Bone marrow failure in pediatric patients

T h e ]ournal of P E D I A T R I C S

683

Bone marrow failure in pediatric patients I. C o r t i s o n e a n d t e s t o s t e r o n e t r e a t m e n t

Nine children with acquired aplastic anemia and [our children with constitutional aplastic anemia were treated for 1 to 19 months with cortisone and testosterone. Five of the nine children in the acquired group demonstrated hematologic improvement which has been sustained without therapy [or 12 to 36 months. Improvement would appear to be permanent in these children. While the [our children with constitutional aplastic anemia demonstrated initial hematologic improvement, prompt relapse occurred following cessation o[ therapy and necessitated maintenance hormonal therapy. Growth per[ormance was not altered during the course of therapy.

Franklin Desposito, M.D.,* Junichi Akatsuka, M.D.,** L. Gilbert Thatcher, M.D.,*** and Nathan J. Smith, M.D.**** MADISON~

WIS.

S ~ v E R E deficits in the numbers of circulating blood cells have been ascribed to the lack of hematopoietic elements in the bone marrow since Ehrlich's original observations in 1888. Since that time, the concept of aplastic anemia has broadened so that now there is recognized a congenital or constitutional type with 1 or without 2 associated malformations and an acquired or toxic form following exposure to an ever expanding list From the Department o[ Pediatrics, University o[ Wisconsin Medical School, Madison, Wis. These studies were supported by a grant-in-aid [rom the Gerber Baby Food Foundation. *Fellow in Pediatric Hematology. **Fellow in Pediatric Hematology. *~*Address, Instructor, Department of Pediatrics, University of Wisconsin Medical School, Madison 6, Wis. ~'~**Al#ed Dorance Daniels Professor oI Pediatrics.

of physical and chemical agents? I n addition, patients with refractory anemia having normal or hypercellular bone marrows are at times included in groups of patients said to have aplastic anemia. However, we would agree with Wintrobe 4 and Clement ~ who think that "it is probably wisest to reserve the term 'aplastic anemia' for patients having hypocellular or acellular marrows" associated with acute or chronic pancytopenias. This distinction becomes even more important in attempting to define response to various therapeutic regimens since pancytopenias associated with cellular marrows have a variable and unpredictable prognosis2 T h e prognosis of both the acquired and congenital forms of aplastic anemia in children has been extremely poor. Table I sum-

684

May 1964

Desposito et al.

TaMe I. Prognosis and survival pretestosterone therapy

Authors

Total No. o[ cases

Survival No.

Survival No. children

No. children ( < 16 years)

Total No. %

Total No. children %

Shahidi and Diamond8 (1961)

40

40

2/40 (5%)

2/40 (5%)

Scott, Cartwright, and Wintrobe7 (1956)

39

9

14/39 (36%)

O/9 (0%)

334

81

11/334 (3.3%)

Not specified

Dreyfusa (1959)

81

18

3/81 (3.7%)

Not specified

Gasser1~ (1961)

20

20

2/20 (lO%)

2/20 (10%)

Wolffs (1959)

marizes the experience of several large series of cases previously reported. Many of these patients received blood transfusions, corticosteroids, splenectomy, and other supportive measures. None received testosterone. Although recovery may occur in as many as 36 per cent of the adult patients, recovery has occurred in less than 10 per cent of children. Thus, while I'4 of the 39 patients reported by Scott, Cartwright, and Wintrobe 7 were improved, there were no survivors in the 9 children whose onset of disease was before 16 years of age. The use of A C T H and corticosteroids in these patients has not been encouraging. Wolff 8 noted no recoveries in the 51 patients treated with A C T H and only 1 remission in the 82 patients treated with cortisone. Similarly, Scott 7 found that only 2 of their 24 patients treated with cortisone showed hematologic improvement. Splenectomy has been of occasional benefit in certain selected patients. T However, results with this procedure have been extremely poor in patients with moderate-to-severe marrow hypocellularity, s In 1942, McCullagh and Jones 11 noted that the anemia present in the hypogonadat male responded to testosterone and suggested that androgens should be considered in the treatment of other anemias not associated with hypogonadism. Kennedy and Gilbertsen 12 in 1957 reported a significant rise in the hemoglobin concentration of women with carcinoma of the breast when treated with

large doses of testosterone. This relationship was also noted in animal studies TM 14 which indicated that androgens had significant erythropoietic activity. Following these and other clinical observations, 1~ Shahidi and Diamond 1G reported the encouraging results of the combined use of testosterone and corticosteroid therapy in a preliminary study of 5 children. Recently, this experienc& was expanded to a total of 24 children with acquired and constitutional types of aplastic anemia. In 9 of 17 patients with acquired disease, a sustained remission had been noted following cessation of therapy. A similar response was also described in 6 of 7 patients with constitutional aplastic anemia, although dependency on hormonal therapy was required in order to maintain these remissions. It is the purpose of this report to present studies concerning 13 children with aplastic anemia treated with cortisone and testosterone and observed at University Hospital since 1959. CLINICAL MATERIAL Since 1959, nineteen children with aplastic anemia have been observed at the University Hospital. Four children died before therapy could be instituted. T w o additional children exhibited only mild pancytopenia and are being observed without treatment. Thus, the 13 remaining patients received combined cortisone and testosterone therapy and comprise the clinical material for this report.

Volume 64 Number 5

Bone marrow failure

For purposes of classification, the clinical material was divided into acquired and constitutional disease depending upon the presence or absence of a positive family history and associated congenital anomalies. Depending upon whether a history of ingestion or exposure to known bone marrow depressants could be obtained, the acquired group was further subdivided into a toxic or idiopathic group. Acquired aplastie anemia. Nine of the thirteen children were thought to be suffering from an acquired form of aplastic anemia. The pretreatment data of these patients are summarized in Table II. In 4 children, a significant history of exposure to known hematopoietic depressants was obtained. T w o children received repeated courses of chloramphenicol for the treatment of minor respiratory infections 6 to 8 weeks prior to the onset of symptoms. A third child experienced repeated exposure to vapor from a gun cleaning solvent used by her grandfather in an enclosed shed where he stored and employed several types of gun cleaning solvents. The vapor from these materials was so strong as to be practically intolerable to the rest of the family, and indeed only the child and her grandfather were able to stay in this shed for any length of time. The fourth child received a prolonged exposure to a fly spray insecticide which she used in liberal amounts to protect a prize 4-H calf from flies during the summer months. The remaining 5 patients were termed Table II. Acquired

I

"idiopathic" since an etiologic agent could not be found. The possibility that some of these patients suffered bone marrow depression from an unknown toxic source certainly exists. While it is theoretically possible that some of the cases of idiopathic etiology represented instances of sporadic, genetically influenced disease, this possibility would seem remote in the absence of family history, associated anomalies, and normal growth. It is also possible that certain patients in the idiopathic group may be manifesting the aplastic phase of acute leukemia. No evidence of leukemia has been encountered in any of these patients during periods of study ranging from 12 to 36 months. T h e age at onset of the disease was recorded as the date of the earliest documented evidence of anemia, leukopenia, or thrombocytopenia. This ranged from 18~2 years to 141,/2 years. Seven children were female and two were male. In only 1 of the nine patients could a history of allergic manifestations be elicited (penicillin allergy) although a strong family history of allergy (eczema and asthma) was obtained in 3 of the parents. The presenting symptoms in these patients were similar; refractory anemia and the combination of easy bruiseability and anemia were the alerting symptoms in 7 patients. One child presented with excessive and severe menstrual bleeding while another had recurrent pneumonitis and chronic upper respiratory infections. Six of the nine patients received either iron, crude liver extracts,

aplastic a n e m i a - - p r e t r e a t m e n t data

G.J. L.P. S.K. T.B. D.L. R.F.

Age at Presumptive Hgb Sex onset etiology (Gin. %) F 4 1/12 Gun cleaning 7.0 solvent M 7 2/12 Chloramphenicol 4.5 F 10 5/12 Chloramphenicol 4.3 F 8 2/12 Insecticide 4.0 F 1 8/12 Idiopathic 4.0 F 3 5/12 Idiopathic 8.8 M 6 2/12 Idiopathic 6.0

P.D. C.F.

F F

Patient S.O.

68 5

4 0/12 Idiopathic 14 1/12 Idiopathic

9.0 8.7

Platelets Bone marrow WBG granulocytes Total hypo(lOS/cu. (lOS/cu. (lOS/cu. ram.) cellularity (%) ram.) mm.) 34.0 Moderate 0.3 4.4 0.4

Retlc

0.1 1.0 0.4 0.1 1.0 0.2

4.4 3.1 2.0 6.0 4.0 2.4

0.4 1.0 0.5 1.5 0.7 0.7

15.0 5.0 5.0 125.0 31.0 10.0

0.3 0.2

5.3 2.1

0.9 1.0

35.0 22.0

Moderate Moderate Severe Mild Moderate Moderate "-~ Severe Moderate Severe

686

Desposito et al.

multivitamins, folic acid, or vitamin BI.~ prior to diagnosis. Five patients received corticosteroids alone for periods of 2 weeks to 3 months without response. All children received blood transfusions prior to cortisonetestosterone therapy and as required to maintain adequate" hemoglobin levels after the start of treatment. T h e criteria used for the diagnosis of acquired aplastic anemia were peripheral pancytopenia, bone marrow hypocellularity, and absence of hepatosplenomegaly, lymphadenopathy, or previous unusual hematologic disorders. These findings were present without exception in all of the patients. The anemia was normocytic or slightly macrocytic in all cases; the mean corpuscular volume ranged from 79 to 125 /~3 and the mean corpuscular hemoglobin concentration ranged from 34 to 39.5 per cent. The initial reticulocyte counts were low in all instances. Initial reticulocyte counts of 1.0 per cent were noted in 2 children (D. L. and L. P.), the remainder being less than 0.4 per cent. The 1.0 per cent reticulocyte counts were also thought to represent inadequate bone marrow response in view of the marked anemia that existed. Total leukocyte counts ranged from 2,000 to 6,000 per cubic millimeter with total granulocytes ranging from 400 to 1,500 per cubic millimeter. The platelet count was below 35,000 per cubic millimeter in 8 of the 9 patients and 125,000 per cubic millimeter in the other child. Bone marrow specimens were obtained by both aspiration and surgical biopsy in all the children. All specimens revealed various gradations of hypocellularity and were classified as mildly, moderately, or severely hypocellular. The designation of marked bone marrow hypocellularity was reserved for those patients in whom the marrow was essentially acellular and contained only islands of lymphocytes, monocytes, plasma cells, or reticulum cells. Moderate hypocellularity was recorded if only occasional islands of granulocyte and erythrocyte precursors were present but no megakaryocytes seen. In the mildly hypocellular group, rare megakaryocytes were also noted. Of the nine children with

May 1964

acquired aplastic anemia, mild hypocellularity was noted in 1 patient, moderate hypocellularity in 5, and severe hypocellularity in 3 patients. One of the children with severe idiopathic disease (R. F.) demonstrated occasional granulocyte and erythrocyte precursors in the marrow at the onset of his symptoms. Subsequent bone marrow aspirations revealed decreasing concentrations of these cells and at autopsy, the bone marrow was noted to be completely acellular. The children were seen in the clinic at 4 to 6 week intervals. Blood transfusions of either packed red blood cells or fresh whole blood were given as required to maintain the hemoglobin concentration above 7 Gm. per 100 rnl. Antibiotics were given only upon specific indication for the treatment of bacterial infections. Prophylactic antibiotics were not employed. All the patients received combined testosterone and cortisone therapy. Testosterone was usually administered as methyl testosterone linguets 1 or 2 mg. per kilogram per day in 3 divided doses sublingually or as the long-acting testosterone enanthate in sesame oil or testosterone cyclopentyl propionate in the dose of 50 to 200 nag. by deep intramuscular injection at 3 week intervals. Prednisone or triamcinolone in dosages of approximately 1 rag. per kilogram per day in 3 divided doses were the usual corticosteroids employed. If no response was noted after 4 to 6 months of the original therapeutic course, alternate analogues of these drugs were used. For example, Patient S. K. received methyl testosterone linguets, oral methyl testosterone, testosterone enanthate, testosterone cyclopentyl propionate, prednisone, prednisolone, triamcinolone, and hydrocortisone at various intervals during the course of her disease. The length of therapy was determined by the child's clinical course and ranged from 1 to 19 months. With the exception of 2 children who died after 1 and 5 months of therapy, the surviving patients were all treated for at least 7 months. Therapy was discontinued when the hemoglobin concentration was above 11 Gm. per 100 rnl. and the total

Volume 64 Number 5

Bone marrow failure

granulocyte count was above 1,800 cells per cubic millimeter. Platelet responses were more varied and a level of at least 40,000 to 50,000 per cubic millimeter in the absence of clinical hemorrhagic manifestations was required before therapy was terminated. Constitutional aplastic anemia. Four of the thirteen children exhibited congenital malformations most frequently reported 1 to be associated with constitutional aplastic anemia. Table III presents a summary of their pretreatment findings. Patient L. E. presented the classical clinical picture of Fanconi syndrome 17 and demonstrated the following anomalies: hyperpigmentation, short stature, mental retardation, absent left thumb, hypoplastic right thumb and thenar eminence, absent right kidney, microcephaly, and strabismus. In addition, a striking incidence of congenital anomalies has appeared in this family. A 5-year-old sister and a 2 ~ year-old brother manifest similar thumb anomalies associated with hematopoietic deficits. Other hematopoietic and congenital anomalies have occurred in this family and will be described fully subsequently. The remaining 3 children all demonstrated hyperpigmentation, primarily about the genitals, nipples, or upper portion of the trunk. Two of these children were of short stature and had heights and weights between the

68 7

third and tenth percentile for their chronologic ages. One child (B. M.) had congenital dysplasia of the hip. Family history revealed that a sibling died in the neonatal period with multiple congenital anomalies which ineluded skeletal defects. Patient R. MeK., in addition to pigment changes, manifested steatorrhea for the first several years of life. A marked reduction in duodenal enzymes was found and he was presumed to have cystic fibrosis of the pancreas. However, recent values for sweat electrolytes were normal and the steatorrhea has completely cleared. This patient would appear to be similar to those cases of pancreatic insufficiency and bone marrow dysfunction recently described by Shwachman and colleagues? 8 With the exception of Patient L. E., no family history of hematologic abnormalities could be obtained in the other children. The age at onset ranged from 5 ~ to 9 ~ years although all patients except L. E. had suggestive histories of hematologic deficits dating 6 to 15 months prior to diagnosis. While all four children manifested mild degrees of easy bruiseability, the presenting symptom was refractory anemia. All received iron and multivitamins prior to their referral to University Hospital. Patients R. MeK. and K. P. received 2 to 4 months of cortieosteroids without hematologic response.

Table IlL Constitutional aplastic anemla--pretreatment data

Age at onset

Associated anomalies

WBC Hgb Retic. (lOS/cu. (Gin. %) (%) ram.)

Total granulocytes (lOS/cu. me.)

Platelets (lOS/cu. mm.)

Bone marrow hypocellularity

Patient

Sex

R. McK.

M

5 9/12 Hyperpigmentation Steatorrhea

10.8

1.4

3.2

0.7

62.0

Mild

K.P.

F

5 6/12 Hyperpigmentation Short stature

9.4

2.3

5.1

1.9

12.0

Mild

B.M.

M

8 7/12 Hyperpigmentation Short stature Congenital dysplasia hip

4.8

1.7

2.6

0.5

25.0

Mild

L.E.

F

9 8/12 Absent thumbs Hyperpigmentation Short stature Microeephaly Strabismus

4.2

0.5

2.2

0.5

14.0

Moderate

688

Desposito et al.

May 1964

Table IV. Pre- and posttreatment laboratory findings

Patient S.O.

Serum iron (l~g %) Pre ] Post 250

G.J.

305

L.P.

244

S.K. T.B.

231

D.L. R.F. P.D.

143

186 188 228 182

Serum iron binding (tLg %) Pre I Post 50 50 50 324 50 120 80 285 106

Serum copper (t*g %) Pre I Post 300 110 98 151 173 215 177

142

93 94 81 229

80 207 105

C.F.

R. McK. K.P. B.M.

182 149 155

206 200 253

L.E.

189

*

150 120 375 319

150 357 396

~Died. In contrast to those children with acquired disease, the marrow findings appeared somewhat more cellular in this group. Three of the four children had mild bone marrow hypocellularity by our criteria. Striking megaloblastoid changes were also noted in these patients. Patient L. E. with the most extensive congenital anomalies demonstrated moderate hypocellularity on an aspirated specimen which was confirmed by bone marrow section taken at the time of autopsy. Follow-up examinations and drug dosages were similar to those employed in the acquired group. The patients were treated from 4 to 14 months and the criteria for cessation of therapy was similar to that previously described. Relapse in the patients was characterized by renewal of symptoms of pallor, weakness, recurrent infections, and easy bruiseability with a concomitant fall in hemoglobin concentration to less than 8 Gm. per cent and total granulocyte count to less than 1,500 per cubic millimeter. Platelet reductions were also noted but were more variable. If relapse occurred, cortisone and testosterone therapy were begun at a lower dosage level. Since long-term maintenance therapy would appear to be necessary for the control of these patients, 6 an attempt was made to reduce the occurrence of side effects by employing a lower dosage regimen. Laboratory methods. Peripheral blood counts were done by standard hematologic

methods? 8 Normal platelet counts by the method used range from 200,000 to 300,000 per cubic millimeter. Serum iron and total iron binding capacity determinations were performed by the methods described by Ramsay? ~ 21 Normal values in our laboratory range from 75 to 150 mg. per 100 ml. and 300 to 400 mg. per 100 ml., respectively. Serum copper determinations were done by utilizing the method of Gubler and co-workers. 22 Normal serum copper values range from 90 to 150 mg. per 100 ml. Free erythrocyte protoporphyrin was performed by the method described by Grinstein and Wintrobe53 The normal value is less than 75 mg. per 100 ml. of packed erythrocytes. Studies of detoxification mechanisms of several of these patients have previously been reported from this laboratory by Wagner and Smith. 24 Fetal hemoglobin determinations were performed by the method of Singer, Chernoff, and Singer. 25 Endotoxin stimulation tests with the use of a Salmonella endotoxin (Lipexal) were performed to determine granulocyte reserve. A description of this test and results in infants and children have been previously reported from this laboratory by Thatcher and Smith. 26 A summary of the pre- and post-treatment laboratory data is presented in Table IV.

Volume 64 Number 5

Free erythrocyte protoporphyrin (#g/lO0 ml. RBC) P re I Post

B o n e m a r r o w failure

Fetal Hgb ( % ) P re [ Post

68 9

Endotoxin stimulation P re I Post

64 4.8

Abnormal Abnormal

Normal

11.4 18.5 16.3

Abnormal Abnormal Abnormal Abnormal

Normal

5.8 59 43 53

3.8 4.5

87

84 38 85

240 RESPONSE

5.3 TO

THERAPY

Acquired group. Five of the nine children in this group demonstrated marked hematologic improvement and have sustained these remissions without therapy for 12 to 36 months. The remaining four patients died 1 to 19 months after the onset of therapy and failed to exhibit any evidence of improvement. A summary of their present clinical status is outlined in Table V. Three of the four patients with toxic etiologies and two of the five patients with unknown etiologies remain clinically asymptomatic. Transfusions or other supportive measures have not been required. Hemoglobin and total granulocyte concentrations have remained within normal limits without therapy. While improvements in the platelet counts have been noted, none of the five responding children have platelet counts above 200,000 per cubic millimeter. Mild peripheral thrombocytopenia has persisted for at least 30 months in four of the five improved patients and has ranged from 50,000 to 166,000 per cubic millimeter. These children have not demonstrated any hemorrhagic manifestations. The first indication of hematologic response was noted in the reticulocyte count which ranged from 4.1 to 7.0 per cent. Reticulocyte responses were evident 1 to 6 months after the onset of therapy. This was followed in 1 to 3 months by a rise in the hemoglobin concentration to above 11.0 Gm.

per cent. Total granulocyte counts were noted to be above 2,000 per cubic millimeter 1 to 6 months after the start of therapy and coincided in most instances with a return of the hemoglobin concentration to normal limits. Sustained platelet responses were not observed before 9 months of therapy and significant rises in platelet number were first noted after 9 to 17 months. The length of treatment varied from 7 to 11 months in those patients demonstrating hematologic improvement. Therapy was discontinued with the attainment of the aforementioned criteria. To date, no patient has required reinstitution of therapy because of hematologic relapse. These five patients have been without therapy for 12 to 36 months with 4 of the five patients being without medication for over 30 months. A transient fall in the hemoglobin level was observed after cessation of therapy in 4 of the five patients in whom adequate documentation could be obtained. The lowest hemoglobin concentration was noted 1 to 3 months following the cessation of therapy and decrements of 2.0 to 4.0 Gm. per 100 ml. were observed. The children were observed further without therapy and a gradual rise in hemoglobin concentration to above 11 Gm. per 100 ml. Occurred over the next 3 to 6 month period. Similar transient mild decreases in total granulocyte and platelet concentrations were also observed. The children

690

Desposito et al.

H q b . q. %

13

May 1964

L

4

5 RETIC. "7, MONTHS TRANSFUSIONS

0 2 II I I

TESTOSTERONE

103/ram 3

6

8

I0

12

14

18

24

METHYL TEST. LINGUETS 30mq.

i

CORTISONE PLATELETS

4

[PREDNISONE20mq.

5C~1~4 901-

T O T A L GRAN.

]

3 I

103/rn m 3

Fig. 1. Toxic aplastic anemia response to therapy.

14

G.J.

12 Hgb" q ' %

IC

8 e

RETIC. ~ MONTHS TRANSFUSION TESTOSTERONE CORTISONE PL^TELE,S

4 3 2 2

4

6

8

I0

12

14

16

1830 3 6

I

CYO_OPENTYLPRDPRIONATE50mq. q, 3WK5. PRIEDNL.~ONE 20mql. J -

-- -- l

Fig. 2. Toxic aplastic anemia response to therapy. remained clinically well during this transient period. Examples of clinical responses are presented in graphic form in Figs. 1 and 2. Constitutional aplastic anemia. All four patients in this group demonstrated rather prompt improvement following institution of combined cortisone and testosterone therapy. However, Patient L. E. died after 4 months of therapy following a generalized monilial septicemia although mild hematologic improvement had been maintained. Two children h a v e been asymptomatic although remaining mildly pancytopenic. Reticulocyte responses up to 4.6 per cent were first observed following 1 to 3 months of therapy. This was followed promptly by a spontaneous rise in the hemoglobin concentration to over i l . 0 Gin. per cent. Platelet counts above 30,000 per cubic millimeter were observed in the three surviving children 5 to 9 months after the onset of therapy although none of

these patients have had platelet counts above 80,000 per cubic millimeter. The length of therapy in the three surviving children ranged from 4 to 14 months with two of the children experiencing hematologic and clinical relapse with cessation of therapy. Thus, Patients R. McK. and B. M. have received an additional course of testosterone and cortisone treatment in lower dosage following relapse. Patient K. P. experienced a drop in hemoglobin level from 15.3 to 10.5 Gm. per 100 ml. 6 weeks after cessation of therapy. A mild fall in total granulocyte count to about 2,000 per cubic millimeter and a decrease in the platelet count from 60,000 to 30,000 per cubic millimeter occurred. Since her bone marrow at this time revealed normal cellularity and a normal granulocyte response to endotoxin stimulation occurred, she was observed further without therapy. During the next 3 months, there was gradual improvement in hemoglobin and platelet concentrations. The granulocytes also increased in the peripheral blood. This child has now been without therapy for 1 year and her hemoglobin has stabilized at I2.5 Gm. per cent. The platelet count has risen to 73,000 per cubic millimeter and she has remained symptom free. The other two surviving children in this group experienced marked decreases in their hemoglobin, granulocyte, and platelet concentrations when therapy was discontinued. Patient R. McK. experienced a fall in hemoglobin concentration from 14.3 to 7.7 Gin. per 100 ml. in the 15 month period following cessation of the first course of therapy. In addition, the. total granulocyte count feI1 below 1,000 per cubic millimeter and the platelets decreased to 12,000 per cubic millimeter. The child experienced several mild respiratory infections, mild bruising, and easy fatigability. At this point he received a second course of cortisone and testosterone in lower dosage than previously. Testosterone was given as the enanthate in sesame oil, 15 mg. every 3 weeks, and prednisone was administered as a 5 mg. oral daily dose. One month following reinstitution of therapy, a reticulocyte count of 1.5 per cent was noted

Volume 64 Number 5

and the hemoglobin concentration rose spontaneously to 12.0 Gm. per cent. Total granulocytes have remained below 1,000 per cubic millimeter although the platelet count has risen to 50,000 per cubic millimeter. He has been maintained with this therapy for the past 14 months and has remained clinically asymptomatic. After experiencing marked clinical and hematologic improvement during a 9 month course of therapy, Patient B. M. promptly relapsed 1 month after cessation of treatment. His hemoglobin fell rapidly from 13.0 to 8.0 Gm. per 100 ml. over the following 4 week period. The granulocyte concentration fell below 1,000 per cubic millimeter and the platelet count decreased from 61,000 to 11,000 per cubic millimeter. In addition, he developed a moderately severe upper respiratory infection, felt extremely weak, and exhibited epistaxis and numerous ecchymoses. Ten milligrams daily of prednisone and testosterone enanthate, 15 mg. intramuscularly every 3 weeks was readministered. Four weeks later, a reticulocyte count of 9.9 per cent was observed with a spontaneous 3 Gm. per 100 ml. rise in the hemoglobin concentration. The child has been maintained with therapy for the past 11 months with the granulocyte concentration approaching normal (1,600 per cubic millimeter) although the platelet count has risen no higher than 30,000 per cubic millimeter. However, he has remained active and asymptomatic during this time. Graphic descriptions of the courses of two of these children are outlined in Figs. 3 and 4. Side effects of therapy. Evidence of virilization and excess corticosteroid effect were noted in all patients. These side effects included cushingoid facies, increased appetite, abdominal distention, moderate-to-marked weight gain, hirsutism, and unusual hair distribution, hyperpigmentation, deep voice, dry skin, acne, and enlargement of the phallus. All of these findings were observed in varying degrees in all thirteen treated patients. Transient hypertension (blood pressure 150/88) was observed in one patient (G. J.) after 7 months of treatment. His blood pressure returned to normal limits shortly after cessation

Bone m a r r o w [ailure

69 1

of therapy. Moderate vertebral osteoporosis and epigastric discomfort were noted in Patient S. K. who was treated for 19 months in full dosage because of severe pancytopenia. Roentgenographic studies failed to reveal a peptic ulcer although marked improvement and disappearance of symptoms occurred following cessation of therapy. This was the only child in whom severe side effects necessitated the stopping of these agents. Of the five patients who died during the course of therapy, the terminal episode in one child was thought in part to be attributable to therapy. This girl (Patient L. E.) with many anomalies associated with moderate bone marrow hypocellularity experienced moderate improvement of her pancytopenia in the 4 months that she received combined therapy. However, she died after a brief R.McK. .

.

.

.

RETIC. % 2 /

i

I

I

I

i

i

i

i

i

i

i

i

i

MONTHS 2 4 6 8 12 16 19 22 28 34 TRANSFUSIONS -~ ENANTHATE TESTOSTERONE CYCLOPEWTYL PROPRIONATEIOOmqR.3W•$ 15m~.q.3WI~S [ I L J CORTISONE IF~E:DNI$ONEj ZOmq" i5mR" PLATELETS @ 103/mm3 50

i

~

I

Fig. 3. Constitutional aplastic anemia response to therapy.

RETIC. ~, ~

t i

MONTHS 0 2 4 6 8 I0 12 14 16 TRANSFUSIONS TESTOSTERONE iT"ENANTHATE~Omq'q'3W~S , 15re~J'q3WI<~ " CORTISONE gO,' PR.E,C ~r....__..___: ~ISONE ] Ioa~

18 21 =

3

,o3.m.o

,

....

,

:, 7.

Fig. 4. Constitutional aplastic anemia response to therapy.

692

Desposito et al.

May 1964

T a b l e V. Acquired aplastic a n e m i a - - p o s t t r e a t m e n t status

Patient

Duration of therapy Hgb (months) (Gm. %)

Retic. (%)

Total granuloWBC Platelets cytes (t03/cu. (103/cu. (lOS/cu. mm,) mm.) ram.)

Present status

Duration of therapy (months)

S.O.

11

13.9

1.2

13,0

4.4

151.0

Asymptomatic

G.J.

7

12.0

0.3

7,1

4.6

50.0

Asymptomatic

34

L.P.

10

11.3

0.3

7.1

4.0

100.0

Asymptomatic

12

S.K.

19

T.B.

9

12,8

1.2

7.2

4.2

I66.0

Asymptomatie

36

D.L.

11

11.5

1.3

8.5

3.6

100.0

Asymptomatic

36

R.F.

8

No response

Died at age of 7~2 Cerebral hemorrhage and sepsis

P.D.

5

No response

Died at age of 4~2 Cerebral hemorrhage

C.F.

1

No response

Died at age of 14~2 Cerebral hemorrhage

No response

36

Died at age of 10 Cerebral hemorrhage

T a b l e VI. C o n s t i t u t i o n a l aplastic a n e m i a - - t r e a t m e n t status

Patient

Duration Interval Duration of before 2nd therapy relapse course (months) (months) (months)

Hgb (Gin. %)

Retic. (%)

Total granuloWBC cytes Platelets Duration (lOS/cu. (lOS/cu, (103/cu. Clinical off mm.) ram.) ram.) status therapy

R. MeK.

4

15

14

12.4

1.5

5.1

0.5

50.0

Asympto- On mainmarie tenance therapy

K.P.

9

--

--

12.7

1.1

8.7

4.0

73.0

Asympto- 12 mos. matic

B. M,

9

11

12.6

1.7

3.5

1.6

30.0

Asympto- On mainmatic tenance

1

therapy

L.E.

4

--

--

11.8

p n e u m o n i t i c a n d septic illness a n d was f o u n d to have generalized moniliasis. Postmortem cultures yielded Candida albicans from the lungs, liver, a n d spleen. I n addition, both adrenal glands were observed to be markedly atrophied. W i t h the exception of the patient just de-

0.3

5.2

4.6

36.0

Expired age 10~ Sepsis

--

scribed, no serious infections, peptic ulcers, hepatic toxicity, glycosuria, or central nervous system symptoms were observed d u r i n g the t r e a t m e n t period. G r o w t h performances were carefully noted d u r i n g the course of thera W. M a r k e d weight gain a n d Cushing-type obesity were observed

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with varying severity in all children. Weight gains of 15 to 40 pounds were noted. Of interest were the height performances of these children on therapy. G r o w t h performances in all patients continued along the same percentile as their pretreatment measurements. Those children who were noted to be shortstatured before treatment continued to grow at a similar rate during treatment and their posttreatment measurements were in the predicted percentiles. No sudden growth spurts or cessation of growth was observed. In 4 patients, pretreatment and 6 month posttreatment bone age roentgenograms failed to reveal any increase in bony maturation over and above that normally expected for their increase in age. DISCUSSION

The present clinical experience would support the findings of Shahidi and Diamond. 6 In those children suffering from acquired toxic aplastic anemia, approximately 50 per cent would appear to obtain significant improvement with the use of corticosteroids and testosterone. Four of the five responding children have remained asymptomatic without therapy for 3 years. In light of the high mortality reported in previous studies (3 to 10 per cent survival), it would appear that the use of cortisone and testosterone may produce a favorable hematologic response in m a n y instances. The exact hematopoietic effect of testosterone is not known. At this time, its effect must be regarded as nonspecific. In contrast to vitamin B12 or iron therapy reticulocytosis appears 1 to 5 months after testosterone administration. In addition, the occurrence of polycythemia reported in patients with adrenogenital tumors 2~ and carcinoma of the breast treated with large doses of testosterone 2s would suggest that androgens can stimulate erythropoiesis beyond the usual physiologic limits. This would again be in contrast to the action of basic nutrients such as vitamin B12 and iron. Indeed, two patients described here had hemoglobin concentrations of over 15.0 Gm. per 100 ml. at the height of their response to therapy. With-

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drawal of therapy produced a transient decrease in the peripheral blood values in all patients. In several instances, the hemoglobin and total granulocyte concentrations fell below normal limits; however, in the acquired group, gradual return of these values to normal occurred within 3 months and did not necessitate re-institution of therapy. While the clinical experience reported here is small, the results would appear to imply that the fastest responses occurred in those children whose pretreatment bone marrows revealed occasional erythrocyte and granulocyte precursors. O f the three children classified as having severe bone marrow hypocellularity on the basis of virtual absence of erythroblasts, granulocyte precursors, and megakaryocytes by aspiration and surgical bone marrow biopsy, none revealed any evidence of improvement and all died. All children classified as having mild bone marrow hypocellularity demonstrated hematologic improvement. In the majority of instances (5 of the nine acquired group), the surgical bone marrow was classified as showing moderate hypocellularity. Four of these five children responded and have become asymptomatic. In general, it may be possible to predict the response in these patients on the basis of bone marrow hypocellularity. In contrast to those children with acquired disease, the patients with congenital aplastic anemia demonstrated a more uniform initial response. However, hematologic relapse following cessation of therapy was noted in 2 of the 3 children and required re-institution of treatment. Second responses occurred with the use of much lower dosages of both drugs. Interestingly, the second responses were again noticeable within 1 month after the re-institution of therapy even though minimal drug dosages were employed. The reasons for dependence on continuing treatment in this group remain unclear, but treatment dependence may be helpful retrospectively in diagnosis. In addition, the prognosis in the genetically controlled group would appear to be more guarded since the hematologic response is not permanent and in the few cases described, the platelet counts revealed only

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minimal improvement. Although the bone marrow was classified as showing only a mild degree of hypocellularity in the three surviving children with constitutional aplastic anemia, the hematologic improvement would appear to be less remarkable than in a similar group of children with acquired disease. The side effects observed during the course of therapy with combined corticosteroid and testosterone therapy were, in general, not severe enough to warrant interruption of treatment. As expected, marked weight gains, hirsutism, acne, and virilization were common. Growth performances, however, remained normal throughout the course of therapy and growth spurts, epiphyseal maturation, and increased bone age were carefully looked for but not observed. This might suggest that the antagonistic effect of cortisone on bony maturation counteracted the androgenic effect on this organ by testosterone. It would appear preferable to administer both steroids orally since intramuscular injections may produce painful hematomas in these children. Of the agents employed, the choice of one analogue over another would not appear to enhance the response as several patients receiving intramuscular testosterone preparations achieved similar responses. In addition, the use of higher dosages of both compounds in nonresponsive patients did not produce hematologic improvement and only increased the side effects. The optimal period of treatment would not appear to be specific. Hematologic improvement occurred within 6 months in the responsive patients. Two nonresponding children were treated for 8 and 19 months and died while receiving therapy. From these and other observations, 6 it would appear that nonresponding patients should be treated for at least 12 months although the remission rate would appear to fall sharply after 6 to 8 months of therapy. However, experience to date is too limited to permit a confident decision as to when treatment can be terminated in any nonresponsive patient. The results of our laboratory data in these patients have been of interest. Increased fetal hemoglobin was found in the eight pa-

May 1964

tients tested and remained slightly elevated even though hemoglobin concentrations returned to normal levels. The finding of elevated fetal hemoglobin in aplastic anemia has been reported by Shahidi, Gerald, and Diamond. 29 They have also reported higher levels of fetal hemoglobin in the constitutional group as contrasted to the acquired group. This finding would appear to be a secondary result of disturbed erythropoiesis and its significance is not clearly understood. The serum iron levels were noted to be elevated in all children and were associated with a marked decrease in the total serum iron binding capacity. Where adequate data were available in the patients demonstrating hematologic improvement, a rise to normal levels of the total iron binding capacity was observed although the serum iron values often remained above normal limits. The elevation of serum iron may be an early manifestation of failing erythropoiesis, 3~ and this determination may be helpful where known bone marrow depressant drugs must be used over a long period of time. In contrast to the recent report of Shahidi, 31 pretreatment elevation of free erythrocyte protoporphyrin was found in two children with constitutional disease while this value was normal in the acquired group. Shahidi has also noted a significant degree of hypochromia and elevation of the free erythrocyte protoporphyrin during the height of testosterone therapy. SUMMARY

Thirteen patients with pancytopenia and hypocellular bone marrows were treated with cortisone and testosterone. Five of the nine patients with acquired aplastic anemia demonstrated hematologic improvement within one to six months after the onset of therapy. Hemoglobin and granulocyte concentrations have remained normal without supportive measures twelve to thirty-six months after cessation of therapy. A moderate degree of thrombocytopenia has persisted in all patients. Four patients with congenital aplastic anemia were treated with cortisone and

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testosterone for four to fourteen months. All d e m o n s t r a t e d evidence of active erythropoiesis within 1 m o n t h of the onset of t h e r a p y and n o r m a l hemoglobin a n d granulocyte concentrations were o b t a i n e d within 3 months. A l t h o u g h d e m o n s t r a t i n g hematologic improvement, 1 child died after 4 months of t h e r a p y from a monilial septicemia. I n contrast to the g r o u p of children with acquired aplastic anemia, m a r k e d decrease in hemoglobin, granulocyte, a n d platelet concentrations occurred following cessation of t h e r a p y in 2 of the 3 surviving children. This relapse required re-institution a n d m a i n t e n a n c e of cortisone a n d testosterone treatment. W i t h the use of lower dosages of both drugs, it has been possible to m a i n t a i n second hematologic responses. I t would a p p e a r that hematologic response m a y be correlated with the severity of bone m a r r o w hypocellularity in the acquired g r o u p since 3 of the 4 failures occurred in children with severe depression of the marrow. Evidence of hypercorticism and virilism occurred with varying severity in all patients, although in only 1 child was this severe enough to w a r r a n t cessation of therapy. Growth performances r e m a i n e d n o r m a I t h r o u g h o u t the course of therapy. W h i l e the responses in the constitutional group w o u l d a p p e a r to occur sooner t h a n in the acquired group, the necessity of m a i n tenance therapy a n d the failure of m a r k e d i m p r o v e m e n t in the granulocyte a n d t h r o m bocyte counts would suggest a more g u a r d e d prognosis in this group. T h e experience in the acquired g r o u p would suggest t h a t remissions m a y be p e r m a n e n t in a p p r o x i m a t e l y 50 per cent of the cases. Appreciation is expressed to Dr. Anthony Pisciotta for the follow-up clinical and laboratory data for his patient, D. L. The technical assistance of Mrs. Mary Joan Kumke and Mrs. Julianne Setzkom in the performance of the various laboratory procedures is gratefully acknowledged. REFERENCES

1. Dawson, J. P.: Congenital pancytopenia associated with multiple congenital anomalies

Bone m a r r o w failure 6 9 5

(Fanconi Type), Pediatrics 15: 325, 1955. 2. Estren, S., and Dameshak, W.: Familial hypoplastic anemia of childhood: Report of eight cases in two families with beneficial effect of splenectomy in one case, Am. J. Dis. Child. 73: 671, 1947. 3. Huguley, C. M., Jr., Erslev, A. J., and Bersagel, D. E.: Council on drugs. Drug related blood dyscrasias, J. A. M. A. 177: 23, 1961. 4. Wintrobe, M. M.: Clinical hematology, ed. 5, Philadelphia, 1961, Lea & Febiger. 5. Clement, D. H.: Aplastic anemia, Pediat. Clin. North America 9: 703, 1962. 6. Shahidi, N. T., and Diamond, L. K.: Testosterone-induced remission in aplastic anemia of both acquired and congenital types: Further observation in 24 cases, New England J. Med. 264: 953, 1961. 7. Scott, J. L., Cartwright, G. E., and Wintrobe, M. M.: Acquired aplastic anemia: analysis of thirty-nine cases and review of pertinent literature, Medicine 38: 119, 1959. 8. Wolff, J. A.: Anemias caused by infection and toxins, idiopathic aplastic anemia and anemia caused by renal disease, Pediat. Clin. North America 4: 469, 1957. 9. Dreyfus, B.: A Propos de 81 observations de pancytopenics idiopathiques chroniques san splenom6galie, Rev. H~mat. 14: 62, 1959. I0. Gasser, C.: Panmyelopathien im Kindesalter Beitrag zur Fanconi-Anemie, Helvet. Paediat. acta 16: 752, 1961. 11. McCullagh, E. P., and Jones, T. R.: Effect of androgens on blood counts of men, J. Clin. Endocrinol. 2: 243, 1942. 12. Kennedy, B. J., and Gilbertsen, A. S.: Increased erythropoiesis induced by androgenic hormone therapy, New England J. Med. 256: 719, 1957. 13. Vollmer, E. P., and Gordon, A. S.: Effect of sex and gonadotrophic hormone upon the blood picture of the rat, Endocrinology 29: 828, 1941. 14. DeBias, D. A.: Effect of testosterone propiohate on the red cell count in ovariectomized and ovariectomized-thyroidectomized rat, Am. J. Physiol. 165: 476, 1951. 15. Gardner, F. H., and Pringle, J. C., Jr.: Androgens and erythropoiesis: I. Preliminary clinical observations, A. M. A. Arch. Int. Med. 107: 846, 1961. 16. Shahidi, N. T., and Diamond, L. K.: Testosterone-induced remission in aplastic anemia, A. M. A. J. Dis. Child. 98: 293, 1959. 17. Fanconi, G.: Familiare Infantile Perniziosartize Anamie (Pernizi6ses Blutbild und Konstitution), Jahrb. f. Kinderh. 117: 257, 1927. 18. Shwachman, H., Diamond, L. K., Oski, F. A., and Khaw, K. O.: Pancreatic insufficiency and bone marrow dysfunction: A new clinical entity. Paper presented at the American Pediatric Society Meeting, Atlantic City, May, 1963. 19. Cartwright, G. E.: Diagnostic Laboratory

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20. 21. 22.

23.

24. 25.

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Hematology. New York, 1958, Grune & Stratton Company. Ramsay, W. N . M.: Determination of iron in blood plasma or serum, Clin. Chem. Acta 2: 214, 1957. Ramsay, W. N. M.: Determination of total iron-binding capacity of serum, Clin. Chem. Acta 2: 221, 1957. Gubler, C. J., Lahey, M. E., Ashenbrucker, H., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism: I. A method for the determination of copper in whole blood. Red blood cells and plasma, J. Biol. Chem. 196: 209, 1952. Grinstein, M., and Wintrobe, M. M.: Spectrophotometric micromethod for the determination of the free erythrocyte protoporphyrin, J. Biol. Chem. 172: 459, 1948. Wagner, H. P., and Smith, N. J.: A study of detoxificafion mechanisms in children with aplastic anemia, Blood 19: 676, 1962. Singer, K., Chernoff, A. I., and Singer, L.: Studies of abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali de-

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naturation, Blood 6: 413, 1951. 26. Thatcher, L. G., and Smith, N. J.: Granulocyte responses to endotoxin in infants and children, J. P~DIAT. 62: 484, 1963. 27. Rathbun, J. C., Plunkett, E. R., and Barr, M. L.: Diagnosis and management of sex anomalies, Pediat. Clin. North America 5: 375, 1958. 28. Kennedy, B. J., and Nathanson, I. T.: Effects on intensive sex steroid hormone therapy in advanced breast cancer, J. A. M. A. 152: 1135, 1958. 29. Shahidi, N. T., Gerald, P. S., and Diamond, L. K.: Alkali-resistant hemoglobin in aplastic anemia of both acquired and congenital types, New England J. Med. 266: 117, 1962. 30. McElfresh, A. E., and Huang, N. N.: Bone marrow depression resulting from the administration of Methicillin, New England J. Med. 266: 246, 1962. 31. Shahldl, N. T. : Morphologic and biochemical characteristics of erythrocytes in testosteroneinduced remission in patients with acquired and constitutional aplastic anemia, J. Lab. & Clin. Med. 62: 294, 1963.