Bone Marrow Fibroblasts Derived from Vitally Frozen Mononuclear Cells As a Source for Germ Line DNA in Acute Myeloid Leukemia (AML)

The introduction of next generation sequencing (NGS) techniques has revolutionized the genetic characterization of malignant diseases. Still, optimal determination of somatic mutations in patients requires not only tumor DNA but also germ line DNA and finding a reliable source for germ line DNA is then crucial. In AML, germ line sources such as skin biopsies and buccal swabs/washes can be hampered by contamination of leukemic cells as well as low amounts of DNA and the use of T-cells is limited by the presence of evolutionary early somatic mutations that are also present in the malignant clone. As AML samples commonly are preserved and biobanked as vitally frozen mononuclear cells, we aimed to find a reliable method using these samples taken at the time of AML diagnosis as a source of germ line DNA. For this purpose, we choose to focus the non-hematopoietic population present in the diagnostic AML bone morrow sample and hypothesized that fibroblasts would fit the purpose. Bone marrow fibroblasts (BMF) were expanded in vitro by thawing vitally frozen mononuclear cells and subsequently grow adherent cells with the aim was to (i) get rid of any leukemic cells (ii) obtain up to 5 µg of germ line genomic DNA. BMFs were cultured from 6 AML patients harboring a monosomy 7 karyotype as well as fibroblast from normal donors. Cells were cultured in MyeloCult™ for up to 6 weeks. After 48 hours, unattached cells washed away while BMF attached to the bottom of the flask and were monitored by light microscopy for their ability to form colonies and retain a flattened shape. At 2, 4 and 6 weeks after initiation of the culture, cells were counted and characterized by a differentiation assay, FISH for monosomy 7, and FACS for surface markers. Doubling time for BMFs was on average 2 weeks although proliferation was age dependent with faster growth in younger patients. After 6 weeks of expansion culture, the number of cells had reached the intended 5.10⁶BMFs and ~5 μg of DNA for most patients. FISH results showed BMF to lack monosomy 7 in contrast to AML to cells. Surface markers were CD73+, CD105+, CD90+, CD45-, CD34- and CD33- and thus showed fibroblast phenotype though also similar with that of mesenchymal stem cells (Table1). For the differentiation assay, BMFs were cultured with accurate types of medium for 10-14 days allowing cells to differentiate into either bone, fat or cartilage cells. The differentiation assay showed that BMFs were able to differentiate to bone and fat but not cartilage cells.

In conclusion, our findings show the feasibility of deriving large amounts of non-hematopoietic cells defined as fibroblasts as a source of germ line DNA from vitally frozen diagnostic AML samples. We consider this as an attractive source of germ line DNA in analyses such as whole genome exome sequencing of AML patients.

Table1: Cell surface markers expressed on cultures bone marrow fibroblasts and AML cells in two AML patient.

CD7

CD 11b

CD13

CD14

CD19

CD33

CD34

CD38

CD45

CD56

CD73

CD90

CD105

CD117

HLA-DR

P1a BMF

+

-

-

+

+

+

P1b AML blast

+

-

+

+

+

+

+

+

+

-

-

-

+

+

P2c BMF

+

-

-

+

+

+

P2d AML Blast

-

-

+

-

-

+

+

+

+

-

-

-

-

+

+

P1: patient1 (^aBMF; ^bblast cells), P2: patient2 (^cBMF; ^dblast cells).