Colony-Forming Unit Cell (CFU-C) Assays in Myelodysplastic Syndrome

Introduction: The myelodysplastic syndromes (MDS) are a group of clonal diseases derived from hematopoietic stem cells (HSC). Colony-forming unit cell (CFU-C) assay is an effective method to study the number and the function of HSC in vitro. In this study, we focus on the characteristics and the prognostic value of CFU-C in patients with MDS.

Patients and Method: CFU-C assays were performed according to the protocol of MethoCult^TM H4435 Enriched (STEMCELL Technologies). A colony was defined as an aggregate of >40 cells.  Clusters consisted of 4 to 40 cells. 560 consecutive newly-diagnosed, untreated subjects with MDS diagnosed from March, 2001 to April, 2013 were studied. All subjects were reclassified according to the 2008 WHO criteria. 535 subjects with evaluable cytogenetics were classified using the International Prognostic Scoring System (IPSS) and the revised International Prognostic Scoring System (IPSS-R) criteria. Follow-up data were available for 470£¨84%£©subjects.  Median follow-up of survivors was 26 months (range, 1-170) months. Subjects receiving an allotransplants were censored in survival analyses. Erythroid and myeloid colonies were isolated from each subject with one cytogenetic abnormality such as del(5/5q-) or +8. Cytogenetic abnormalities of each colony were analyzed by fluorescence in situ hybridization (FISH). SPSS 17.0 software was used to make statistical analysis.

Results: Frequencies of burst-forming units-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocytes/macrophages (CFU-G/M) were significantly lower than normals (P<0.05) (Table 1).  Subjects classified as lower risk in IPSS and IPSS-R had significantly higher numbers of BFU-E and CFU-E (P<0.05) but similar numbers CFU-G/M and clusters-G/M compared with higher risk subjects (Table 2).  In 11 subjects with del(-5/5q-) or +8 identified by G- and/or R-banding, both normal and abnormal CFU-Cs were identified in 8 subjects studied by FISH. A high ratio of cluster- to CFU-G/M (>0.6) was associated with poor-risk cytogenetics (Table 2) and with worse overall survival in univariable (Figure 1, P=0.001) and multivariable analyses (HR 1.748, [1.01-3.0]; P=0.046) after adjusting for IPSS. 

Conclusions: These data suggest abnormalities of proliferation and differentiation of erythroid and myeloid precursor cells in vitro parallel the ineffective hematopoiesis typical of MDS and may be useful in predicting outcomes of patients with MDS.

Table 1.  CFU-C in MDS subtypes

	N	BFU-E	CFU-E	CFU-G/M	N	Ratio of cluster- to CFU-G/M
RA	21	8 (0-44)	40 (0-134)	14 (0-127)1	6	0.25 (0.40-1.00)
RT	4	18 (4-55)	75 (60-90)1	30 (18-70)1	2	2
RARS	27	12 (0-33)	35 (1-140)	12 (0-70)1	10	0.45 (0.17-0.80)
RCMD	275	10 (0-80)	33 (0-178)	14 (0-100)	126	0.35 (0-0.83)
RAEB1	112	10 (0-258)	32 (0-312)	14 (0-89)	53	0.47 (0-1.00)
RAEB2	103	9 (0-46)	25 (0-120)	13 (0-72)	42	0.37 (0-1.00)
MDS-U	15	4 (0-58)	25 (0-161)	10 (0-43)	3	2
Del(5q)	3	2 (2-4)	15 (0-20)	5 (5-41)1	1	2

¹ No significant difference compared with normals.

² Too few cases to analyze.

Table 2.  Associations between CFU-C and clinical and laboratory variables

	N	BFU-E	P	CFU-E	P	CFU-G/M	P	Number	Ratio of cluster- to CFU-GM	P
IPSS			0.064		0.006		0.361			0.089
Low	30	13 (0-44)		60 (0-169)		19 (0-45)		10	0.44 (0.24-0.70)
Int-1	361	10 (0-258)		33 (0-312)		14 (0-127)		150	0.33 (0-1.00)
Int-2	115	9 (0-61)		30 (0-137)		14 (0-72)		52	0.45 (0-1.00)
High	29	7 (0-34)		21 (0-93)		12 (0-67)		12	0.44 (0-1.00)
IPSS-R			0.003		0.003		0.125			0.209
Very low	7	16 (9-25)		30 (15-120)		18 (5-33)		2	0.29 (0.10-0.49)
Low	130	14 (0-80)		42 (0-178)		17 (0-70)		48	0.31 (0-0.77)
Intermediate	173	10 (0-66)		34 (0-161)		13 (0-127)		81	0.37 (0-1.00)
High	139	9 (0-259)		29 (0-312)		11 (0-89)		51	0.33 (0-1.00)
Very high	86	8 (0-61)		25 (0-137)		14 (0-91)		42	0.47 (0-1.00)
Cytogenetics (IPSS)			0.867		0.055		0.290			0.007
Good	327	10 (0-258)		36 (0-312)		15 (0-89)		133	0.33 (0-1.00)
Intermediate	133	10 (0-69)		30 (0-162)		12 (0-127)		63	0.45 (0-1.00)
Poor	75	10 (0-61)		25 (0-137)		14 (0-91)		28	0.42 (0-1.00)
Cytogenetics (IPSS-R)			0.990		0.090		0.676			0.022
Very good	7	11 (4-20)		48 (1-110)		14 (8-28)		2	0.49 (0.43-0.56)
Good	324	10 (0-258)		35 (0-312)		15 (0-89)		132	0.33 (0-1.00)
Intermediate	129	10 (0-69)		30 (0-162)		12 (0-127)		62	0.45 (0-1.00)
Poor	27	10 (0-61)		35 (0-137)		16 (0-48)		8	0.36 (0.15-1.00)
Very poor	48	11 (0-42)		22 (0-120)		14 (0-91)		20	0.53 (0-1.00)

Figure 1. Overall survival in subjects with cluster- to CFU-G/M ratios ¡Üor > 60%.

Disclosures: No relevant conflicts of interest to declare.

See more of: 637. Myelodysplastic Syndromes – Clinical Studies: Poster II
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