC skin	MDS/AML	RUNX1/ETV6/ANKRD26: MDS/AML or ALL ALL > ETV6, MDS/AML > RUNX1/ANKRD26

Abbreviations: AR; autosomal recessive, AD; autosomal dominant, XR; x-linked recessive, MDS: myelodysplastic syndrome, AML; acute myeloid leukemia, SCC; squamous cell carcinoma, BCC; basal cell carcinoma, AVM; arteriovenous malformation, VSD; ventricular septal defect, ASD; atrial septal defect, HPV; human papilloma virus, CAMT; congenital amegakaryocytic thrombocytopenia, TAR: thrombocytopenia absent radii, ICH; intracranial hemorrhage, RP; ribosomal protein

Figure 1:

Features of a BMF patient’s clinical history, physical examination, and basic laboratory testing that are typically more consistent of either a constitutional or acquired BMF. Abbreviations: TBD; telomere biology disorder, PNH; paroxysmal nocturnal hemoglobinuria, DEB; diepoxybutane, LGL; large granular lymphocytosis.

Immune marrow failure

Immune AA is a diagnosis of exclusion. Although no specific disease markers exist for AA, its immune pathophysiology has been inferred from the identification of active cytotoxic T-cells in AA patients, and most importantly, its responsiveness to IST(6). Clues to diagnosis include association with other immune diseases such as non-viral hepatitis(7) and eosinophilic fasciitis(8), and the presence of glycosyl-phosphatidylinositol (GPI)-negative paroxysmal nocturnal hemoglobinuria (PNH) clones, which are common in AA, observed in MDS, and rare in constitutional BMF(9).

Pure red cell aplasia (PRCA) is an immune mediated anemia characterized by absolute reticulocytopenia and absence of maturing erythroid precursors on marrow. When PRCA is diagnosed, secondary causes must be ruled out including autoimmune disorders, classically rheumatoid arthritis, lymphoproliferative disorders such as chronic lymphocytic leukemia or t-cell large granulocytic leukemia (T-LGL), solid malignancy (classically thymoma), and infection such as parvovirus B19(10). T-LGL can mimic BMF by causing pancytopenia, but neutropenia is most common. T-LGL is diagnosed by the identification of LGLs on peripheral blood smear, a clonal population of activated T cells on immunophenotyping, and evidence of clonality by T-cell gene receptor rearrangement(11, 12).

Fanconi Anemia

Fanconi anemia (FA) is due to defects in DNA repair and characterized by the presence of congenital malformations, BMF, and a predisposition to development of both leukemia and solid malignancies. Typically, it is autosomal recessive (AR) and related to mutations in now 22 known FANC genes (FANCB is X-linked recessive and FANCR is autosomal dominant [AD]). Physical abnormalities are present in most and may be subtle, but their absence does not exclude FA. Short stature, café au lait spots, and characteristic limb abnormalities such as short or absent radii and abnormal thumbs are most common(13). Additionally, microcephaly, microphthalmia, and renal anatomical deformities(14) may be seen. BMF is common and occurs at a lifetime cumulative incidence of 50%. Patients with congenital abnormalities are at higher risk of early onset marrow failure, while patients with a normal physical phenotype are more likely to develop malignancy at an older age(15). Squamous cell carcinoma (SCC), particularly of the skin, head and neck, and vulva, occurs at high rates in FA; this type of cancer presenting at a young age warrants consideration of constitutional BMF, particularly FA(16).

Telomere Biology Disorders

Telomere biology disorders (TBD) are a spectrum of diseases due to mutations in telomere maintenance genes, causing an increased rate of attrition and short telomeres. Inheritance can be AD, AR, or XR depending on the specific gene involved, with AR disease tending to have a more severe phenotype. Specific clinical syndromes characterize the TBD spectrum. Dyskeratosis congenita (DC), the classic TBD, occurs in childhood and is characterized by the triad of leukoplakia, skin abnormalities, and nail dystrophy. Other TBD syndromes include Hoyeraal-Hreidarsson (HH), characterized by growth retardation, cerebellar hypoplasia, ataxia and microcephaly, Revesz syndrome (RS) defined as HH + exudative retinopathy, and Coats plus (CP), characterized by retinal vasculopathy, cerebral calcification and leukodystrophy. HH, RS, and CP are most commonly AR presenting in early childhood. A subset of patients, most typically with TERC/TERT mutations, present later in life with subtler findings, typically progressive BMF and co-existing lung or liver fibrosis.

Bone marrow failure is common in TBD and may present initially as unilineage cytopenia (particularly thrombocytopenia) with later progression. In patients with DC, risk of BMF was found to be 94% by age 40(17), along with increased cancer risk, particularly SCC of the head and neck and myeloid malignancies(16). Early hair greying is a distinctive feature of TBD and should be interrogated for as part of the clinical history of both patient and family.

Lung involvement (classically pulmonary fibrosis [PF]) is common and may occur in the absence of BMF. Mutations associated with TBD have been identified as the underlying etiology for 20–25% of familial PF cases(18, 19). Hepatic involvement occurs in 40% of patients ranging from liver enzyme abnormalities to cirrhosis(20). The co-occurrence of BMF, PF, or unexplained liver disease is highly suggestive and should prompt investigation for TBD. Other clinical manifestations include vascular disorders such as pulmonary arteriovenous malformations(21) and gastrointestinal telangiectasia(22), as well as immunodeficiency(23).

Diamond-Blackfan Anemia

DBA is a rare congenital anemia caused by multiple mutations in ribosomal protein (RP) genes as well as TSR2 which plays a role in ribosome pathogenesis. Genetic inheritance is heterogenous but most commonly AD or sporadic. Diagnosis usually is made early (95% at ≤ 2 years of age)(24). Disease characterization includes macrocytic anemia, reticulocytopenia and absent or markedly reduced red cell precursors in the bone marrow. Due to young age at presentation, failure to thrive, pallor, and difficulty feeding are usual initial clinical manifestations. Patients can have intrauterine growth retardation and continue to have short stature in later life(25). Limb abnormalities can be present, particularly the characteristic triphalangeal thumb, as well as congenital cardiac defects, most commonly ventricular and atrial septal defects, and cephalic malformations, including microcephaly, and characteristic facies(24, 26, 27). DBA patients have increased cancer risk, in particular MDS/AML, colon cancer, and osteogenic sarcoma(28).

Less classical types of DBA have been recently described. They present in late childhood or adulthood, with a less severe phenotype such as mild anemia, or isolated macrocytosis(29). Rare “DBA-like” disorders are due to mutations in non-RP genes (such as GATA1, EPO, and ADA2). Patients with mutations in EPO have normocytic anemic and lack the congenital malformations seen in classic DBA, whereas in GATA1 mutations, patients may have either congenital dyserythropoiesis or a more classic DBA phenotype(30, 31). Mutations in ADA2 result in deficiency of adenosine deaminase 2 (DADA2), a syndrome of vasculitis with recurrent strokes, autoinflammation and immunodeficiency(32).

Shwachman Diamond Syndrome

Shwachman Diamond Syndrome (SDS) is also a disorder of ribosomal biogenesis almost always due to biallelic mutations in the SBDS gene. SDS classically presents with both BMF and exocrine pancreatic dysfunction. Neutropenia is the first and commonest cytopenia, though normocytic anemia is present in up to 80%, and patients may progress to pancytopenia and severe marrow failure(33). First presentation is typically at <1 year of age and SDS is clinically suspected in children with cytopenias, recurrent infections, and evidence of pancreatic dysfunction, such as failure to thrive, poor feeding(34), and steatorrhea. However, less typical presentations can occur later in life, as pancreatic dysfunction may improve with age and only be apparent on testing. Skeletal abnormalities include short stature, variable metaphyseal widening, delayed development of secondary ossification centers, thoracic abnormalities and osteoporosis. Hepatomegaly and raised liver enzymes that often improve with age, intellectual disability, congenital heart defects, and endocrinopathy may also be present(35–39).

SAMD9/SAMD9L

SAMD9/SAMD9L disorders are AD and due to gain of function mutations in sterile alpha motif domain containing 9 (SAMD9/SAMD9L) genes located on the long arm of chromosome 7; penetrance is variable and de novo mutations are common(40, 41). There are different clinical phenotypes within the spectrum of SAMD9/SAMD9L disorders, with a unifying feature of cytopenias, risk of marrow failure, and predisposition to early onset MDS with associated monosomy 7 or 7q deletion. SAMD9/SAMD9L patients are classified either syndromic or non-syndromic, with non-syndromic patients presenting predominantly with hematologic disease.

Specific syndromes are associated with the particular gene mutated. SAMD9 causes “MIRAGE” syndrome; myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital problems, and enteropathy, while SAMD9L causes Ataxia Pancytopenia Syndrome; neurologic (predominantly cerebellar) symptoms and pancytopenia,(42–44) and SAMD9L-associated autoinflammatory disease (SAMD9L-SAAD); nodular neutrophilic panniculitis, interstitial lung disease, basal ganglia calcifications, cytopenia(45). SAMD9/SAMD9L disorders have two unique genetic adaptive features that may help identify it. Transient monosomy 7 is seen in affected children, and somatic reversion by second-site mutation or copy neutral loss of heterozygosity occurs, neutralizing the gain of function mutation and resulting in long term normalization of once low blood counts(46).

GATA2 deficiency syndrome

GATA2 deficiency is a recently identified AD disorder with marrow failure, and a predisposition to myeloid malignancy. Important associated features include immunodeficiency, lymphedema, and pulmonary alveolar proteinosis (PAP). Patients often have normal blood counts at birth developing cytopenias later in life, or may first present to medical attention with MDS or leukemia. Profound monocytopenia, and low B and NK cells are typical of GATA2 deficiency(47). Unusual infections include non-tuberculous mycobacterial and recurrent warts due to HPV. GATA2 is expressed in lymphatic vessels and the vasculature, and its deficiency leads to lymphedema and increased thrombotic risk(48). PAP, due to overaccumulation of alveolar surfactant, presents as progressive dyspnea and cough, an isolated DLCO or restrictive pattern on pulmonary function tests, and “crazy paving” on CT chest. The combination of BMF or MDS with atypical infections, lymphedema, or PAP strongly suggests GATA2 deficiency(49, 50).

Inherited platelet disorders (CAMT, TAR, RUNX1, ETV6, ANKRD26, MECOM)

Congenital amegakaryocytic thrombocytopenia (CAMT) is due to AR or compound heterozygous mutations in the c-MPL. CAMT clinically presents at birth with severe thrombocytopenia, without syndromic features, and absent or markedly reduced megakaryocytes on marrow. Some neurological abnormalities can occur with CAMT but may relate to intracranial bleeding. Patients typically progress to pancytopenia with aplasia by 1 year of age(51).

Thrombocytopenia with absent radii (TAR) is due to biallelic mutations in RBM8A and characterized by thrombocytopenia and bilaterally absent radii with thumbs present, and other skeletal defects. In contrast to CAMT, thrombocytopenia typically improves with age. Cow’s milk intolerance, renal tract abnormalities, cardiac defects and other congenital malformations are associations(51, 52).

Three similar rare familial platelet AD disorders, RUNX1, ETV6 and ANKRD26, feature mild-to-moderate thrombocytopenia, increased platelet size, and predisposition to hematologic malignancy(53). RUNX1 familial platelet disorder with predisposition to myeloid leukemia (FPDMM) is mainly associated with increased risk of MDS/AML but also T-cell acute lymphoblastic leukemia (ALL)(54). Platelet function defects are present in most patients, resulting in a moderate-to-severe bleeding disorder. Patients with germline ETV6 mutations more frequently develop ALL than myeloid malignancies(55). ANKRD26 more frequently leads to myeloid malignancy and bleeding phenotype is milder without platelet function defect(56). MECOM (MDS1 and EVI1 complex locus) mutations have been associated with a rare recently discovered syndrome characterized by congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Progressive BMF has been seen in MECOM, along with decreased B-cells, cardiac and renal defects, and congenital deafness(57).

Specialized testing in constitutional BMF

The use of specialized testing is guided by the clinical suspicion for a particular constitutional BMF syndrome. In our BMF clinic, we routinely perform DEB testing in children and young adults, and telomere length (TL) testing in all patients, as FA and TBD are the commonest constitutional BMF, and these functional assays will ultimately aid in interpretation of germline genetic results. Missed diagnosis of FA or TBD has profound implications for HSCT conditioning. Avoidance of alkylating agents and irradiation in FA and use of non-myeloablative regimens in TBD have led to improved long-term outcomes. Chromosome instability is a hallmark of FA cells. Induction of chromosomal breakage using either DNA crosslinking agents such as diepoxybutane (DEB) or mitomycin C (MMC) is the best functional evidence of FA. Testing is typically performed on peripheral blood and reported as either positive or negative, determined by the amount of induced chromosome breakage. However, FA has a relatively high rate of revertant mosaicism in blood cells (15–25%), meaning the proportion of FA affected hematopoietic cells may be low enough to produce to a false negative result(58). If FA remains a strong clinical consideration, testing should be performed on fibroblasts, best obtained from skin biopsy, and genetic germline testing should be obtained.

Telomere length measurement is performed using flow-FISH and reported as a percentile for age. Both granulocytes and lymphocytes are assessed; short telomeres in granulocytes may occur in any type of BMF due to marrow stress, but short telomeres in lymphocytes is typical of a germline defect in a telomere gene. TL <1^st percentile in lymphocytes is highly suggestive telomere disease, but TBD patients can also have lengths in <10^th; percentile; in these cases the clinical context is important.

Disease specific testing exists for both DBA and SDS, though in clinical practice this may be superseded by germline mutation screening. Erythroid adenosine deaminase (eADA) activity can aid in diagnosing DBA; high levels have a high positive and negative predictive value of 91% for disease(59, 60), but may be falsely low after blood transfusion. eADA can be used as a familial screen to identify mild or silent RP gene carriers. Demonstration of pancreatic dysfunction by reduced pancreatic enzyme levels (trypsinogen if <3 years and isoamylase >3 years), or low levels of fecal elastase remains part of SDS diagnostic criteria. Other supportive testing includes abnormal 72-hour fecal fat analysis, reduced levels of fat-soluble vitamins (A, D, E, K), or evidence of pancreatic lipomatosis by either imaging or histology.

Assessment of the immune system by serum immunoglobulin and levels and assessment of peripheral blood T and B cell lymphocyte subsets may uncover a primary immunodeficiency syndrome with associated features of BMF. If suspected, referral to immunology is warranted.

Hematopathology

Careful examination of the bone marrow aspirate and biopsy is required to differentiate aplastic anemia from hMDS. In both, marrow is paucicellular. In hMDS cellularity is reduced, in contrast to typical MDS where cellularity is normal or increased. Dyserythropoiesis is common in AA and cannot be used to distinguish AA from MDS, but myeloid and megakaryocytic dysplasia are not seen in AA. Peripheral blood or marrow blast counts are not increased in AA, and the presence of increased reticulin marrow fibrosis is suggestive of a myeloid neoplasm.

An abnormal karyotype indicates a diagnosis of MDS but is present in only about 50% of MDS cases. Certain cytogenetic abnormalities such as 13q deletion and trisomy 8 are regarded by some experts as consistent with AA (in the absence of morphologic dysplasia) as patients demonstrated transience of the aberrant chromosome and responsiveness to IST(61, 62), but 13q deletion is also diagnostic of MDS by WHO criteria. Monosomy 7 is the commonest chromosomal aberration associated with evolution to myeloid neoplasms in both immune AA and most constitutional BMF. Transient monosomy 7 is classically associated with SAMD9/9L. Certain chromosomal abnormalities have specific associations: +1q in FA, Iso7q in SDS, and monosomy 7 or der(1;7) in GATA2.

Patients with RUNX1 and ANKRD26 germline mutations have atypical megakaryocytes on baseline bone marrow examination characterized by small hypolobated megakaryocytes with eccentric nuclei or megakaryocytes with separated nuclear lobes. In patients with isolated thrombocytopenia, megakaryocytic atypia should not be used as a sole criterion for MDS. Evolution to MDS in these familial platelet disorders is associated with the development of additional cytopenias, multilineage dysplasia, cytogenetic abnormalities, somatic mutations, ringed sideroblasts or increased blasts. Flow cytometry analysis can be diagnostically helpful in discriminating different etiologies of marrow failure(4). A subset of GATA2 patients present with pancytopenia and markedly hypocellular marrows, overlapping morphologically with aplastic anemia. Flow cytometry can reveal an underlying immunodeficiency in GATA2 deficiency evident by disproportionate loss of bone marrow B-cells, dendritic cells, NK-cells, and/or monocytes. In contrast, in AA lymphoid populations are usually well represented(50). Identification of PNH clones by peripheral blood flow cytometry is helpful in discriminating immune AA vs. constitutional BMF. Immunophenotypic assessment of myeloid and erythroid lineages can reveal abnormal patterns of maturation typical of MDS, such as expression of CD7 or loss of CD38 on myeloblasts. T-cell clonality assessed by V-beta receptor family flow cytometry analysis can identify clonal LGL.

Somatic mutations of genes seen in myeloid cancers are commonly seen in both AA and MDS though with differing clonal landscapes. They have also been found in constitutional BMF, in the absence of MDS/AML, though their frequency and significance are not as well defined. In AA they are typically at low variant allele frequency (VAF) in BCOR/BCORL1, DNMT3A and ASXL1. Spliceosome, RAS, TET2, and RUNX1 mutations are uncommon in AA and their presence, particularly at high VAF, and with another somatic mutation, points to MDS(63, 64). Somatic mutation testing can be useful in diagnostically difficult BMF cases but is not necessary in clear cases of immune-mediated disease.

Germline genetic testing

Germline genetic sequencing is the ideal test to detect constitutional BMF. However, it is not currently feasible to perform in all centers due to resource constraints and high cost. In such circumstances, it is reasonable to restrict testing to patients in whom a suspicion for constitutional BMF exists (Figure 2). Using machine learning models, immune AA can be predicted at >90% specificity using a combination of common clinical variables: age, sex, blood count severity, family history and telomere length (unpublished data). Immune AA can be reliably discerned by physicians in most cases using routine clinical data according to this model. Germline targeted sequencing can take 4–6 weeks, and treatment of adult SAA patients with IST need not be delayed while awaiting results unless there is a clinical suspicion for constitutional BMF. Decision to treat prior to genetic testing results will be guided by the clinical context, in particular severity of neutropenia, presence or absence of infection, and potential for upfront HSCT.

Figure 2:

An algorithm for further evaluation of BMF patients as practiced in our BMF clinic as a linear approach. Patients who do not have clinically suspicious features and who have normal telomere length are unlikely to have constitutional BMF (specificity >90% using machine learning model). Treatment for patients with likely immune SAA need not be delayed while awaiting results from specialist testing or genetic sequencing. Abbreviations: TBD; telomere biology disorder, AA; aplastic anemia, WES; whole exome sequencing, WGS; whole genome sequencing, NGS; next generation sequencing, TL; telomere length, IST; immunosuppressive therapy.

Fibroblasts cultured from a skin biopsy are the best source of DNA for germline testing. Peripheral blood testing may result in false positive (i.e. detection of somatic variants in hematopoietic cells within the VAF range for germline [40–60%]) or false negative (i.e. somatic reversion seen in FA or SAMD9/SAMD9L) results. However, for practical reasons most hematologists first perform blood testing and then confirm with fibroblasts, depending on the initial results and clinical suspicion. A mutation found in multiple family members supports a germline predisposition.

Interpretation of germline genetic results can be complicated. Variants are reported according to criteria from the American College of Medical Genetics and Genomics (ACMG) and can be classified as benign, likely pathogenic, pathogenic or variants of unknown significance (VUS), using criteria that incorporates incidence of the variant in population databases, functional and computational studies, and evidence of familial segregation or de novo occurrence. A reported VUS may be clinically significant for a particular patient, and the status of a VUS can be updated and reclassified over time as more information becomes available. Interpretation depends on the patient’s clinical characteristics, and family pedigree, in addition to the criteria used by ACMG (Figure 3)(65). If a VUS is detected in the setting of clinically suspected constitutional BMF syndrome, advice should be sought from someone with disease-specific expertise. Identification of potential germline mutations is particularly critical for marrow failure patients undergoing HSCT, if related donors are under consideration. Donor derived MDS/AML has been reported in a number of patients with unrecognized germline mutations (e.g. TERT, RUNX1, GATA2) transplanted using healthy matched related donors who harbored the same mutation (66, 67). If a VUS is ultimately determined to be possibly disease causing, reporting it to a genomic database can aid in future classification of the variant’s pathogenicity.

Figure 3:

Considerations for interpretation of a germline variant of uncertain significance (VUS).

Some patients in whom constitutional marrow failure is clinically suspected have negative targeted sequencing for genes currently known to cause BMF. In such cases, if strong suspicion exists for BMF, whole exome or whole genome sequencing of both the patient and first-degree relatives can be performed to assess for novel variants.

Conclusion / Summary:

Distinguishing acquired from constitutional BMF is challenging but important given the clinical implications. Although heterogenous in presentation, specific syndromes can be suspected based on history and physical findings. Diagnostic clues that suggest constitutional BMF include young age, family history, chronic onset, as well as the presence of multi-organ involvement. Telomere length and DEB are valuable to exclude FA and TBD. Genetic testing is the ideal way to assess for constitutional BMF and can provide definitive answers when a pathogenic variant is present, however, interpretation of VUS can be tricky and may require specialist input.

Practice Points:

• Distinguishing acquired from constitutional bone marrow failure is critical as it affects choice of therapy. Missed diagnosis of constitutional marrow failure has a profound impact on hematopoietic stem cell transplantation, both in the selection of donors and the chosen conditioning regimen.

• Diagnostic clues pointing towards constitutional marrow failure include young age, multi-organ disease, chronic onset of cytopenia, and positive family history.

• Genetic germline targeted sequencing is ideally performed in all cases of AA, but this may not be possible due to resource constraints. In such circumstances, testing adult SAA patients without clinical features of constitutional BMF and normal telomere length can be considered low priority.

• Treatment with immunosuppression need not be delayed while awaiting genetic results unless a reasonable clinical suspicion for constitutional BMF exists.