DISCUSSION
We report the results of detailed genomics studies on four families in
which pairs of siblings developed myeloid neoplasms and, in particular,
AML in 7 out of 8 cases. According to the current World Health
Organization (WHO) classification of hematological neoplasms with
germline predisposition and the last published recommendations from ELN
for diagnosis and management of AML, we identified germline mutations
both in genes with a well-established role in predisposing to the
development of MDS/AML and genes with no defined role in this context.
Indeed, CEBPA and DDX41 are included in myeloid neoplasms
with germline predisposition without a pre-existing platelet disorder;FANCA is listed in the category of genes mutated in myeloid
neoplasms associated with bone marrow failure syndromes; JAK2 is
included in emerging disorders with germline predisposition; and,
finally, LYST and ERBB4 have not been reported yet in such
classifications. None of the identified mutations is currently annotated
as pathogenic in cancer databases; however, they could all play an
important role in hematological diseases development.
In particular, the variant identified in CEBPA is a truncating
mutation and, importantly, it is located at the 5’-end of the gene
(c.62, p.S21). N-terminal mutations in CEBPA are known to have a
penetrance close to 100% of leukemia development; however, they
correlate with a favorable prognosis. Due to the unavailability of AML
cells, we could not assess if in our patients there was acquisition of a
somatic CEPBA mutation; nonetheless, both siblings are currently
in CR and under clinical monitoring. The frameshift indel identified inDDX41 tumor suppressor gene is currently annotated as VUS and it
is not listed either in COSMIC or cBioportal. This mutation is expected
to truncate the protein before its functional helicase domain and likely
causes a loss of function. Truncating mutations in DDX41 have
been shown to increase the risk of developing myeloid neoplasms and are
associated with faster progression to leukaemia. Both our patients are
males who, in presence of DDX41 mutations, are expected to
develop myeloid malignancies more frequently than females. Moreover,
patient ID3 harbored the hotspot mutation p.R525H in DDX41 ,
frequently acquired as somatic mutation in carriers of germlineDDX41 variants. No pathological DNA was available for the other
sibling, who however developed a Ph+ CML.
The LYST gene is mutated in autosomal recessive mode in Inborn
Errors of Immunity syndromes and, in particular, in Familial
Hemophagocytic Lymphohistiocytosis (FHL) syndromes with
Hypopigmentation: Chediak-Higashi syndrome and Hemophagocytic
Lymphohistiocytosis (HLH). LYST is a lysosomal trafficking regulator and
a key effector of cytotoxic granules’ biogenesis. It is involved in the
modulation of cytotoxic T-lymphocytes (CTL) and natural killer (NK)-cell
functions by regulating degranulation. LYST-deficient CTLs and NK-cells
display impaired ability to kill target cells and accumulate giant
cytotoxic granules. Patients with Chediak-Higashi syndrome display
oculocutaneous albinism, easy bruising, recurrent pyogenic infections
and exhibit abnormal functions of NK-cells and alterations in
neutrophils, leading to neutropenia. We have no evidence to infer a
direct role for the identified LYST mutation in predisposing to
development of myeloid neoplasms; however, we can envision a possible
role for a deregulated immune system in controlling the homeostasis of
hematopoietic differentiation.
In the fourth family we scored a more complex landscape with three
germline VUS shared by both siblings. The most noteworthy is mutation
p.S858R in FANCA gene. It is annotated as VUS in cancer
databases; however, notably, it is annotated as pathogenic in FAMutdb, a
database of variants identified in Fanconi Anemia (FA)
(http://www2.rockefeller.edu/fanconi/) and it is reported in
several FA patients of different origins (Italian, German,
Indian-Jewish). FA is an autosomal recessive disease usually associated
to mutations of other FANC members for its manifestation. The
penetrance and phenotypic manifestations of the syndrome are highly
variable. Our patients had no signs of FA and we did not identify
pathogenic mutations in other sequenced members of FANC gene
family. However, carriers of heterozygous FA mutations present increased
risk for development of MDS and AML. Therefore, we can envision an
incompletely penetrant phenotype imposed by p.S858R FANCAmutation, which requires cooperation with other germline lesions.
Germline predisposition to myeloid neoplasms due to pathogenic or likely
pathogenic variants of JAK2 gene are emerging as new disorders;
however, in both siblings of Family4, we identified a germline variant
currently annotated as VUS: p.G571S. This mutation is located in exon 13
and, as the most common oncogenic variant in JAK2 , p.V617F,
within the region encoding for the autoinhibitory JH2 pseudokinase
domain of the protein. Although the biological significance of this
variant is not well established yet, in vitro assays in Ba/F3 cells
suggest no significant impact on the JAK2 protein functions. This
variant has been reported associated to MPN with a frequency of around
0.01% and as germline both in sporadic and in familial cases of
Essential Thrombocythemia (ET),. Moreover, it is
listed in COSMIC (COSM29107, COSM142855: 10 mutations, 7 of which in the
Haematopoietic and lymphoid category).
Finally, the missense variant in the receptor tyrosine kinaseERBB4 has an unknown biological significance and it has not been
reported in COSMIC. We currently have no clues on its possible role in
our clinical context.
Although functional characterization of these mutations is still
required, we speculate that in Family4 the full MDS/AML phenotype may
result from the cooperation between the FANCA and JAK2missense mutations. Interestingly, in a WES analysis of mutations in a
cohort of patients with BM failure syndromes of suspected inherited
origin, 11.6% of patients carried 2 co-occuring potential alterations.
In Family4, in siblings sharing this same complex germline landscape,
the clinical history of the disease followed independent pathways, with
acquisition of independent somatic mutations except for a common
prevalent clone (VAF>30% in both siblings) with p.K700E
mutation in SF3B1, suggesting a selective pressure imposed by the
germline variants on acquisition of this somatic mutation. In a cohort
of 16 FA patients, this same mutation in SF3B1 was identified in
a patient with a germline FANCA mutation, who developed
refractory anemia with ring sideroblasts and mutations in SF3B1and JAK2 seem to co-occur in myelodysplastic/myeloproliferative
neoplasms with ring sideroblasts and thrombocytosis ,.
Notably, clonal evolution reconstruction by single cell sequencing in a
FA patient harboring a mutation in FANCA , showed appearance, in
the early stage of MDS, of a clone with the SF3B1 p.K700E
mutation, together with a mutation in RUNX1 , which expanded to
become dominant with progression of the disease to AML.
Currently, a field of intense investigation is the study of clonal
hematopoiesis (CH), a premalignant state in which hematopoietic stem and
progenitor cells clonally expand due to acquisition of somatic mutations
in genes that confer selective growth advantages. CH has been associated
to increase risk of development of a number of diseases that include
both hematological tumors and non-malignant conditions such as ischemic
cardiovascular, inflammatory and autoimmune diseases. The analysis of CH
is performed on peripheral blood in several clinical contexts,
independent from hematological disease, by NGS that include both WES and
targeted gene panels. The search for CH in an ever-growing number of
non-hematological patients will generate a critical mass of genomic data
on the hematopoietic system that could prove extremely helpful in the
field of germline predisposition to myeloid neoplasms, significantly
increasing the wealth of information available for this traditionally
under investigated cancer type.
In conclusion, we show that thorough genomic analysis using large gene
panels appositively targeted on cancer predisposing genes allow
identification of novel germline variants. Indeed, in each family of our
cohort, we identified at least one novel variant, affecting also genes
not included in the current ENL guidelines for AML management. According
to The American College of Medical Genetics and Genomics (ACMG)
guidelines for interpretation of sequencing variants, a critical
component for understanding of significance of a VUS is the observed
clinical phenotype. On these basis, we suggest reclassification as
pathogenic of the likely pathogenic p.S21Tfs*139 in CEPBA and the
VUS p.K392Afs*66 in DDX41 . Finally, considering that myeloid
neoplasms with germline predisposition often display clinical and
morphological characteristics similar to sporadic cases and that age at
diagnosis in the two groups often overlap, it is really challenging to
suspect familiarity in the context of hematological tumours. Indeed,
based exclusively on their clinical parameters, none of the cases of our
cohort would have been suspected of familiarity. However, studying
affected siblings, we identified potential germline predisposition in
each family. Our data underline how current clinical practice
underestimate familial cases within hematological neoplasms and calls
for implementation of novel clinical practices that should include
thorough reconstruction of personal and family history and genetic
testing with large gene panels targeted for predisposing genes.