Annals of Hematology (2020) 99:913–924 https://doi.org/10.1007/s00277-020-03954-2 REVIEW ARTICLE Mosaicism in Fanconi anemia: concise review and evaluation of published cases with focus on clinical course of blood count normalization Eileen Nicoletti 1 & Gayatri Rao 1 & Juan A. Bueren 2,3,4 & Paula Río 2,3,4 & Susana Navarro 2,3,4 & Jordi Surrallés 3,5,6 & Grace Choi 1 & Jonathan D. Schwartz 1 Received: 25 July 2019 / Accepted: 6 February 2020 / Published online: 17 February 2020 # The Author(s) 2020 Abstract Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1–6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investi- gational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs. Keywords Fanconi anemia . Bone marrow failure . Autologous stem cell transplantation . Gene therapy . Mosaicism Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00277-020-03954-2) contains supplementary material, which is available to authorized users. * Eileen Nicoletti 4 Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz en@rocketpharma.com (IIS-FJD), Madrid, Spain 5 Genome Instability and DNA Repair Syndromes Group, Department 1 Rocket Pharmaceuticals, Inc., New York, NY, USA of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain 2 Hematopoietic Innovative Therapies Division, Centro de 6 Investigaciones Energéticas, Medioambientales y Tecnológicas Servicio de Genética e Instituto de Investigaciones Biomédicas del (CIEMAT), Madrid, Spain Hospital de Sant Pau, Barcelona, Spain 3 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain 914 Ann Hematol (2020) 99:913–924 Fanconi anemia and mosaicism most likely emanating from reversion or other compensatory mutations in long-term repopulating HSC populations. Fanconi anemia overview Mosaicism in FA patients was noted prior to initial identi- fication of the first FANC gene (FANCC) in 1992 [11], with Fanconi anemia (FA) is a deoxyribonucleic acid (DNA) repair initial reports describing FA patients in whom 60–80% of disorder resulting from mutations in genes encoding for cultured lymphocytes (either PHA-stimulated T cells or protein components of the FA/BRCA DNA repair pathway. EBV-transformed B cells) displayed resistance to More than 20 identified proteins interact as components of the concentrations of alkylating agents typically toxic to FA FA core, FANCI, FANCD2 and downstream complexes. In patient cells [12, 13]. Several mechanisms by which an conjunction with other DNA repair elements, FA proteins additional genetic event may result in a functional FA gene facilitate repair of interstrand crosslinks (ICLs) and other and protein have been identified and include gene conversion, forms of DNA damage [1, 2]. FA proteins also contribute to intragenic crossover, back mutation, and second-site mutation other cellular processes beyond DNA repair, including – each of which may result in a restoration of the affected gene cellular responses to oxidative stress and inflammatory to wildtype [8, 14, 15]. Second-site mutations involve either a stimuli [3–5]. compensatory point mutation (insertion or deletion) at a dis- FA is characterized by frequent bone marrow failure tinct site within a mutated gene, resulting in a functional gene (BMF), myeloid malignancies, and epithelial cancers [1, 6]. and protein that may be nonetheless distinct from wildtype The BMF and neoplastic manifestations of FA are frequently [15–17]. The mechanisms by which these various mutations but not universally accompanied by congenital abnormalities may result in cell populations expressing a functional gene including growth retardation/short stature, microcephaly, and and/or protein are illustrated in Fig. 1. abnormalities of skin pigmentation, upper limbs (especially thumbs and radii), gonads, kidneys, and other organ systems. Manifestations of BMF are frequently noted during the first Mosaicism in other genetic disorders decade of life; the incidences of myeloid and solid organ malignancies increase during the second and third decades of Reversion mutations and resultant mosaicism have been life, respectively, such that the cumulative incidence of acute reported in Bloom syndrome, X-linked and adenosine myeloid leukemia (AML)/myelodysplastic syndrome (MDS) deaminase severe combined immunodeficiency (X-SCID and is approximately 30% by age 30 and the cumulative incidence ADA-SCID), Diamond Blackfan anemia (DBA), Wiskott- of solid organ cancers approaches 40% by age 40 [6, 7]. Aldrich syndrome, epidermolysis bullosa, tyrosinemia, and dyskeratosis congenita (DC) [18–25]. The extent of phenotypic correction in any clinical setting is dependent on Fanconi anemia mosaicism both the chronology of a reversion event either during embryonic or postnatal development and the degree by which Somatic mosaicism in FA (hereafter referred to as mosaicism) a disorder manifests in hierarchical (as opposed to heterarchical) arises from reversion or other compensatory mutations in hema- organ systems. Disorders predominantly affecting blood cells topoietic stem cell/progenitor cells (HSPCs) from which arise a may be particularly reversible because of the hierarchical population of bone marrow and blood cells with a functional nature of hematopoiesis, with partial or comprehensive DNA repair capacity. Patients in whom a reversion in a phenotypic corrections identified most frequently in the pathogenic FA mutation has occurred frequently have two context of normalization within a single lineage [19–21]. distinct blood cell populations. One population is sensitive to Multilineage mosaicism has been reported in DC [25]. In DNA-damaging agents (mitomycin-C [MMC] or several of these conditions and in FA in particular, the diepoxybutane [DEB]) and consistent with an FA diagnosis and underlying disorder predisposes to heightened genetic another population resistant to these DNA-damaging (or instability, enhancing the potential for additional mutations, clastogenic) agents – hence the “mosaic” label indicating the many of which are potentially deleterious (resulting in coexistence of phenotypically distinct cellular components characteristic frequent malignancy in Bloom syndrome, DBA, [8–10]. The purpose of this review is to summarize the existing DC, and FA), but in more limited instances may confer a literature regarding FA mosaicism, with particular emphasis on beneficial phenotypic reversion. A particularly unstable persistent diagnostic and prognostic uncertainties. We provide an intragenic duplication was recently characterized in a FANCB evaluation of case series in which FA mosaic patients were patient with mosaicism associated with mild hematologic followed clinically over years (and at times decades), with deficits. The instability of this duplication was evident not emphasis on the clinicopathologic relevance of laboratory only from a decreasing percentage of aberrant blood cells over diagnostic evaluations of these cases. Additionally, we 11 years of observation but from the presence of a modest conducted a literature-based assessment of FA mosaic cases population (8%) of fibroblasts displaying a wild-type allele, Ann Hematol (2020) 99:913–924 915 Fig. 1 Several mechanisms by which additional chromosomal mutations within a single chromosome (and gene). Gene conversion, rearrangements or mutations may facilitate correction of an inherited intragenic crossover, and back mutations result in genetic correction in recessive genetic disorder. The yellow and blue bars indicate an one allele within a daughter cell (indicated by the green border and individual gene, with black hashes indicating a disease-causing background). Second-site mutations (white hash) result in a gene mutation. Reversion mutations may arise either during or subsequent to capable of generating a functional protein (indicated by the green DNA replication and may involve transfer of genetic material between dashed border and green background) in one allele, although the gene paired chromosomes (gene conversion or intragenic crossover) or itself may differ from wildtype. Adapted from Pasmooij et al. [17] representing the first identification of reversion in a non- of cultured fibroblasts. Analysis of bone marrow progenitors hematopoietic cell population in FA [26]. indicated multilineage presence of the compensatory mutation, consistent with a hypothesized prenatal reversion FA mosaicism: selective growth advantage in a long-term HSC, resulting in multilineage engraftment and prognostic uncertainty via shared in utero circulation [28, 29]. This example of gene reversion and selective growth advantage enabling compre- In the abovementioned diseases, mosaicism has been de- hensive and sustained hematopoiesis has represented a natu- scribed as “natural gene therapy” [27, 28], and a selective rally occurring proof of concept for the potential hematologic growth advantage of revertant cells in selected disorders and correction of FA via gene therapy. A selective advantage of organ systems (including the hematologic component of FA) gene-corrected FA cells has been demonstrated in murine FA has long been hypothesized. The potential for a marked models [30–32], human embryonic stem cells [33], induced proliferative advantage at the HSC level has been most pluripotent stem cells [34], and gene-corrected cells from FA profoundly illustrated by a case of monozygotic twin sisters patients transplanted in immunocompromised mice [35]; diagnosed with FA early in life who nonetheless had these observed in vivo selective advantages have been largely demonstrated hematologic stability during 28 years of facilitated by administration of cytotoxic conditioning agents. reported follow-up, with clastogen resistance evident in A selective advantage has also recently been demonstrated in peripheral blood lymphocytes despite characteristic fragility nonconditioned FA-A patients receiving gene-corrected 916 Ann Hematol (2020) 99:913–924 autologous CD34+ cells, by means of progressive increases in additional information regarding the number of breaks per cell gene-corrected and clastogen-resistant lymphocytes and mar- further contributing to diagnosis [10]. Specific DNA- row progenitors during 18–30 months of follow-up [36]. damaging agents, concentrations, other culture and cell selec- The presence of a revertant FA hematopoietic cell popula- tion conditions, and threshold aberrancy levels have varied tion has often been associated with a favorable hematologic among investigative centers such that no single standard for course in FA patients. However, the presence of clastogen- a determination of mosaicism exists. The presence of diver- resistant blood and marrow cells may not uniformly be asso- gent DEB resistance between a patient’s lymphocytes and ciated with long-term BMF- or AML-free survival. Gregory cultured fibroblasts is considered consistent with FA mosai- and colleagues reported a male patient with hematologic sta- cism, as is an increasing percentage of DEB-resistant lympho- bility during the initial 9–17 years of life and a stable popula- cytes over sequential intervals. A German-led international tion of DEB-resistant peripheral blood lymphocytes (61– consortium of investigative centers has also identified mosai- 74%). Analysis of bone marrow progenitors at age 15 indicat- cism by decreased clastogen stimulation of G2-phase arrest ed multilineage presence of the revertant mutation (including [15]; French investigators have described normalized lymphoid, myeloid, and erythroid progenitors). At ages 15 FANCD2 ubiquitination in the presence of clastogens as in- and 17, this patient nonetheless developed evidence of an dicative of mosaicism [38]. As will be discussed subsequently, 11q deletion in a population of non-revertant bone marrow results of DEB-chromosomal stability evaluations in periph- cells – a cytogenetic abnormality potentially associated with eral blood lymphocytes may not necessarily be indicative of myeloid malignancy [37]. The prognostic uncertainty regard- reversions in other hematopoietic compartments, including ing the presence of a hematopoietic population containing a multipotent HSPCs [14]. reversion mutation is rendered additionally complex because Gene sequencing has become an increasingly valuable and the diagnostic methods utilized in FA are most frequently utilized modality for the confirmation of an FA diagnosis; directed at a differentiated T-lymphoid cell population that however, sequencing of distinct blood and marrow progenitor may be genetically divergent from multilineage stem and pro- cell populations has been performed infrequently and con- genitor populations. The diagnosis and clinical prognosis of tinues to be a predominantly research-focused modality [28, FA mosaic patients during recent decades are detailed 37]. Evaluation of the MMC resistance of cultured bone subsequently. marrow-derived colony forming cells (CFCs) has emerged as a complementary test for identifying phenotypic reversion Mosaicism diagnosis in progenitor populations. The number of investigations in which this parameter has been correlated with long-term clin- Diagnosis of mosaicism in FA is contingent on demonstration ical outcomes is extremely limited [28, 37, 39]; however, the of a population of blood and/or bone marrow cells that display increasing percentage of MMC-resistant bone marrow CFCs resistance to concentrations of DNA-damaging agents that are has been identified in recent autologous gene therapy clinical typically toxic to FA hematopoietic cells. Diagnostic challenges trials and has been recognized as a relevant indicator of genet- persist because mosaicism may be restricted to specific cell ic and phenotypic reversal in pluripotent hematopoietic pro- lineages and evaluation of multiple blood and marrow lineages genitor cells [36]. Ongoing discussions involving internation- is frequently not clinically feasible. The percentage of resistant al multidisciplinary investigators have increasingly identified cells within a single lineage may not be indicative of the overall MMC resistance of bone marrow progenitors as meaningful hematologic milieu and (especially at a single timepoint) may and potentially objective clinical phenomena in either FA mo- not provide clinically meaningful prognostic information. saicism or autologous hematopoietic gene therapy settings A critical component of an FA diagnosis and of potential [40, 41]. Ongoing consensus discussions have also indicated FA mosaicism involves evaluation of cultured T lymphocytes the potential importance of evaluations employing multiple in the presence of DNA-damaging agents, most frequently MMC concentrations, serial evaluations of MMC resistance DEB or MMC. Lymphocytes from FA patients frequently over time, and corroboration of clastogen resistance with func- display marked chromosomal instability (also referred to as tional assays such as FANCD2 ubiquitination. When feasible, fragility, evidenced by chromosomal breaks and radial fig- genetic sequencing and multilineage assessment are addition- ures) in the presence of clastogen concentrations that result ally relevant for comprehensive mosaicism assessment. in minimal abnormalities in non-FA cells [9, 10]. Some cen- ters, including those involved in the evaluation of a large and Clinical outcomes in FA mosaic patients rigorously followed Spanish FA mosaic population, have pro- posed diagnostic algorithms classifying individuals with few- In addition to the two illustrative and deeply investigated FA er than 20% aberrant cells cultured in 0.1-μg/ml DEB as non- mosaic cases described previously, several additional series FA patients, 20–40% as consistent with FA mosaics, > 60% as published over a 16-year period (1997–2013) have detailed consistent with FA, and 40–60% as possible mosaicism, with the clinical course and essential laboratory correlates in FA Table 1 Fanconi Anemia Mosaicism: Publications Detailing Clinical Outcomes of Multi-patient Cohorts Ann Hematol (2020) 99:913–924 Reference Cohort (incl. Compln. Age & Duration Clinical Observations Laboratory Analyses & Clinical Correlates Group) of Follow-up Lo Ten n=8 Age 9-30y Mild hematologic deficits in 6 of 8 patients ▪ MMC-res. in 37-100% of PB lymphocytes Foe FANCC n = 3 n = 6 single ▪ Pt EUFA-192 followed age 3→16y with MMC-res. 0% (age 3y) → 95% 1997 unknown n = 5 eval. (age 16y); clonal hematopoiesis demonstrated by PCR n = 2 f/u ▪ Gene conversion in 2 siblings (EUFA 449/50) with MMC-res 0% & 90% 22→28y (respectively) & divergent hematologic course & 3→16y Gross n=5 Age 6-21y ▪ Hematologic improvement in 4 of 5 patients ▪ Lymphoid-restricted reversion suspected in pt without hematologic 2002 FANCA n = 4 All followed ▪ PB increases in some pts noted over 3-6y period (platelet ↑ most improvement FANCC n = 1 over 3-6y delayed in several cases) ▪ % of MMC-res PB lymphocytes not described (only breaks/cell) interval Soulier n=8 Age 4-34y ▪ No BMF or AML/MDS in 8 mosaic patients (median f/u 5y) ▪ Normal PB lymphocyte FANCD2 ubiquitination pattern (immunoblot): 2005 FANCA n = 5 FANCD2 Median f/u 5y ▪ BMF/aplasia in 31 of 45 non-mosaic FA pts 1° determinant of reversion n = 1 unknown n = 2 (1-27 y) ▪ AML/MDS in 2 of 45 non-mosaic FA pts ▪ Mechlorethamine 0.05μg/ml chromosomal breakage in PB lymphocytes: n = 45 non-mos. FA Age 2-36y ▪ Normalization of Hb/Plt over 1&3 y w/ subsequent stabilization in 1 20-56% (n = 4); negative (<20%; n = 3); ambiguous result in (n = 1) contr. (non-mos.) FANCA pt (27y f/u) Kalb n = 5 FANCD2 Age 9-34y ▪ Mild/protracted hematologic course in 3 pts (1 received androgens) ▪ Limited description of MMC-res of PBL/LCL 2007 (median 20y) although eventual BMF requiring Xfusn at age 17 & 18y in 2 pts ▪ 9-10% MMC-induced G2 phase arrest in in LCLs from n = 3 mosaic pts n = 23 FANCD2 F/u 0-18y ▪ Aggressive hematologic course in 2 pts (BMF age 4, 5) & death prior (8% in non-FA controls) non-mos. contr. (median 4y) to age 10 despite HSCT (n = 1) or androgens (n = 2) ▪ Focus of publication was aggressive clinical course of FANCD2 & hypomorphic nature of many FANCD2 mutations Trujillo n = 34 Detailed heme ▪ For n = 14 with detailed f/u: ▪ n = 34 PB T-lymphocyte chromosomal fragility: 10-58% cells with 2013 FANCA n = 25 f/u for n = 14: n = 6: tri-lineage normalization w/o BMF/AML age 14-26.5y (median aberrancies FANCD2 n = 3 over 3-22.5y 22-25y) ▪ n = 9 w/ evaluation of BM MMC-resistance: FANCE n = 1 (median n = 2: trilineage ↑ trend (age 12, 18y); plts < nl 5 of 5 pts w/MMC-res alive at last f/u unknown n = 5 9.5-10y) n = 3: bi-lineage normalization (↓plt n = 2) 2 of 4 pts w/ MMC-sensitivity died at ages 11, 18y Age 0-26.5y n = 3: death or BMF/HSCT (n = 1: no normalization; n = 2 some ↑/nl w/subsequent ↓↓) ▪ Normalization of decreased lineages over 2-6y, most frequently over 2-3 years 917 918 Ann Hematol (2020) 99:913–924 mosaic patients. These include 4 publications and a published doctoral thesis, describing outcomes in FA mosaic patients An additional investigation based on these and other pub- (range: n = 5–14 per publication) [8, 14, 15, 38, 39]. lished FA mosaic cases is described in a subsequent section of Detailed information regarding these series is provided in this review. We undertook this literature-based investigation Table 1. Essential themes emerging from these cohorts include with an intent of identifying a subset of FA mosaic patients the following: most likely to harbor reversion mutations in long-term repopulating HSCs. 1. FA mosaicism has largely been reported in cases arising from more frequently observed mutations in FA core and Hypomorphic mutations in FA ubiquitination complex genes (predominantly FANCA, FANCC, and FANCD2); there is limited published infor- Relatively little has been reported regarding the incidence or mation on FA mosaicism arising from mutations in down- clinical significance of hypomorphic mutations in FA, in stream or more recently discovered FA genes/proteins which the impacted protein is expressed with reduced quantity [26, 42, 43]. and/or function. Hypomorphic mutations have been described 2. Hematologic stability and improvement have been noted in FANCD2 and were not associated with any reduction in the in a single lineage, or frequently in bi- and trilineage set- typically aggressive hematologic manifestations associated tings, consistent with the hypothesis that reversion muta- with this complementation group; the incidence of mosaicism tions arise across a spectrum of pluripotent or more com- in FANCD2 also appears comparable to that in FANCA or mitted HSPCs [39]. other core complex FA groups [15]. 3. A broad range of peripheral blood lymphocyte resistance Recently, a cohort of 11 Sicilian patients with FANCA to DEB (or other clastogens) has been reported, with a hypomorphic mutations were reported, in which mutations limited number of studies showing correlation between resulted in FANCA proteins that localized to cytoplasm and the percentages of resistant PB cells and clinical out- facilitated more normalized mitochondrial function (relative comes. (Of note, T lymphocyte mosaicism has been re- to cells lacking FANCA), with peripheral blood-derived lym- ported in up to 15–20% of FA patients and had been phoblast sensitivity comparable to non-mosaic FA patients. frequently associated with HSCT engraftment failure pri- The clinical course of this cohort ranged from indolent (n = or to the incorporation of fludarabine-based conditioning) 3) to severe (n = 2), with the majority of patients (n = 6) en- [44]. countering moderate single-, bi-, or trilineage cytopenias and 4. FA mosaicism has been frequently but not universally receiving allogeneic hematopoietic transplant at ages 9–21 associated with stable or increasing hematologic parame- [45]. It is likely that as more comprehensive genetic sequenc- ters over prolonged follow-up. ing becomes clinically feasible, additional hypomorphic mu- 5. In the limited settings where normalization of blood tations may be recognized, including some with potential for counts has been observed over time, increases in blood more indolent clinical progression resemblant of mosaicism, lineages have been reported over a 1- to 6-year interval but without the clastogen resistance associated with compen- (most frequently over 2–3 years), and multilineage in- satory mutations in FA. Additional investigations into the mi- creases have been observed in non-contemporaneous tochondrial and other “non-canonical” functions of FA pro- (i.e., staggered) sequences as indicated in Table 1 [14, teins may also elucidate potential prognoses associated with 38, 39]. these uncommon variants [46]. 6. In the limited settings where normalization of blood counts has been observed over time, platelet normaliza- tion has at times been the most indolent and incomplete Review of FA mosaic publications relative to other lineages, with some increases resulting in with emphasis on blood count normalization levels below normal limits but not requiring clinical inter- vention, as indicated in Table 1 [14, 39]. Overview and objectives 7. Evaluation of MMC resistance in bone marrow CFCs has been undertaken infrequently. In the one series in which The clinical significance of FA mosaicism remains uncertain BM MMC resistance was evaluated, 5 of 5 patients with more than 20 years after this phenomenon was initially report- documented MMC resistance were alive and without he- ed. We sought a comprehensive literature-based understand- matologic complications at last follow-up (with stable ing of FA mosaicism with emphasis on cases most likely em- clinical course over 9–15 years in 3 patients for whom anating from reversion mutations in long-term repopulating this information was available) and 2 of 4 patients in HSC populations. Focusing on the minority of FA mosaic whom marrow CFCs were sensitive to MMC died at ages cases in which normalization of all peripheral blood lineages 11 and 18 of BMF and unspecified complications [39]. was reported, we identified a population of FA mosaic patients Ann Hematol (2020) 99:913–924 919 in whom there appears to be high likelihood of multi-decade maintenance of these levels over multiple intervals in settings BMF-free and leukemia-free survival. where earlier counts were not available or presence of these Our objectives were to identify FA mosaicism cases report- levels at a single timepoint with prior/subsequent hematologic ed in the medical literature for which there was information stability reported by the investigator or blood counts normal- regarding individual patient clinical outcomes and to further ized or within normal range per investigator without precise identify the subset of FA mosaic cases in which there was values specified. normalization of all peripheral blood lineages (because such cases likely result from reversion mutations in long-term Results repopulating HSC populations) and to determine clinical course in this clinical subpopulation. In particular, we sought Figure 2 details the literature search results in which 51 pub- to quantify the incidences of BMF, hematologic malignancy, lications describing FA mosaicism were identified between and other adverse outcomes in FA mosaic patients, including 1976 and 2018, of which 23 publications (n = 123 patients) those with blood count normalization and those with no or contained clinical information. A subset of 37 patients (30%) incomplete normalization. had normalization of blood counts (including 19 for whom detailed blood count information over multiple timepoints Methods was reported). Fifteen FA mosaic patients were reported in whom no or incomplete normalization (including normaliza- We identified published FA mosaic cases via PubMed tion of 1 or 2 lineages) was described. Blood count informa- searches, reference citations, and a publicly available doctoral tion was not reported in 71 patients. The subgroup of 37 FA thesis on this specific condition. A database was constructed mosaic patients with normalized blood counts were reported from cases in which there was sufficient clinical information from investigators in Spain (n = 13), Germany (n = 10), enabling evaluation of age at FA and mosaic diagnosis, dura- France (n = 8), the USA (n = 5), and Japan (n = 1). tion of follow-up and development of BMF, malignancy, al- Complementation groups of patients in this cohort were logeneic hematopoietic stem cell transplant (HSCT), survival, Group A (n = 24), Group B (n = 1), Group C (n = 1), Group and cause of death. Blood count normalization was defined as E (n = 1), Group T (n = 1), and not reported/known (n = 9). improvement in absolute neutrophil count (ANC) to at least Publications detailing these patients are shown in 1000/mm3, hemoglobin to at least 9 g/dL, and platelets to at Supplemental Table 1. As indicated previously, there is limit- least 50,000/mm 3 after previously lower levels or ed published information on FA mosaicism arising from Fig. 2 In the cohort of FA mosaic patients (n = 37) with normalized blood groups of patients identified in this cohort included: Group A (n = 24), counts and information regarding clinical outcomes, individuals were Group B (n = 1), Group C (n = 1), Group E (n = 1), and Group T (n = 1); included from the following cohorts: Spain (n = 13), Germany (n = 10), in n = 7 patients, complementation group was unknown France (n = 8), the USA (n = 5), and Japan (n = 1). Complementation 920 Ann Hematol (2020) 99:913–924 Fig. 3 Swimmer plot depicting clinical course for 37 FA mosaic patients of 37) were alive without BMF, malignancy, or transplant at last follow- with normalized blood counts, including age at diagnosis (when up. Criteria for normalization are provided in the text and are in general available), last follow-up, and occurrences of BMF, AML/MDS, more stringent (exclusive) than those applied by investigators in Table 1 allogeneic HSCT, and solid organ cancers. The majority of patients (34 mutations in downstream or more recently discovered FA (lung cancer), and 29 (head and neck cancer). BMF was re- genes/proteins. ported in one patient (3%, age 20) and leukemia (AML/MDS) The clinical outcomes for the 37 FA mosaic patients with in one patient (3%, age 19). An additional patient developed normalized blood counts are depicted in Fig. 3. Median an 11q deletion (MLL rearrangement) in a clonal population follow-up within this subgroup was to age 18 (range 4– persistent over 2 years of follow-up but without overt evi- 34 years). Thirty-four patients (92%) were alive at last fol- dence of leukemia. Solid organ malignancies were reported low-up. Death in 3 patients occurred at ages 19 (AML), 29 in 4 patients (11%, ages 24–33) including 2 patients with Table 2 Clinical outcomes for Fanconi anemia mosaic patients A. Mosaic with blood count normalization (n = 37) Status n (%) Age (y) Alive at last follow-up 34 (92%) At last follow-up: Median: 18 Range: 4–34 Deceased at follow-up 3 (9%) At death: 19, 29, 29 BMF 1 (3%) At BMF diagnosis: 20 AML/MDS 1 (3%) At AML/MDS diagnosis: 19 Solid organ malignancy 4 (11%) At cancer diagnosis: Median: 29 Range: 24–33 B. Mosaic with no/incomplete blood count normalization (n = 15) Status n (%) Age (y) Alive at last follow-up 8 (53%) At last follow-up: Median:14 Range: 8–47.5 Deceased at follow-up 7 (47%) At death: Range: 9–39 BMF 12 (80%) At BMF diagnosis: Median: 9.5 Range: 0–47.5 AML/MDS 2 (13%) At AML/MDS diagnosis: 17.5, 47.5 Solid organ malignancy 2 (13%) At cancer diagnosis: 38, 39 Ann Hematol (2020) 99:913–924 921 multiple sequential cancer diagnoses. Additional details re- result of reversions in committed progenitor (lymphoid- garding these outcomes are provided in Table 2. The clinical inclusive) lineage or short-term repopulating progenitor cells, outcomes for the 15 FA mosaic patients in whom no or in- but more detailed genetic evaluation of progenitor lineages complete normalization was reported were more adverse and will be needed to validate this hypothesis. Additional nonge- are provided in Table 2; 8 of these patients (53%) were alive at netic elements (including epigenetic factors, stromal microen- last follow-up with 7 deaths occurring between ages 9 and 39. vironment, telomeric depletion, and aldehyde exposure and BMF was reported in 12 of 15 patients (80%, median age metabolism) may also contribute to hematopoietic stability 9.5 years); 2 of 15 patients (13%) developed AML/MDS, in settings of genetic correction [5, 47, 48]. It is plausible that and 2 (13%) developed solid organ malignancies. reversion events may only result in favorable clinical out- FA mosaic patients with blood count normalization were comes when these take place in long-term repopulating less likely to develop BMF (p < 0.0001, Fisher’s Exact Test, 2- HSCs and additional favorable metabolic or other nongenetic sided) and more likely to remain alive (p = 0.0034) relative to conditions. A case of mosaicism emerging subsequent to ini- FA mosaics without normalization. A lower percentage of tiation of androgen therapy has been reported, with blood mosaic patients with normalization developed hematologic count normalization persisting subsequent to androgen dis- malignancy (3% vs. 14%), but this difference was not statis- continuation (duration undetermined); it is plausible that an- tically significant. The incidences of solid organ malignancies drogens may selectively enhance hematopoiesis in dormant were similar between these subgroups. revertant HSCs and that some cases of prolonged androgen- Based on these findings, FA mosaicism with normalized attributed hematologic benefit were in fact the result of unde- blood counts appears to occur in a minority (30%) of patients tected mosaicism [49]. Prospective translational studies will whose peripheral lymphocyte assessments indicate resistance be required to enable optimal understanding of genotype/ to DNA-damaging agents via standard testing; the potential phenotype correlations in settings of reversion mutations or for reporting bias is such that the actual incidence may be therapeutic genetic correction – these are planned in the con- lower; it is likely that fewer than 5% of all FA patients expe- text of the next generation of gene therapy clinical rience blood count normalization as a result of reversion/ investigations. compensatory mutations. FA mosaicism with normalized Clinical trials of autologous gene therapy in FA have been blood counts is associated with very limited incidences of conducted during the prior decades, with most recent results BMF or hematologic malignancy, limited requirement for al- indicating genetic correction and reversal of sensitivity to logeneic HSCT, and relatively limited mortality during the DNA-damaging agents at levels resembling those seen in FA initial 2–4 decades of life. Solid tumors arise during the 3rd mosaic patients [35, 50, 51]. Our findings also provide ratio- and 4th decades of life in FA mosaic patients regardless of nale for further clinical evaluation of gene therapy because blood count normalization, as is the case with non-mosaic they indicate that there is a likely threshold of genetic and FA patients. Two of four FA patients with normalized blood phenotypic correction above which multi-decade BMF-free counts who developed solid tumors had a second malignancy survival may be likely. These comparisons between FA mo- diagnosed within several years of the initial cancer diagnosis; saic and gene therapy treated patients are limited in that mo- solid tumors were fatal in at least 2 of 4 reported patients. saicism likely results from a single-cell mutation (or mutation within a very finite subset of cells), whereas gene therapy Discussions and summary involves introduction of a likely higher number of gene- corrected HSPCs with oligoclonal hematopoietic repopulation These findings indicate that there is a population of FA mosaic observed to date [36]. The long-term significance of the patients with very limited risk of BMF or hematologic malig- oligoclonal hematopoiesis identified in nonconditioned gene nancy, likely as a result of reversion mutations in primitive therapy recipients (and suspected in FA mosaic patients) is HSPC populations. Limitations for conclusive analysis result uncertain, although there exist similarities to the clonal hema- from the absence of bone marrow-based genetic or topoiesis that has become increasingly recognized as a distinct mitomycin-resistance evaluation in the overwhelming major- phenomenon, particularly in elderly individuals. Clonal hema- ity of FA mosaic cases. Analysis is also limited because se- topoiesis in the elderly occurs frequently and, even in the quential chronologic assessment of peripheral blood or bone absence of mutations identified with hematologic malignancy, marrow clastogen resistance has been performed infrequently. is associated with increased risk of hematologic malignancy It is very likely that FA mosaicism with normalized blood and cardiovascular events, although the absolute risk in indi- counts is the result of mutations in long-term HSPCs, but it vidual patients is relatively limited [52, 53]. Long-term fol- is not known what percentage of HSPC with reversion events low-up studies of the initial gene therapy treated FA patients is required to enable stable or normalized peripheral blood cell are underway, although multi-year observation will likely be counts [28, 37]. Presumably those mosaic patients in whom required to enable definitive conclusions regarding the rami- no or incomplete normalization occurred were diagnosed as a fications of oligoclonal hematopoiesis. 922 Ann Hematol (2020) 99:913–924 In summary, FA mosaicism is a condition in which varied Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, and frequently divergent degrees of resistance to DNA dam- adaptation, distribution and reproduction in any medium or format, as age are present within the hematopoietic system of an individ- long as you give appropriate credit to the original author(s) and the ual patient, or (more rarely) the entire hematopoietic system source, provide a link to the Creative Commons licence, and indicate if displays evidence of normal or near-normal resistance to DNA changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated damage despite clastogen sensitivity in non-hematopoietic tis- otherwise in a credit line to the material. If material is not included in the sues. These findings are at times accompanied by normalized article's Creative Commons licence and your intended use is not or stable peripheral blood counts, with some patients surviv- permitted by statutory regulation or exceeds the permitted use, you will ing into adulthood without bone marrow failure or hematolog- need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. ic malignancy. Some degree of mosaicism is likely present in 15–20% of FA patients, although the incidence of reversion mutations in primitive HSCs resulting in sustained hematolog- ic stability likely occurs in fewer than 5% [9, 10, 15, 38]. 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