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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
Hepatitis C Virus Serum Markers and Liver Disease in Children With
Leukemia During and After Chemotherapy
By Anna Locasciulli, Daniela Cavalletto, Patrizia Pontisso, Luisa Cavalletto, Elena Scovena, Cornelio Uderzo,
Giuseppe Masera, and Alfredo Alberti
The pattern of hepatitis C virus (HCV) serum markers and
liver disease was investigated in 11 leukemic children
showing anti-HCV reactivity at least once during long-term
observation to define the role of HCV infection and the
behavior of HCV serologic markers in this patient cohort.
Antibodies to HCV by first- and second-generation enzyme-linked immunosorbent assay (ELISA)and by secondgeneration (four antigens) recombinant immunoblotting
assay (RIBA) and HCV-RNA by nested polymerase chain
reaction (PCR) were serially examined in serum. Liver disease was defined according to transaminase levels. Seven
of 11 patients were found HCV-RNA positive during chemotherapy and after blood transfusion, 3 of 11 became
viremic during follow-up, and 1 of 11 was always HCVRNA negative. Seroconversion to anti-HCV positivity by
second-generationELISA occurred in all the HCV-RNA positive children either during or after chemotherapy. Alanine
aminotransferase (ALT) levels were elevated in all the
HCV-RNA positive patients during antileukemictreatment
and normalized in seven of them after therapy withdrawal,
despite persisting viremia. These results indicate that
HCV-RNA testing by polymerase chain reaction is required
to correctly identify HCV infection in patients with leukemia while on chemotherapy. Viremia did not correlate with
ALT levels and anti-HCV patterns.
0 1993 by The American Society of Hematology.
B
ble with the one observed in Italian volunteer blood donors
(0.7% to 0.8%).*
All of the patients treated in our institution are entered in
prospective study of liver disease that is based mainly on
periodic clinical and biochemical assessment, including
tests for hepatitis virus markers during and after chemotherapy. We observed that some patients did develop antiHCV
positivity during follow-up, a finding that became particularly evident after the second generation enzyme-linked immunosorbent assay (ELISA) test was introduced for screening in May 1991.
Here we investigate the pattern of HCV serum markers,
including the anti-HCV reactivities as detected by the firstand second-generation ELISA and by the recombinant immunoblotting assay (RIBA) confirmatory test and serum
HCV-RNA measured by PCR, having selected for this
study those cases who showed serum anti-HCV reactivity at
least once during long-term observation.
The aim is to determine the contribution of HCV infection to biochemical evidence for liver disease in our patients
with leukemia and the behavior of HCV diagnostic markers
in this particular patient population.
IOCHEMICAL and clinical signs of liver disease occur
frequently during chemotherapy in patients with leukemia and correct recognition of etiologic factors is needed
to define the most rational treatment.’
Besides the toxic effect of drugs, parenterally acquired
viral hepatitis, mainly of the nonA, nonB type, plays a significant role in this clinical setting.*s3The ability to diagnose
the infection as caused by hepatitis C virus, the main bloodborn nonA, nonB agent,“ has dramatically improved in recent years. Indeed, first- and second-generation immunoassays have been introduced to test for the different antiviral
antibodies in serum and the polymerase chain reaction
(PCR) can also be used to measure viremia d i r e ~ t l y .Anti~.~
HCV screening by means of the first-generation test has
been available in our institution since January 1990, and
soon after its introduction all children receiving chemotherapy for leukemia were studied for serum anti-HCV reactivity. Although more than two thirds of these patients had
evidence of ongoing chronic liver disease, according to persistent alanine aminotransferase (ALT) elevation, only 1 of
1 14 patients (0.7%) had detectable anti-HCV reactivity in
serum when tested once during the on-therapy period,
usually at the time of significant abnormalities in liver enzyme levels (unpublished data). This figure was far below
the prevalence of anti-HCV positivity seen in our transfusion-dependent thalassemic children (76%),’being compara-
From the Division of Pediatric Hematology, University of Milano, Monza; and the Clinica Medica 11, University of Padova,
Italy.
Submitted February 8, 1993; accepted June 21, 1993.
Supported by Comitato “ML Verga”per lo studio e la cura delle
leucemie infantili.
Address reprint requests to Anna Locasciulli, MD, Ematologia
Pediatrica, Nuovo Ospedale “S. Gerardo, Via Donizetli, 106,
20052 Monza (Milano) Italy.
The publication costs of this article were defayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solelyto
indicate this fact.
0 1993 by The American Society ofHematology.
0006-49 71/93/S208-0007$3.00/0
”
2564
MATERIALS AND METHODS
Patients. Patients with leukemia treated at the Department of
Pediatric Hematology, S. Gerardo Hospital, Monza, are prospectively followed for liver disease since presentation. Antileukemic
treatment is performed according to the Italian Cooperative Protocols for Acute Lymphoblastic Leukemia (ALL)’ and Acute Myeloid Leukemia (AML).9 Briefly, therapy for ALL, consisting of
induction, consolidation, and maintenance phases, for a duration
of 2 years, includesthe following drugs: vincristine (VCR), daunorubicin (DNR), L-asparaginase, prednisone (PDN), cyclophosphamide (CPM), cytosine arabinoside (Ara-C), 6-mercaptopurine (6MP), methotrexate (MTX) high (5,000 mg/m2) and standard (20
mg/m)’ dose; prophylaxis of central nervous system (CNS) involvement is performed with intrathecal (IT)MTX alone or in combination with Ara-C and PDN, with or without 1,800 rad cranial irradiation. Polychemotherapy for AML is based on anthracyclines,
Ara-C standard (200 mg/m2)and high (3,000 mg/m2) dose, etoposide, CPM, and 6-thioguanine for induction and consolidation
phases lasting 4 months. CNS prophylaxis is performed with Ara-C
IT. In the absence of an HLA-identical donor, patients are subseBlood. Vol 82,No 8 (October 15). 1993: pp 2564-2567
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2565
HCV INFECTION IN CHILDHOOD LEUKEMIA
quently randomized to receive autologous bone marrow (BM)
transplantation or maintenance therapy with 6-TH, Ara-C, and
anthracyclines for a duration of 2 years.
For this study, we selected patients diagnosed from 1988 to 1989,
as all of them had sera stored both at -20°C and -70°C and could
be observed after chemotherapy withdrawal.
Among 89 children with various forms of leukemia diagnosed in
our institution in this 2-year period, 57 (64%)completed chemotherapy treatment in first complete remission. Eleven of 57 (19.2%)
were found anti-HCV positive in serum at least at one occasion
during follow-up and were thereby included in this study.
There were six boys and five girls, with a mean age at diagnosis of
8.2 years (range: 2.2 to 12.9 years). Ten patients had ALL and one
AML, according to the French-American-British (FAB) classification."
All children were treated with antileukemic drugs for a median
period of 24 months (range 4 to 28 months). Median follow-up was
32 months (range: 28 to 5 1 months) from diagnosis and 10 months
(range: 4 to 27 months) from treatment withdrawal. All children
had been transfused and the average number oftransfusions was 15
per patient (range: 6 to 107). None received intravenous (IV) immune globulin, which may produce passive ELlSA and RIBA positivity, during the entire period of observation. None had a positive
history of liver disease or had received blood or blood products
before the onset of leukemia.
Assessment of liver disease. The I 1 children included in this
study were monitored after the diagnosis of leukemia for clinical
and biochemical evidence of liver disease, using the following parameters: serial testing of serum ALT, alkaline phosphatase (AP),
bilirubin, albumin, and prothrombin time. Serum samples were
regularly stored at -20°C and -70°C.
Assessment of hepatitis B (HBV and C (HCW serum
markers. HBV markers, including HBsAg, anti-HBs, and antiHBc, were tested in serum by commercially available radioimmunoassay (Abbott Laboratories, North Chicago, IL).
Antibodies to HCV (anti-HCV) were detected by first- and second-generation Ortho-ELISA tests (Ortho Diagnostic Systems,
Raritan, NJ) and the methods and evaluation of results were performed according to the manufacturer's instructions. To define the
specificity of the results obtained by ELISA, reactive sera were also
investigated by RIBA. For this purpose, sera were analyzed by second-generation (four antigens) RIBA (Chiron Corp, Emeryville,
CA and Ortho Diagnostic Systems), following the manufacturer's
instructions.
Table 1. Hepatitis C Virus Serum Markers and Transaminase
Profile in 11 Children With Acute Leukemia
Within 6 mo
From Diagnosis
No. of patients:
Anti-HCV positive by firstgeneration ELISA"
Anti-HCV positive by
second-generation
ELISA'
HCV-RNA positive by PCR
No. of patients with raised
ALTt
Meanlmedian ALT value$
A t The End of
Treatment
At Latest
Follow-Up
011 1
011 1
211 1
311 1
211 1
911 1
611 1
11/11
711 1
1111 1
10111
311 1
4041400
2121146
1371134
Confirmed by RIBA.
t ALT, serum alanine aminotransferase.
t Normal value < 40 IU/L.
Table 2. Serum HCV-RNA and Liver Disease in 10 Children
With Leukemia and Evidence of HCV Infection
During treatment:
Mean ALTO value
Type of liver disease
Resolved hepatitis
Chronic hepatitis
Outcome of Liver
disease when off
therapy:
Normalization s6 mo
Ongoing hepatitis
Group A'
(6 cases)
Group B t
(1 case)
Group CS
(3cases)
101.5 L -37
160
125 f 58
-
-
-
6
1
4
2
1
3
3
-
Difference among groups A, B and C were not significant.
Group A, early HCV-RNA detection (< = 2 months from presentation).
t Group B, late HCV-RNA detection (9 months from presentation).
t Group C, seroconversionfrom HCV-RNA- to HCV-RNA+.
§ ALT, serum alanine aminotransferase; normal value < 40 IU/L.
HCV-RNA was detected in serum using the nested PCR technique. Two sets of primers specific for the 5' untranslated region
(5' UTR) were used. External primers: 5'-GCCATGGCGTTAGTATGAGT-3' (sense) and 3'-TCCAGAGCATCTGGCACGTA-5'
(antisense). Inner primers: 3'-TATCCCACGAACGCTCACGG-5'
(antisense) and 5'-GTGCAGCCTCCAGGACCCC-3'(sense). Amplification products were visualized by ethidium bromide staining.
Two normal sera and water were used as negative control in each
experiment and a known positive reference sample was also included."
Blood components administered to the patients were obtained
from HBsAg-negative volunteer donors, who were also tested for
the presence of anti-HCV since January 1990.
This investigation was approved by local institutional human
research committees.
RESULTS
The results of serum HCV markers and of transaminases
during the observation period in the 1 1 patients with leukemia are summarized in Table 1. Six patients were found
HCV-RNA positive in serum early in the course of leukemia, usually I to 2 months after diagnosis and blood transfusion. None of them was positive for anti-HCV by firstgeneration ELISA while 50% (three cases) were positive by
second-generation ELISA. All six patients had evidence of
chronic hepatitis during chemotherapy with persistent (four
cases) or intermittent (two cases) elevation of ALT. Four of
them (Table 2, group A) normalized ALT after cessation of
chemotherapy, although viremia persisted, whereas two
maintained abnormal ALT up to the end of follow-up. Only
1 of the 6 children had jaundice during follow-up. At the
end of the chemotherapy period (Table l ) , seven patients
were HCV-RNA positive. All of them had raised ALT, as
the four additional cases who were HCV-RNA negative.
Anti-HCV was not found in any of the cases by first-generation ELISA whereas it was positive in two and negative in
five HCV-RNA-positive cases by second-generation
ELISA. However, all but one HCV-RNA positive patients
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2566
LOCASCIULLI ET AL
became anti-HCV positive after cessation of chemotherapy.
Overall, 10 of 11 patients (90.9%)were HCV-RNA positive
in serum at latest follow-up (Table 1).
The patient HCV-RNA negative had a unique false positivity for anti-HCV by second-generation ELISA not confirmed by RIBA.
The behavior of liver disease after chemotherapy withdrawal in the 10 HCV-RNA positive cases was as follows
(Table 2): seven patients showed normalization of ALT
within 6 months although with persistent viremia, whereas
three children continued to have elevated ALT levels.
Among the 7 children with normal biochemical profile at
6 months, 3 had experienced a sharp peak in ALT levels
shortly after cessation of treatment, followed by rapid and
complete normalization. In the other 4 cases, biochemical
improvement was concomitant with chemotherapy withdrawal. The relation between the outcome of liver disease
and anti-HCV reactivity by second generation assay was
analyzed after chemotherapy withdrawal (Table 3). Ofthe 3
patients with ongoing hepatitis off-therapy, 2 were persistently anti-HCV positive and t showed intermittent reactivity. Among the 7 cases showing ALT normalization, 5 resulted persistently positive whereas in the remaining 2
anti-HCV appeared only transiently.
HBsAg remained negative in serum during follow-up in
the 11 children, whereas anti-HBs or anti-HBC and antiHBs were transiently positive in 5 and 4 patients, respectively.
DISCUSSION
Signs of liver damage, mainly represented by elevation of
transaminase levels, are frequently detected in patients with
leukemia when on chemotherapy and, among the various
possible etiologic factors, an important role is attributed to
infection with hepatitis viruses, whose expression and replication is certainly favored by immunosuppression.',12We
have previously documented the role of hepatitis B
v i r ~ s , ' ~but
, ' ~many patients have chronic liver disease in the
absence of a positive hepatitis B serology. Therefore, we
have analyzed the possible role of hepatitis C virus using
available serologic assays for the detection of anti-HCV and
the PCR for the study of serum HCV-RNA. Screening of
patients sera by anti-HCV with second-generation assays
showed a group of patients who became antibody positive
during follow-up. These results were compared with those
obtained with first-generation ELISA and all patients were
also analyzed for HCV-RNA in serum by the sensitive PCR
to assess the prevalence and behavior of viremia. The results
obtained during and after the period of chemotherapy show
Table 3. Pattern of Liver Disease and Anti-HCV Profile in 10
Leukemic Children With Viremia After Therapy Withdrawal
Anti-HCV
Positivity:
ALT Normalization
(7 cases)
Ongoing Hepatitis
(3 cases)
Persistent
Transient
Intermittent
517
217
213
-
-
113
that first-generation assays are insensitivein detection of the
infection, either in the early phase or during further followup, as most HCV-RNA positive patients were negative by
ELISA for anti-C100-3 during the observation period.
Other studies have shown that immunosuppression has a
more profound influence on the ability of HCV-infected
patients to produce a n t i c 100 compared with production of
anti-C22I5and our findings are in agreement with the conclusion that anti-C 100 testing greatly underestimates HCV
infection. Second-generation ELISA was more sensitive in
this respect and several patients developed anti-C22 and/or
antLC33 either during chemotherapy or after its withdrawal. Overall, when on chemotherapy, 7 of 11 patients
were HCV-RNA positive: none of them were anti-HCV positive by first-generation assay and three were anti-HCV positive by second-generation assay, leaving four patients with
viremia but without detectable anti-HCV. All four patients
became anti-HCV positive when off therapy. These results
clearly confirm that HCV-RNA testing by PCR is required
to correctly identify HCV infection in patients under immunosuppression. However, it should be mentioned that there
are still limitations on the availability of HCV-RNA testing
and that the reliability of HCV-RNA detection by PCR
among laboratories has been reported to be poor.I6
Interestingly, serum HCV-RNA remained detectable
after chemotherapy withdrawal in all the patients, whereas
transaminases, which were elevated while on antileukemic
treatment, normalized in several of them during follow-up.
This would suggest that either HCV was not the main cause
of liver damage, which could have been related to drug toxicity, or that the cytopathic effect of the virus was potentiated
by immunosuppression, then being attenuated after restoration of immunocompetence. Another possibility is that the
association of antileukemic drugs and HCV infection could
lead to complementary liver-damaging processes, as is well
established in alcoholics with severe liver disease.",''
Efforts in this study to identify factors that might predict
progression of liver disease were unrewarding. Neither the
severityof the liver disease, defined by the mean ALT levels,
nor the pattern of anti-HCV reactivity during and after chemotherapy had a clear-cut profile. In agreement with other
st~dies,'~-'~
the observation that patients with biochemical
resolution remained positive for HCV-RNA could suggest
that persistent infection in the absence of liver inflammation may occur similarly to that observed in HBsAg camers.
On the other hand, the apparent absence of liver disease
among patients with viremia might be caused in part by the
strict criteria based on ALT levels we commonly use to define liver disease. Indeed, progressive liver disease has even
been documented in patients with normal ALT.'pL9
Interestingly, we observed no severe hepatitis reactivation
after chemotherapy withdrawal, despite the fact that all the
children remained viremic. Three of them showed symptomless ALT peaks shortly after cessation of chemotherapy,
followed by enzyme normalization within 6 months. This
observation is in agreement with previous studies performed in larger series of children with leukemia followed
0ff-thera~y.I~
Indeed, we have never observed a fulminant
hepatitis following immune reconstitution after chemother-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
HCV INFECTION IN CHILDHOOD LEUKEMIA
apy withdrawal also in children with hepatitis B,22in contrast to other experiences.23324
In regard to anti-HCV profiles, we had serial sera only in a proportion of patients,
because in leukemic children it is often very difficult to take
blood samples. However, in the cases with serial testing, we
observed that a persistent anti-HCV reactivity was present
not only in patients with ongoing hepatitis, but also in cases
with ALT normalization. This observation is in contrast
with a previous
in which we established a close
correlation between persistence of anti-HCV and progression of liver disease.
The need for prolonged follow-up of patients surviving
leukemia with chronic HCV infection is emphasized by the
fact that the natural history of HCV infection has not been
fully clarified and that additional late consequences are
likely.Ig Thus, our observation in this study cohort continues.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1993 82: 2564-2567
Hepatitis C virus serum markers and liver disease in children with
leukemia during and after chemotherapy
A Locasciulli, D Cavalletto, P Pontisso, L Cavalletto, E Scovena, C Uderzo, G Masera and A
Alberti
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