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Erythropoiesis in Fanconi’s Anemia
By Blanche P. Alter, Mary Ellen Knobloch, and Rona S.Weinberg
Fanconi’s anemia (FA) is an autosomal recessive condition in
which greater than 90% of the homozygotesdevelop aplastic
anemia. To determine the relation betweenerythroid progenitors and clinical status, blood and marrow mononuclear cells
were cultured in methyl cellulose with erythropoietin, plus
other hematopoietic growth factors, and growth in normal
oxygen (20%)was comparedwith growth in low, physiologic
oxygen (5%). Peripheral blood cultures were performed from
24 patients, and marrows from six. Patients were classified
into six clinical groups. Group 1: Severe aplasia, transfused;
one patient; no erythroid progenitors. Group 2: Severe,
transfused, androgen unresponsive; one patient; no blood
burst-forming units-erythroid (BFU-E). Group 3: Androgen
responsive; eight patients, with decreased blood BFU-E.
Group 4: Aplastic, about to start treatment; two patients;
below normal numbers of colony-forming units-erythroid
(CFU-E) and BFU-E. Group 5: Stable, with mild anemia,
andfor thrombocytopenia, and/ or macrocytosis; seven patients; with below normal numbers of blood BFU-E. Group 6:
Hematologically normal; five patients; blood BFU-E low
normal to normal. One marrow had normal numbers of
CFU-E and BFU-E. Incubation in 5% oxygen doubled CFU-E
and BFU-E only in the patients with close t o normal or normal
growth in 20% oxygen. Hemin and interlgukin-3 increased
growth slightly in those cultures where there was some
growth with erythropoietin alone. Our data show that there
is a correlation between current clinical status and in vitro
erythropoiesis. Cultures of erythroid progenitors may also be
useful predictors of hematologic prognosis in FA, although
our follow-up period is too short to prove this hypothesis.
0 1991by The American Society of Hematology.
F
We wished to determine whether there is any predictive
value regarding hematologic status of in vitro erythroid
cultures. Table 1 outlines a classification of FA patients
according to hematolagic severity. Hematopoietic culture
data were previously reported from 23 patients studied in
10 laboratories, and the results are summarized in Table 2
according to the classification scheme in Table l.’3317-z
Although seven patients had normal hemoglobin levels at
the time of the study, including two patients who also had
normal platelet counts, none had normal numbers of
erythroid progenitor cells.”26 However, our own results
differ in that we were able to identify FA patients with
normal blood counts who also had normal erythroid progenitors, indicating that there is a correlation between current
hematologic status and in vitro erythropoiesis, and suggesting the potential for a predictive value for this assay.
In an effort to stimulate erythropoiesis in vitro from
samples of blood or marrow from FA patients with impaired hematopoiesis, we examined several variables. Although it has been suggested that oxygen-free radicals
increase chromosome breakage, the role of toxic oxygen
products in hematopoiesis in FA is not clear.27We performed parallel cultures in incubators in room air, and in
incubators with the oxygen reduced to 5%, which is closer
to physiologic levels. Other variables included the addition
of potential growth factors, such as higher concentrations of
erythropoietin (Ep), hemin, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF),
growth hormone, and androgens. Some of these manipulations led to small increases in burst-forming units-erythroid
(BFU-E)-derived colonies, but none completely compensated for the erythropoietic defect.
ANCQNI’S ANEMIA (FA) is an autosomal recessive
chromosome breakage disorder in which the incidence
of aplastic anemia exceeds 90% in homozygotes. The
predicted median survival age is 16 years from the literature,’!’ and 25 years from the International Fanconi Anemia
Registry (IFAR).3 The IFAR includes many patients who
were diagnosed as FA before the development of significant
hematologic disease. These families are particularly concerned about whether such a patient will develop aplastic
anemia, and if so, when, as well as the potentials for
therapy. Currently, the only FA patients who can be
considered “cured” are those who have received successful
transplants of hematopoietic stem cells from HLA-matched
bone marrow or placental bl00d.4,~Although the initial
response rate to androgens of aplastic anemia in FA is
greater than 50%, the survival is only prolonged by approximately 5 years. Less than a dozen patients were able to
discontinue androgen treatment without relapses of their
aplastic anemia by the time they were reported, and their
long-term outcome is unknowm6l6 Patients with FA are also
at risk for leukemia (lo%), liver tumors (5%), and other
malignancies (5%), but aplastic anemia remains the major
and earliest threat, with an average age of onset of 8 to 9
years.
From the Departments of Medicine and Pediatiics, Pol&Annenberg
Levee Hematology Center, Mount Sinai School of Medicine, New
York, Ny.
Submitted January 4,1991; accepted March 26,1991.
Supported in part by grants from the National Institutes of Health
(HL26132), the Fanconi Anemia Research Fund, the National
Organization for Rare Disorders, and a General Clinical Research
Center grant to the Rockefeller University Hospital (Mol-RROO102)
from the National Institutes of Health.
Address reprint requests to Blanche P. Alter, MD, Division of
Hematology, Mount Sinai School of Medicine, One Gustave Levy
Place, New York, NY10029.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 sole& to
indicate this fact.
0 I991 by TheAmerican Society of Hematology.
0006-4971/91/7803-0007$3.OOJO
602
MATERIALS AND METHODS
Blood and bone marrow samples were obtained from patients
with FA according to protocols approved by the Institutional
Review Boards at Mount Sinai Medical Center and Rockefeller
University Hospital (New York, W ) .The diagnosis of FA was
made in all patients because of increased chromosome breakage
after culture of peripheral blood lymphocytes with diepoxybutane
(performed by the laboratory of Arleen Auerbach, PhD, Rockefeller
University Hospital). Thirty-two studies were performed from 24
Blood, VOI 78, NO3 (August 1). 1991: pp 602-608
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ERYTHROPOIESISIN FANCONI’S ANEMIA
603
Table 1. Clinical Classification of FA Patients
status
Group Transfusions Androgens
1
Yes
No
2
Yes
Yes
3
No
Yes
4
No
No
5
No
No
6
No
No
Severe aplastic anemia, failed or
never received androgens
Severe aplastic anemia, currently on,
but not responding to, androgens
Responding to androgens, previously severe or moderate aplasia
Severe or moderate aplastic anemia,
needs treatment
Stable,with some sign of marrow
failure, eg, mild anemia, mild neutropenia, thrombocytopenia, high
the cell yield was sometimeslow, not all parameters could be tested
in each experiment.
All cultures were performed in humidified room air (20%
oxygen) with 4% CO, at 37°C. In some experiments, cultures were
also incubated in 5% oxygen, using either a Bellco box (Bellco
Glass Inc, Vineland, NJ) gassed with 5% oxygen balanced with
nitrogen (before August 1989), or a Hereaus incubator (Hereaus
Inc, South Plainfield, NJ) in which 5% oxygen was maintained by
nitrogen (after August 1989). EIythroid colonies were identified by
their red color, and were counted on day 6 for colony-forming
units-erythroid (CFU-E), and 9, 13, 16, and 20 for BFU-E, using a
Bausch and Lomb (Rochester, NY) stereozoom dissecting microscope. Triplicate wells were counted and averaged.
MCV, high Hb F
Normal hematology,
RESULTS
normal Hb F
patients; three patients were studied twice and one was studied six
times within a 2-year period. This patient underwent a successful
pregnancy with red blood cell (RBC) and platelet support, and
subsequently returned to her usual group 5 status,” while the other
patients had no change in their clinical features. These repeated
studies of the same patients gave similar results. Blood counts were
obtained with a Coulter S + IV (Coulter Electronics,Hialeah, FL)
or a Technicon H1 (Technicon Industrial Systems, Tarrytown,
NY),and hemoglobin (Hb) F was measured by alkali denaturatioamEp levels were obtained in 14 patients, by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA) from SmithKline Laboratories (Philadelphia, PA) or Roche Laboratories
(Raritan, NJ).
Erythroid cultures were performed as described previously.M
Ten to 30 mL of blood was collected in heparinized vacutainer
blood collection tubes (Becton Dickenson, Rutherford, NJ), and
0.5 mL of bone marrow was added to-5 mL of (Y medium (GIBCO
Laboratories, Grand Island, NY)containing heparin (Liquaemin;
Organon Inc, West Orange, NJ) 50 U/mL. Mononuclear cells were
recovered after centrifugation on Ficoll-hypaque (Pharmacia Fine
Chemicals, Piscataway, NJ), 300,000 mononuclear cells plated per
milliliter, and 0.3 mL miniwells (Nunc, Intermed, Roskilde, Denmark) were used in triplicate. Recombinant human Ep, kindly
provided by Ortho Pharmaceuticals (Raritan, NJ) and by Amgen
Biologicals (Thousand Oaks, CA), was used at 2, 4,and 8 U/mL.
Additions to cultures, which contained Ep at 2 U/mL, included
hemin at 100 and 200 pmol/L (Sigma Chemical Co, St Louis, MO),
IL-3 at 50 and 100 U/mL (Amgen), human growth hormone at 50,
100, and 200 ng/mL (HGH; kindly provided by Genentech Inc,
South San Francisco, CA), GM-CSF at 50 and 100 U/mL (Amgen),
mol/L (Sigma). Because
and etiocholanolone at lo-’, lo-’, and
According to the classification in Table 1, our group of
patients included one patient in group 1, one in group 2,
eight in group 3, two in group 4, seven in group 5, and five in
group 6. Laboratory data are summarized in Table 3. Six of
the eight in group 3 had Hbs of at least 10 g/dL, all had
mean cell volumes (MCVs) above 100 fL,and seven had
increased Hb F at the time of the study. Only one had a
platelet count above lOO,OOO/pL. Three of these eight
patients had a history of severe aplastic anemia before
beginning androgens. Both patients in group 4 were anemic, macrocytic, thrombocytopenic, and had Hb F levels of
12%; one was “severe” and the other “moderate.” Six of
the seven in group 5 had Hbs above 10 g/dL, three had
platelets above lOO,OOO/pL, but all had increased Hb F and
were macrocytic for age. By definition, all five patients in
group 6 had normal blood counts and normal MCVs for
age, but two patients in this group had increased Hb F
(3.8% in 2-year-old patient 24, and 5% in 4-year-old patient
21).
Serum Ep was measured in 14 patients, and log [Ep]
correlated inversely with the Hb level (Fig 1). Ep was very
high (>500 mU/mL) in those patients whose Hb was less
than 10.5 g/dL, above the range expected in patients with
similar degrees of anemia due to iron deficiency, and even
higher than reported previously for FA patients?’ Renal
anomalies may provide a partial explanation for E p levels
lower than expected (Table 3), eg, patient 12 with a single
kidney had an E p of 566 mU/mL at an Hb of 8.2 g/dL, while
patient 17 with normal kidneys had an E p of 7,980 mU/mL
at an Hb of 8.7 g/dL.
Table 2. Hematopoietic Cultures From FA Patients in Literature
Bone Marrow
No. of
Group
Patients
1
2
3
4
5
6
1
3
4
2
10
1
1
1
No data
AML
CFU-C
0 (1)
dec (3)
dec (4)
dec (10)
dec (1)
0 (1)
0 (1)
CFU-E
Peripheral Blood
BFU-E
dec (1)
dec (3)
CFU-C
0 (1)
BFU-E
0 (1)
dec (2)
nl (2)
dec (2)
dec (7)
0 (1)
0 (1)
0 (1)
0 (1)
dec (6)
dec (1)
Comments
2 With nl Hbs
dec (3)
0 (1)
4 With nl Hbs
1 With nl Hb
References
18
13,18
18-22
13,17,18,20,24
13,26
25
23
Numbers in parentheses indicate number of patients for whom study was done. All patients exceptthe one in group 6 had decreased platelets.
Abbreviations: CFU-C, colony-forming unit, myeloid; CFU-E, colony-forming unit. erythroid (matureerythroid progenitor);BFU-E, burst-forming
unit, erythroid hmature erythroid progenitor);dec. decreased: nl, normal; Hb, hemoglobin.
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ALTER, KNOBLOCH, AND WEINBERG
604
Table 3. FA Patient Data
Rx
(Y)
No. Group
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
2
3
3
3
3
3
3
3
3
4
4
5
5
5
5
5
5
5
6
6
6
6
6
F
M
M
M
19
22
19
5.5
11
37
12
8
3
M
8
11
F
M
4
3
11
11
4.5
21
19
9
3
3.9
35
32
1.9
M
F
M
M
F
F
F
M
F
M
M
F
M
M
M
M
M
6.8
12.2
14.4
7.9
8.6
15.4
10
13.9
12.1
11.7
7
8.2
10.5
12
10.7
12.8
8.7
10.9
11.5
12.8
11
16.3
14.7
11.7
83.8
91.5
110.5
115.2
101.3
110
108
101
104
101
105.6
113
107
95.8
105.1
99.2
114.9
114
97.6
83
79
96.5
89.9
78.9
TXD 1,400 476
TXD 3,600 1,224
0.4 4,200
27.4 4,600
9.7 3,700
1.7 2,900
11.9 5,200
8 5,500
15.3 5,100
6.4 4,000
12.6 4,700
12.9 4,100
26.2 4,000
4 6,500
6.9 2,930
7.2 6,400
7.6 4,600
4.9 2,600
2.2 2,500
1
7,600
5 6,800
1.1 6,300
0.1 5,900
3.8 6,800
184
1,443
1,131
728
1,760
900
2,776
1,410
1,189
1,120
3,120
1,143
1,024
1,610
1,014
600
2,736
2,448
4,599
4,543
1,510
28
22
70
17
38
73
130
61
33
55
45
25
41
184
73
112
63
62
107
343
167
269
224
248
Renal Status
Outcome
BMT, died AML 1 y, age 21
Died sepsis and CNS bleeding, age 24
0.5
1
8
0.2
5
23 Ectopic
2,400
39
3,700
110
657
79
3,108
566
BMT
8
0.3
0.4 Pelvic
1.1
10.4
7,980
300
43.3
5
BMT
Umbilical cord tx
Single
Horseshoe
Single
5
Currently off androgens
1 Son
Reflux
3 Sons
1 Son
37
Pelvic, ectopic, reflux
Abbreviations: Hb. hemoglobin; MCV, mean cell volume; Hb F, hemoglobin F; WBC, white blood count; ANC, absolute neutrophil count; Plat,
platelet count; Ep, erythropoietin; Rx. duration of treatment with androgens; BMT, bone marrow transplant; AML, acute myelogenous leukemia;
CNS, central nervous system; F,female; M, male.
Standard erythroid cultures are usually performed with 2
U/mL of Ep, incubated in 20% oxygen (room air), and
examined on day 13 or 14. Normal individuals have 5 to 60
colonies derived from BFU-E per 100,000 peripheral blood
mononuclear cells. Some normals reach maximal erythroid
colony number later than day 13.” Figure 2 shows the
results of blood BFU-E cultures with Ep at 2 U/mL from
our FA patients, on the day of maximal growth for each
patient. Patients in groups 1 through 4 had less than one
EP IN FA
10000
1000
a
*
I
t
1
2 0 % 0,
-
-I
5
\
=
r
colony per 100,000 cells at all times, in both room air and
low oxygen. In group 5, one patient (patient 14) had three
colonies in 20% and two colonies in 5% oxygen. However,
in group 6, the group with normal blood counts, three of the
five had normal numbers of colonies in room air and
showed an increase in low oxygen to the same extent as we
have observed in normals.33The two patients (patients 21
and 24) who had no blood BFU-E growth with Ep 2 U/mL
in either oxygen concentration were the ones with slightly
elevated levels of Hb F.
Higher concentrations of Ep, 4 or 8 U/mL (Fig 3) were
slightly more effective than 2 U/mL, increasing colony
numbers by a small amount in two of the patients in group
100
-
10
-
30
-
25
-
,
I
#
5% 0,
,
EP 2 U/ML
I
0
,
,
,
HB <10
HB >10
,
,
- 30
- 25
a
W
I
1 1
6
* 5
A
I
I
8
* * I
5 c
A 6
10
12
14
16
HB G/DL
GROUP
Fig 1. Semi-log plot of serum Ep versus Hb in 14 FA patients. The
linear regression equation is: Ep mU/mL = -570 (Hb) 7,743. R =
-.6,P= .01.
+
GROUP
Fig 2. Blood BFU-E-derived colonies/lV cells on the day of
Hb < 10 g/dL. (*I,
maximalgrowth (day 13 or 16) with Ep 2 U/mL. (0),
Hb > 10 g/dL. Left, room air (20% OJ. Right, decreased 0, (5%).
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605
ERYTHROPOIESIS IN FANCONI'S ANEMIA
5% 0,
20% 0,
30
-
25
-
I
I
I
I
I
I
HB
- - e HE
EP HIGH
0
<
>
I
I
5% 0,
20% 0,
I
10
10
- 30
'
-25
30
-
25
-
,
,
#
I
I
,
IL3
- 0
__
4
I
I
I
I
I
HB <10
HB >10
In
0
:
5 -
4
4
4
I
,
GROUP
0 10
4
4
4
- 5
5
A -
0
a
'
1
'
2
8
A Q e 3
4
5
I
6
1
2
GROUP
GROUP
-- m4
A
3
5
+
6
- 30
- 25
1;:
10
5
a
GROUP
Fig 3. Blood BFU-E-derived colonies/105 cells on the day of
maximal growth (day 13 or 16) with Ep 4 or 8 UlmL. (0).
Hb < 10 g/dL.
(4),Hb > 10 g/dL. Left, room air (20% 0,).Right, decreased 0, (5%).
Fig 5. Blood BFU-€-derived colonies/lOs cells on the day of
maximal growth (day 13 or 16) with Ep at 2 U/mL, plus IL-3 50 or 100
U/mL. (0).
Hb < 10 g/dL. I+),
Hb > 10 g/dL. Left, room air (20% 0,).
Right, decreased 0, (5%).
3, one in group 4,and one in group 5, as well as in one of the
two patients in group 6 (patient 21) in whom there was no
growth with 2 U/mL. As in the cultures in 2 U/mL, only
those in group 6 had higher numbers of colonies in low
oxygen than in room air with high Ep.
Hemin, which increases the number of BFU-E in cultures
from normals,34increased growth when added to cultures
containing Ep at 2 U/mL only from the group 6 patients,
now including one of the two patients from whom there was
no growth with Ep alone (Fig 4). The hemin effect was more
dramatic in low oxygen than in room air, as we have seen in
normal cultures.33
IL-3 was also tested in cultures containing Ep at 2 U/mL
(Fig 5). There was a slight increase in the number of blood
BFU-E from group 6 samples, again more dramatic in low
oxygen than in room air. BFU-E were now seen in the low
oxygen cultures containing IL-3 from the two patients in
this group in whom there was no growth under standard
conditions.
The results with GM-CSF added to Ep-containing cultures are shown in Fig 6. GM-CSF increased BFU-E growth
slightly from group 6 samples, but only in room air, not in
low oxygen.
Another potential hematopoietic growth factor tested
with Ep was an androgen, etiocholanolone (Fig 7). Although androgens are effective in vivo in more than 50% of
FA patients, including all eight of our patients in group 3,
etiocholanolone did not augment BFU-E growth in any of
the groups of patients. A similar failure of androgens to be
effective in vitro was noted previously." HGH also failed to
increase the numbers of BFU-E in cultures from patients in
groups 1,2,3, and 5 (data not shown).
Time course data are shown in Fig 8 for blood BFU-E
from two patients in group 6 and one in group 5. The group
6 data in the top panels (patient 20) are indistinguishable
from those of normals in our laboratory,33with 10 to 15
BFU-Merived colonies per 100,000 cells plated in Ep, an
Ep plateau of 2 U/mL, increased growth in hemin, and
substantially increased growth with all factors in low oxygen
compared with room air. In contrast, the growth patterns in
the middle panels, from another group 6 patient (patient
21), resemble the patterns in the bottom panels, from a
group 5 patient (patient 14). The patterns from two other
group 6 patients (patients 22 and 23) resemble those in the
top, and from one other group 6 patient (patient 24) those
in the middle. This segregation of group 6 BFU-E growth
into normal and abnormal patterns suggests that there may
be a correlation between in vitro erythropoiesis and in vivo
30
25
- HEMIN
,
,
I
-
,
,
I
__
0
4
I
I
I
HB < l o
HB >10
I
I
4
30
-30
- 25
25
tn
z
5
0
u
5
-
-
I
,
GM-CSF
GROUP
Fig 4. Blood BFU-€-derived colonies/105 cells on the day of
maximal growth (day 13 or 16) with Ep 2 U/mL, plus hemin 100
Hb < 10 g/dL. (4),Hb > 10 g/dL. Left, room air (20% OJ.
amol/L. (0).
Right, decreased 0, (5%).
,
I
0
--*
20
15
10
+
0
GROUP
5% 0,
20% 0,
5 % 0,
20% 0,
,
It
I
HB <10
HE >10
I
I
I
- 30
- 24
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ALTER, KNOBLOCH, AND WEINBERG
606
20% 0,
- ETlOC H0L A N 0 LONE
-
30
25
fn
g
5% 0,
30
25
20
z
9
15
0
0 10
S
0
GROUP
GROUP
Fig 7. Blood BFU-E-derived colonies/lOs cells on the day of
maximal growth (day 13 or 16) with Ep 2 U/mL, plus etiocholanolone
IO-',
or lo-' mol/L. (0).
Hb < 10 g/dL. (+), Hb > 10 g/dL. Left,
room air (20% 0,).Right, decreased 0, (5%).
20% 0,
30
20
-0
15
C
I
I
I
I
5% 0,
I
I
A
I
I
B
I
a
I
DISCUSSION
I
I \
The serum Ep levels that we monitored correlate with the
Hb, rather than with the classification group. Ep was close
to normal (<50 mU/mL) only in all of the group 6 patients,
in group 5 patient 14 who had a single kidney and patient
19, both of whom had Hbs greater than 11.5 g/dL, as well as
group 3 patient 6 whose Hb was 15.4 g/dL and who resides
at altitude. The inverse relation between log Ep and Hb is
similar to the finding of McGonigle et al," although our
patients had higher values. Because patients in groups 4
and 5, all not on androgens, had Ep levels as high as
patients in group 3 who were on androgens, it is apparent
that the elevated Ep levels are related to impaired erythropoiesis, and not to androgens per se.
25
.-m
0
I
status: the two group 6 patients with poor growth were the
ones who had elevated levels of Hb F.
Figure 9 summarizes the limited data available from bone
marrow cultures. Marrow CFU-E were present, although in
decreased numbers, in the only patient in group 3 who had
a marrow study, patient 6. Low oxygen led to an increase in
the number of CFU-E, from 19 to 34 per 100,000 cells, still
below our normal range of 75 to 150. There were essentially
no marrow or blood BFU-E. Both patients from group 4
had very low numbers of marrow CFU-E ( < 7 per 100,000
cells), and only one had any marrow BFU-E (5 per 100,000
cells, significantly below our normal range of 10 to 75);
blood BFU-E were below one per 100,000 cells in both.
Only one marrow was performed in a group 5 patient,
patient 14. Marrow CFU-E were slightly below normal (40
per 100,000 cells), while marrow BFU-E were below normal
(6 per 100,000 cells), and blood BFU-E were also low,
below 3 per 100,000 cells. Only one group 6 patient, patient
20, had a bone marrow, with normal numbers of marrow
CFU-E and BFU-E, which doubled in low oxygen, associated with normal numbers of blood BFU-E.
:
10
5
0
5
2 0 % 0,
.-:3
3
C
2
2
2
0
0
5 % 0,
1 4
.Vt
0
0
1.
tn
W
1
1
CFU-E
0
wz
5
150
-
100
-
50
-
-
- 100 0
E
--
m
- 50
4
0
EP 2,
HEM,
Day
EP HI,
+ GM,
0
A IL3,
HGH
: :
I ? :
0
I
BFU-E
BFU-E
,
I
t
1:;
v)
O
75
0 ET10
Fig 8. Time courses of the growth of blood BFU-E-derivedcolonies/
106 cells from three FA patients. (A and B) 3-year-old group 6 patient
number 20, with completely normal colony numbers, and a normal
increase in 5% 0,. (C and D) 3-year-old group 6 patient number 21,
with below normal colony numbers. (E and F) 11-year-old group 5
patient number 14, with below normal colony numbers. In this one
experiment, 5% 0, was provided in a Bellco box that was gassed
daily, rather than the Heraeus incubator used for all other 5% 0,
cultures. (O),Ep 2 U/mL. (V),
Ep 8 U/mL. (A),IL-350 U/mL. (W), Hemin
GM-CSF 100 U/mL. (0).Etiocholanolone
mol/L.
100 pmol/L. (e),
(0).
HGH 200 ng/mL.
00
r-
4
0
Day
110
0
0
0
200
CFU-E
0
0
r
4
Fig 9. Bone marrow CFU-E- and BFU-E-derived colonies/106cells
with Ep 2 U/mL. (O),Hb < 10 g/dL. (+), Hb > 10 g/dL. Left, room air
(20% 0,). Right, decreased 0, (5%).
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ERYTHROPOIESIS IN FANCONI’S ANEMIA
607
Their blood counts were normal, including MCV for age.
The culture data presented here represent the investigaHowever, patients 21 and 24 had increased Hb F, and
tion in a single laboratory of 24 patients ranging from
patient 24 had a borderline neutrophil count. The only
complete aplasia (groups 1 and 2) to hematologically
group 6 patient reported in the literature had decreased
normal (group 6). This is the largest single study of this
progenitor^.'^,'^ However, our patients are different. Three
type. The small number of patients with severe aplasia
had totally normal blood BFU-E under routine conditions,
(groups 1, 2, and 4) is unfortunate from the perspective of
with a normal increase in low oxygen, as well as augmented
experimental analyses, but is a realistic representation of
growth in hemin or IL-3 (Fig 8). Patients 22 and 23 are 32the clinical spectrum of FA. Our observation of decreased
and 35-year-old brothers, whose sister is patient 18 in group
or absent erythroid progenitors in these patients is consistent with the reports on such patients in the literat~re.’~.’~5. The third group 6 patient with normal erythroid progenitors, patient 20, has no signs of a hematologic problem. The
These patients had severe aplastic anemia, unresponsive to
normal progenitor growth suggests, clearly without proof,
androgens; the in vitro results indicate that they had
that these three patients may not develop aplastic anemia;
reduced or absent erythroid progenitor cells that were
much longer follow-up time is necessary to prove this
sufficiently mature to produce colonies in 7 or 14 days in
suggestion.
response to Ep, alone or associated with an extensive
There remain two patients in group 6 (patients 21 and 24)
variety of manipulations.
whose erythroid progenitors were decreased, particularly
Patients in group 3 previously had either severe or at
under standard conditions. Several of the manipulations
least moderate aplastic anemia, which responded to androdid elicit growth of some BFU-E, but the numbers were less
gen treatment. At the time of study, two of the four patients
than normal. However, their in vitro erythropoiesis was in
in the literature had Hb levels of 10 g/dL or more, as did six
general better than seen in group 5. These group 6 patients
of our eight patients. Nevertheless, none had normal
erythroid colony numbers. In our studies, the combination
were young when studied, ages 2 and 4. Although their
blood counts and MCVs were normal for age, they did have
of Ep plus IL-3 led to erythroid colony growth in the largest
slightly increased Hb F. Further cultures and clinical
number of patients, but the growth was poor. All of the
patients had at least two clear signs of impaired hematopoiefollow-up will be important to determine whether the
pattern of their erythroid progenitor growth defines the
sis, such as macrocytosis, increased Hb F, thrombocytopenia, and neutropenia. The only patient in this group whose
lower end of the spectrum for group 6, or whether it is in
fact predictive of a deteriorating hematopoietic status.
marrow was studied (patient 6) did have CFU-E, although
below normal numbers, but essentially no marrow or blood
It is not clear why FA patients with normal Hb levels in
BFU-E. Patient 3 had the most blood BFU-E, particularly
groups 3 and 5 have reduced numbers of blood and marrow
with high Ep, hemin, IL-3, and GM-CSF, but the numbers
BFU-E, as well as marrow CFU-E. The progenitors that are
were still below normal.
measured in normal individuals may be an unused excess,
The two patients in group 4, who had moderate-severe
not required to sustain normal levels of in vivo erythropoiesis. FA patients with normal Hbs on androgens presumably
aplastic anemia and were about to begin treatment, had
absent or very decreased numbers of both CFU-E and
have sufficient erythroid progenitors to sustain blood proBFU-E. The two group 4 patients in the literature also had
duction in vivo, but lack the excess, reserve pool that is
decreased marrow BFU-E, although CFU-E were present
detected in normals with the in vitro assays. Patients with
in those cases.” Thus, patients with decreased erythropoiesome abnormality in erythropoiesis, eg, macrocytosis and
sis in vivo also had decreased erythropoiesis in vitro. While
increased Hb F, signs of “stress erythrop~iesis,”~~
may also
they did have some mature erythroid progenitors (CFU-E),
lack a “reserve” progenitor pool.
they lacked the earlier progenitors, BFU-E. None of the
Our examination of erythroid progenitors in a large
factors that we tested restored normal erythropoiesis in
group of FA patients confirms the general statement that
vitro.
these progenitors are reduced or absent in most FA
The patients in group 5 comprise the most diverse group.
patients. However, we have identified an important excepWhile none were on treatment, their Hbs ranged from 8.7
tion to this in group 6, patients with normal blood counts.
to 12.8 g/dL (six of seven had Hbs 2 10 g/dL). All had RBCs
Even here there appears to be a segregation of patients.
that were macrocytic for age, with increased Hb F, and most
One subset had totally normal colony growth, and totally
had at least mild thrombocytopenia as well as neutropenia.
normal blood counts. The other subset had poor colony
All patients were clinically well at the time of study,
growth, but with better response to hematopoietic factors
although one (patient 13) subsequently had an elective
than in group 5. These patients also had normal blood
umbilical cord blood transplant. Despite their mild course,
counts, although their Hb F was increased. Studies of group
none of these patients had normal in vitro erythropoiesis.
6 patients will be infrequent, because these patients may
Patient 14, whose blood counts were normal, although
not be identified as FA because their hematology is normal.
macrocytic with increased Hb F, had the best growth of
They include siblings of more severely affected FA patients,
blood BFU-E with all tested variables (Fig 8, panels E and
as well as children identified because of their physical
F). The number of BFU-E-derived colonies in the blood of
anomalies, before the development of aplastic anemia.
group 5 patients was generally slightly better than in those
Serial studies of these patients may provide prognostic
of group 3.
information regarding this evolution. Additional in vitro
The patients in group 6 are perhaps the most interesting.
manipulations with combinations of hematopoietic growth
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ALTER, KNOBLOCH, AND WEINBERG
608
factors may indicate potential therapeutic directions as
well.
ACKNOWLEDGMENT
We are grateful to the Rockefeller University Hospital laboratory of Arleen D Auerbach, PhD, for the chromosome breakage
studies, to all the FA patients and their families, who provided
specimens, and to the companies which provided factors (AMGEN,
ORTHO, Genetics Institute, and Genentech). We thank Richard
A. Drachtman, MD, for performing the bone marrow aspirations,
and Liya He, MB, for her technical expertise.
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1991 78: 602-608
Erythropoiesis in Fanconi's anemia
BP Alter, ME Knobloch and RS Weinberg
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