B C R R e a r r a n... L e u k e m i a R e...

Transcription

B C R R e a r r a n... L e u k e m i a R e...
BCR Rearrangement–Negative Chronic Myelogenous
Leukemia Revisited
By Razelle Kurzrock, Carlos E. Bueso-Ramos, Hagop Kantarjian, Emil Freireich, Susan L. Tucker, Michael Siciliano,
Susan Pilat, and Moshe Talpaz
Purpose: To document the characteristics of patients
with major breakpoint cluster region (M-bcr) rearrangement–negative chronic myelogenous leukemia
(CML).
Patients and Methods: The hematopathologist, who
was blinded to patients’ molecular status, reviewed the
referral bone marrows and peripheral-blood smears
from 26 patients with Philadelphia (Ph) translocation–
negative CML who lacked Bcr rearrangement (and
other evidence of a Bcr-Abl anomaly) and 14 patients
(controls) with chronic-phase Ph-positive CML. Clinical
data was ascertained by chart review.
Results: Among the 26 M-bcr rearrangement–negative CML patients, three pathologic subtypes emerged:
(1) patients indistinguishable from classic CML (n ⴝ 9),
(2) patients with atypical CML (n ⴝ 8), and (3) patients
with chronic neutrophilic leukemia (n ⴝ 9). Among the
14 patients with Ph-positive CML who were included in
the blinded review, 13 were classified as classic CML,
and one was classified as atypical CML. The only statis-
tically significant difference between M-bcr rearrangement–negative subgroups was in the proportion of
patients having karyotypic abnormalities, an observation common only in patients with atypical CML (P ⴝ
0.008). However, the small number of patients in each
subgroup limited our ability to differentiate between
them. Interferon alfa induced complete hematologic
remission in five of 14 patients; four of these remissions
lasted more than 5 years. Only one of 26 patients
developed blast crisis. The median survival of the 26
patients was 37 months.
Conclusion: Patients with M-bcr rearrangement–
negative CML fall into three morphologic subgroups.
Disease evolution does not generally involve blastic
transformation. Instead, patients show progressive organomegaly, leukocytosis, anemia, and thrombocytosis. Some patients in each subgroup can respond to
interferon alfa.
J Clin Oncol 19:2915-2926. © 2001 by American
Society of Clinical Oncology.
HE HALLMARK OF chronic myelogenous leukemia
(CML) is the Philadelphia (Ph) translocation t(9;
22)(q34;q11), which, at the molecular level, results in the
transfer of the 3' end of the ABL gene next to the 5' end of
the disrupted BCR gene.1-5 The product is a chimeric
BCR-ABL gene. In the majority of cases of CML, the BCR
breakpoint occurs in the central, major breakpoint cluster
region (M-bcr). Exon b2 or b3 of BCR is joined to exon a2
of ABL, resulting in a b2-a2 or b3-a2 junction. This
transcript encodes a 210-kd protein (p210Bcr-Abl).4,5 The
creation of this anomalous protein is believed to play a
critical role in the pathogenesis and phenotypic manifestations of CML.
Patients with Ph-positive CML have typical clinical and
morphologic features: splenomegaly, neutrophilia, basophilia, frequent thrombocytosis, a low leukocyte alkaline
phosphatase (LAP) score, and a hypercellular bone marrow
with an increased myeloid:erythroid ratio and granulocytic
hyperplasia. The disease course inevitably evolves from a
chronic phase to a terminal blast transformation phase. A
minority of patients (5% to 10%) do not have cytogenetic
evidence of the Ph chromosome. Over the last few years,
M-bcr rearrangement has been well documented in a subset
of Ph-negative patients.6-11 It is now established that M-bcr
rearrangement–positive CML patients have a molecular
fingerprint (ie, the p210Bcr-Abl protein) and a clinical phe-
notype and outcome that is indistinguishable from that of
their Ph-positive counterparts.6,7 The existence of a Phnegative, M-bcr rearrangement–negative CML has, however, remained a matter of debate. Some investigators have
suggested that these individuals represent part of the spectrum of chronic myelomonocytic leukemia (CMMoL).12
Others have reported that their features differ subtly, but
recognizably, from classic CML, and that they should be
designated atypical CML.13 A subset of these patients
seems to have a disease highly reminiscent of CML.14,15 An
entity called chronic neutrophilic leukemia has also been
described and is characterized by a marked predominance of
mature neutrophils (shift to the right, in contrast to the
immature forms and shift to the left, which appear in the
other subsets).16-22
T
From the Departments of Leukemia, Bioimmunotherapy, Pathology,
Biomathematics, and Molecular Genetics, University of Texas M.D.
Anderson Cancer Center, Houston, TX.
Submitted August 11, 2000; accepted March 6, 2001.
Address reprint requests to Razelle Kurzrock, MD, Department of
Bioimmunotherapy, Box 302, 1515 Holcombe Blvd, Houston, TX
77030.
© 2001 by American Society of Clinical Oncology.
0732-183X/01/1911-2915
Journal of Clinical Oncology, Vol 19, No 11 (June 1), 2001: pp 2915-2926
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2915
2916
KURZROCK ET AL
In this article, we review the characteristics of 26 patients
with CML who lack the Ph chromosome and M-bcr rearrangement. Our results suggest that these patients can be
further subgrouped into three pathologic categories: (1)
those with disease indistinguishable from CML, (2) those
with disease classifiable as atypical CML,13 and (3) those
with disease suggestive of chronic neutrophilic leukemia.
The phenotypic characteristics, response to therapy, and
outcome of these patients are reported.
PATIENTS AND METHODS
Patient Population
All patients referred to the University of Texas M.D. Anderson
Cancer Center with a tentative diagnosis of CML had a full work-up—
including blood, bone marrow aspirate, and biopsy—and molecular
studies, at our center. Data on all patients were recorded in the
Leukemia Department computer database. Informed consent was obtained according to institutional guidelines.
Criteria for diagnosis of M-bcr rearrangement–negative chronic
myelogenous leukemia were (1) a hypercellular bone marrow with
granulocytic hyperplasia, (2) persistent, unexplained, peripheral granulocytic leukocytosis of ⱖ 20 ⫻ 109 cells/L, (3) absence of the Ph
chromosome, and (4) no M-bcr rearrangement. Additional molecular
tests for BCR-ABL, as outlined below, were also performed on selected
patients and proved negative in all cases.
To rule out the presence of CMMoL, other myeloproliferative
disorders (essential thrombocythemia, myelofibrosis, polycythemia
vera), or leukemoid reaction, patients who had any of the following
features were excluded: (1) monocytosis (bone marrow monocytes ⬎
3%, peripheral blood monocytes ⬎ 6%), (2) polycythemia, (3) persistent thrombocytosis ( ⬎ 1,000 ⫻ 109/L) in the presence of only
moderate leukocytosis (⬍ 30 ⫻ 109/L), (4) significant bone marrow
myelofibrosis or dysplasia, (5) bone marrow blasts ⬎ 5%, (6) coexisting significant thrombocytopenia and anemia (hemoglobin ⱕ 11 g/dL
coexisting with platelets ⱕ 100 ⫻ 109/L), (7) significant leukoerythroblastosis, (8) significant numbers of tear-shaped RBCs (⬎ three per
high-power field), or (9) concurrent infection or other neoplasm.
Pathology Review
All initial bone marrow aspirate and biopsy slides and peripheralblood smears were re-examined by our hematopathologist (C.E.B.R.) for this study. Forty patients were reviewed. These 40 patients
included 14 individuals with classic, chronic-phase, Ph-positive
CML and 26 patients who presented with characteristics of CML but
lacked the Ph chromosome and M-bcr rearrangement. The hematopathologist was blinded to the karyotypic or molecular status of the
patients being reviewed.
DNA Analysis by Southern Blot
Fifteen micrograms of DNA was digested with restriction endonucleases under conditions recommended by the supplier (International
Biotechnologies, Inc, New Haven, CT), electrophoresed on 0.8%
agarose gel, blotted, and hybridized according to the Southern blot
method. The probes were labeled by oligo primer extension to a
specific activity of 1 to 3 ⫻ 109 cpm/␮g of DNA. After hybridization,
filters were washed at 60°C for 1 hour with a solution of 0.1 ⫻ silver
sulfadiazine and chlorhexidine (SSC; 1 ⫻ SSC ⫽ 0.15 mol/L sodium
chloride ⫹ 0.015 mol/L sodium citrate) and 0.1% sodium dodecyl
sulfate. Filters were then dried and autoradiographed.
Probes used to determine M-bcr rearrangement status were a 3' bcr
(1.2-Kb HindIII/BG/II) genomic probe (bcr[PR-1]) and a larger universal bcr probe encompassing most of the 5.8-kb M-Bcr (Phl/bcr-3)
(Oncogene Science, Inc, Manhasset, Long Island, NY).
RNA Analysis by Northern Blot
Total cellular RNA was isolated by a modified guanidinium isothiocyanate– cesium chloride method.23 Polyadenylated RNA was selected
by chromatography on oligo (dT)-cellulose columns. The polyadenylated RNA (5 ␮g per lane) was size fractionated by electrophoresis in
1.1% agarose gels containing 2.2 mol/L of formaldehyde and blotted
with 20 ⫻ SSC. The filters were hybridized to P-radiolabeled DNA
probes, washed, and radiographed under the same conditions as DNA
filters. The probes used to detect BCR and ABL transcripts were a 5'
EcoRI/Pst I fragment (Oncogene Science, Inc) of the BCR gene and a
EcoRI/BamHI fragment derived from the human ABL gene.
Polymerase Chain Reaction
The amplification method, primers, and precautions to prevent
false-positive and false-negative results have been previously described
in detail.24-27 Amplification was performed to look for the BCR exon
b3/ABL exon a2 (b3-a2) junction, the BCR exon b2/ABL exon a2
(b2-a2) junction, and the BCR exon c3 (exon 19) and ABL exon a2
junction (e19-a2).24,25 The probes used were a b2-a2 probe (28 mer
with 22 bases in BCR exon b3), a b3-a2 probe (25 mer with 11 bases
in BCR exon b2),26 and a c3-a2 junctional probe as previously
described.25 Amplification was performed for 40 cycles.
Southern blot analysis and hybridization of all amplified samples
were performed. Ten microliters of the amplified product was run on
3% Nusieve/1% Seakem (FMC, Rockland, ME) composite gels,
transferred overnight to Genescreen Plus membrane (New England
Nuclear, Boston, MA), and baked at 80°C for 2 hours. Oligonucleotide
probes complementary to the junctional BCR-ABL sequences24 were 5'
end labeled with phosphorus-32, and hybridization was performed
overnight. The membranes were washed as recommended by the
manufacturer and exposed to Kodak XAR film (Eastman Kodak Co,
Rochester, NY) for 3 to 48 hours. To confirm the integrity of the cDNA
and to confirm that the amplification procedure worked, all samples
were also amplified for the normal ABL product using primers
encompassing ABL exon 2 and ABL exon 1b and a probe that spans this
region as previously described.27
Western Blot
Western blot analysis to detect Bcr-Abl proteins was performed per
methods previously reported.28 Briefly, extracted proteins were electrophoresed through 6.5% polyacrylamide gels and transferred to
Immobilon P filters (Millipore, Bedford, MA). Enhanced chemiluminescence detection system and the 8E9 anti-Abl mouse monoclonal
antibody were used for detection (Amersham, Arlington Heights, IL).
Cytogenetic Analysis
Chromosome studies were performed on bone marrow cells from all
patients at the time of referral and every 3 to 6 months during
follow-up. A minimum of 20 metaphases were analyzed from each
sample, and the karyotype was reported according to the International
System for Human Cytogenetic Nomenclature. The bone marrow
samples were cultured overnight without mitogenic stimulating in
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2917
BCR REARRANGEMENT–NEGATIVE CML
Ham’s F10 medium supplemented with 10% fetal calf serum. Standard
cytogenetic procedures were used on the samples, and the slide
preparations were stained with Giemsa after a trypsin pretreatment,
which yield G-banded chromosomes.
Interphase Fluorescent In Situ Hybridization
Details of this procedure have been previously described.29 Briefly,
buffy coat was removed from heparinized blood sample, and the
interphase cells were used to detect BCR-ABL using the Vysis LS1
BCR-ABL translocation probe directly labeled with SpectrumOrange
fluorophore (Downers Grove, IL). This was a mixture of a BCR probe
directly labeled with SpectrumGreen fluorophore and an ABL probe
directly labeled with SpectrumOrange fluorophore. When viewed
through a multiple-pass filter (Omega, Brattleboro, VT), the BCR probe
showed green fluorescence whereas the ABL probe showed red fluorescence. In regions where both probes overlapped (the Ph translocation), an intermediate yellow color was detected.
Hypermetaphase Fluorescent In Situ Hybridization
30
Details of this procedure are contained in a prior publication. This
technique allows in situ analysis of large numbers of metaphases
(approximately 500). Briefly, after 24-hour culture of bone marrow
aspirates, 1 ug/mL of Colcemid was added for another 24 hours of
culture. Cells were fixed in acid alcohol and dropped on slides. The
E6B probe from 5 MB of human DNA spanning the breakpoint on
chromosome 9q34 involved in the Ph translocation was labeled with
biotin for visualization with fluorescein for fluorescent in situ hybridization (FISH) detection of the Ph chromosome. Criteria for evaluating
Ph-positive and Ph-negative cells were as previously published30: two
chromosome regions of equal green fluorescence indicated normal
cells, and three chromosome regions (one clearly less robust than the
other two) indicated Ph-positive cells.
Response Definition
Complete hematologic remission (CHR) required that all of the
following be present: bone marrow blast count ⱕ 5%, no circulating
blood blasts, no splenomegaly or evidence of extramedullary involvement, absolute neutrophil count ⱖ 1.5 ⫻ 109/L, platelet count ⱖ 100
⫻ 109/L, WBC count at or below the upper limits of the normal range.
Partial hematologic remission (PHR) required the WBC count to be
ⱕ 20 ⫻ 109/L and at least a 50% decrease in spleen size in the presence
of ⱕ 5% marrow blasts. Responses had to last at least 4 weeks before
being designated CHR or PHR.
Statistical Analysis
The statistical estimation of distribution of survival times was
performed using the Kaplan-Meier method.31 Survival was calculated
from the time of initial diagnosis. For survival analysis, all patients still
alive were censored at the date of last follow-up. Comparisons between
survivals were made using the log-rank method. Categoric data were
compared using the ␹2 test or Fisher’s exact test as indicated, and
continuous data were compared using the Mann-Whitney test or
Kruskal-Wallis as appropriate.
RESULTS
Patient Characteristics
A total of 26 patients meeting our criteria for M-bcr
rearrangement–negative CML were identified in the Leuke-
Table 1.
Patient Characteristics
No. of
Patients
No. of patients
No. of men/women
Age, years
Median
Range
Symptoms and signs at presentation
Splenomegaly
Fever
Weight loss ⱖ 10 lb in 6 months
Fatigue
Night sweats
Sweet’s syndrome
%
26
14/12
63
23-88
16
4
4
4
3
1
62
15
15
15
12
4
mia Department computer database (Table 1). The present
database includes long-term follow-up on the 11 patients
initially reported by us in 199014 and 15 new individuals.
All consecutive patients who met the criteria were included.
Fifty-four percent of the patients were men. Although
their median age at diagnosis was 63 years, ages ranged
widely; the youngest patient was 23 years old and the oldest
patient was 88 years old. Most patients had few symptoms
on presentation. A minority of the group presented with
fever, weight loss, and night sweats (Table 1). One individual presented with biopsy-confirmed acute febrile neutrophilic dermatosis (Sweet’s syndrome).32,33 Physical examination was generally normal except for splenomegaly,
which was discerned in 62% of patients at diagnosis.
The median WBC count at presentation was 36 ⫻ 109/L
(range, 22 to 300 ⫻ 109/L); mature neutrophils constituted
45% to 90% of the total WBC count. By definition, patients
had ⱕ 6% peripheral blood monocytes. The median monocyte percentage in the peripheral blood was 2%. The median
platelet count was 251 ⫻ 109/L (range 50 to 1,046 ⫻
109/L); median hemoglobin was 11.8 g/dL (range, 9.0 to
15.0 g/dL). Seven patients (27%) had blood basophil counts
more than 2% (range, 3% to 5%). The median LAP score
was 21 U (range, 1 to 372 U; normal range, 25 to 139 U).
Twelve (55%) of the 22 patients with available data had
LAP scores below the normal range; six (27%) had LAP
scores above the normal range; and four (18%) had scores
within the normal range.
Bone Marrow Pathology
All referral bone marrow aspirates and biopsies were
re-examined for this study by our hematopathologist. Bone
marrows were hypercellular (median cellularity ⫽ 95%),
except for patient no. 8, whose marrow was 35% cellular.
There was a marked elevation in myeloid:erythroid ratios
(median ratio ⫽ 10:1) and no increase in blasts.
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2918
KURZROCK ET AL
Table 2. Summary of Diagnostic Features Proposed for BCR-Positive
CML, BCR-Negative Atypical CML (aCML), and CMMoL*
Peripheral blood
Monocytosis
Basophilia
Immature granulocytes
Blasts
Bone marrow
Granulocytic dysplasia
Erythroid cells
Other
LAP score
Abnormal karyotype other than
Ph chromosome
BCR-Positive
CML (%)
BCR-Negative
aCML (%)
CMMoL
(%)
⬍3
⬎2
⬎ 20
ⱕ2
3-8
ⱕ2
10-20
⬎2
⬎8
⬍2
⬍ 10
⬍2
⫺
⬍ 10
⫹/⫺
10-15
⫹/⫺
⬎ 15
Decreased
Rare
Decreased
Frequent
Normal
Frequent
*Summarized from references13,34-36.
To ascertain the differences between the study patients
and those with classic CML, bone marrows from 14
patients with chronic-phase Ph-positive CML were included in the review. The pathologist was blinded to the
cytogenetic and molecular status of the patients. Thirteen
of the 14 patients with Ph-positive CML were given a
diagnosis of CML on pathologic review. However, one
patient was given a diagnosis of atypical CML (based on
criteria in Table 2).13,34-36
With regard to the 26 patients with M-bcr rearrangement–
negative CML, three pathologic patterns emerged. First,
there were nine patients whose bone marrow pathology was
not distinguishable from that of classic Ph-positive CML
(patients no. 3, 12, 14, 15, 16, 18, 21, 24, and 25)(Table 3
and Fig 1). However, two of these patients had abnormally
large megakaryocytes, a feature that was believed to be
unusual for classic CML (but has not been included in the
criteria for diagnosis of atypical CML).13,34,35 The second
category of patients could be classified as atypical CML
according to previously established criteria (Table 2 and Fig
2).13,34,35 There were eight patients in this subgroup (patients no. 1, 2, 4, 6, 9, 11, 17, and 22). These patients
generally showed granulocytic dysplasia in the bone marrow as well as increased erythroid cells. In the peripheral
blood, there was a shift to the left but not as pronounced as
that in classic CML. Basophilia was absent. However,
within this group of eight patients, a spectrum existed from
those clearly distinguishable from CML because of the
presence of most of the above features in those in whom
only very subtle distinguishing findings were present. For
example, patient no. 6 was classified as atypical CML but
only on the basis of lack of basophilia. The third group of
patients could best be classified as chronic neutrophilic
leukemia based on bone marrow and peripheral-blood
pathology (n ⫽ 9; patients no. 5, 7, 8, 10, 13, 19, 20, 23, and
26).16-21 These patients had marked blood and bone marrow
neutrophilia with a shift to the right (Figs 3 and 4).
Karyotype
Cytogenetic studies were performed on all patients.
Neither the Ph translocation t(9;22) nor its variants were
seen in any patient. Indeed, no patient had an abnormality of
chromosome 22. At the time of presentation, 15 patients
(60%) had a normal diploid karyotype. One patient had no
mitotic cells. The following karyotypic abnormalities were
seen in the other 10 patients: trisomy 21 (patients no. 2 and
4), trisomy 8 (patients no. 2 and 9), loss of the long arm of
chromosome 20 (patients no. 7 and 22), loss of the long arm
of chromosome 13 or translocation involving this region
(patients no. 16 and 17), trisomy 19 (patient no. 2), t(9;14)
(p13;q32) (patient no. 8), loss of chromosome 17 (patient
no. 11), and trisomy 14 (patient no. 6).
Most of these patients had cytogenetic anomalies in a
minority of the metaphases with the majority of metaphases
remaining diploid. In addition, some of the above patients
had several distinct clonal anomalies that coexisted. For
instance, patient no. 2 showed 22 diploid metaphases, one
metaphase with trisomy 8, one metaphase with trisomy 21,
and one metaphase with trisomy 19.
Eighteen patients also had follow-up karyotype analysis
performed one or more times after the baseline test. Three
patients showed clonal evolution (patients no. 9, 10, and
11). The abnormal karyotypes that emerged with time in
these individuals included a translocation between the short
arm of chromosome 1 and the long arm of chromosome 6,
loss of the long arm of chromosome 20, and trisomy 21
combined with loss of the long arm of chromosome 15.
Molecular Studies
Rearrangement in the M-bcr was not observed in any of
the 26 patients (Southern blot analysis of DNA; Table 3).
Northern blot analysis of mRNA was performed on four
patients (patients no. 2, 4, 11, and 22) and demonstrated
only normal BCR and ABL transcripts. PCR was performed
on four patients (patients no. 18, 19, 20, and 24); no
evidence of the b2-a2, b3-a2, or e19-a2 BCR-ABL junctions
was discerned. Western blot was performed on 10 patients
(patients no. 3, 9, 14, 15, 16, 18, 19, 20, 24, and 26) and
showed no evidence of p210Bcr-Abl(product of b2-a2 and
b3-a2 BCR-ABL mRNAs),3-5 p190Bcr-Abl(product of e1-a2
BCR-ABL mRNAs),26 or p230Bcr-Abl(product of e19-a2
BCR-ABL mRNA).37 FISH analysis was performed on three
patients (patients no. 14, 15, and 24) and showed only
normal chromosomes 9 and 22.
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2919
BCR REARRANGEMENT–NEGATIVE CML
Table 3.
Patient No.
Molecular Characteristics
M-Bcr*
mRNA
PCR and FISH
Western Blot
1
2
Germline
Germline
ND
ND
ND
ND
3
Germline
ND
Only normal BCR and ABL
transcripts seen
ND
ND
4
Germline
ND
5
6
7
8
9
Germline
Germline
Germline
Germline
Germline
Only normal BCR and ABL
transcripts seen
ND
ND
ND
ND
ND
No p210Bcr-Abl, p190Bcr-Abl,
or p230Bcr-Abl
ND
10
11
Germline
Germline
12
13
14
Germline
Germline
Germline
ND
Only normal BCR and ABL
transcripts seen
ND
ND
ND
15
Germline
ND
16
Germline
ND
17
18
Germline
Germline
ND
ND
19
Germline
ND
20
Germline
ND
21
22
Germline
Germline
23
24
Germline
Germline
ND
Only normal BCR and ABL
transcripts seen
ND
ND
25
26
Germline
Germline
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
I-FISH: normal chromosomes 9 and
22
Hypermetaphase FISH: normal
chromosomes 9 and 22
ND
ND
PCR: No b2-a2, b3-a2, or e19-a2
junction
PCR: No b2-a2, b3-a2, or e19-a2
junction
PCR: No b2-a2, b3-a2, or e19-a2
junction
ND
ND
ND
PCR: No b2-a2, b3-a2, or e19-a2
junction
Hypermetaphase FISH (500 cells):
normal chromosomes 9 and 22.
ND
ND
ND
ND
ND
ND
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
ND
ND
ND
ND
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
ND
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
No p210Bcr-Abl, p190Bcr-Abl,
or p230Bcr-Abl
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
ND
ND
ND
No p210Bcr-Abl, p[190Bcr-Abl,
or p230Bcr-Abl
ND
No p210Bcr-Abl, p190Bcr-Abl,
or p230Bcr-Abl
Abbreviations: BM, bone marrow; I-FISH, interphase FISH; ND, not done; PB, peripheral blood; PCR, polymerase chain reaction.
*Both a 3⬘ bcr probe and a probe encompassing almost all of the 5.8-kb M-bcr region were used.
Treatment
A wide variety of therapies were used in these patients.
The most common drug administered was hydroxyurea. Of
17 patients who were given this compound, 13 achieved a
PHR, three achieved a CHR, and two showed no adequate
response. The median duration of response was, however,
only 5 months (range, 1.5 to 80⫹ months).
Fourteen patients were treated with interferon alfa
(IFN-␣; dose range, 3 ⫻ 106 units/d to 9 ⫻ 106 units/d). Six
of these patients (43%) responded (five CHRs and one
PHR). The durations of the CHRs were 1, 63, 84, 101, and
100⫹ months.
Other management strategies were used in only a very
small number of patients. Three patients were treated with
busulphan; they achieved short-lived PHRs (3, 7, and 12
months). Two patients had splenectomy, and neither improved. One patient received low-dose cytarabine with no
response, and another was given high-dose cytarabine
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2920
KURZROCK ET AL
Fig 1. CML, typical (patient no. 24). Bone marrow smear shows increased granulocytic elements in all stages of maturation with predominance
of progranulocytes, myelocytes, and metamyelocytes. Basophils are increased. (Wright-Giemsa stain, original magnification ⴛ600)
combined with cisplatin with no response. The one individual who received fludarabine attained a PHR that lasted 4
months. One of the two patients who underwent allogeneic
bone marrow transplantation responded; this patient
achieved a 6-month PHR.
Disease Evolution
To date, 18 patients have died. The cause of death was
unknown in three patients, and two died because of cardiac
disease. Ten patients developed progressive anemia and
thrombocytopenia. This was generally accompanied by
marked leukocytosis and hepatosplenomegaly. Four of
these patients succumbed to sepsis, pneumonia, or both, and
Fig 2. CML, atypical (patient no. 4). Bone marrow smear shows increase
in neutrophils and precursors. Rare neutrophils have pseudo-Pelger-Huet
nuclei (upper left). Occasional dyserythropoiesis (above center) and dysplastic micromegakaryocytes (lower right) were also identified. Basophils were
not increased. (Wright-Giemsa stain, original magnification ⴛ600)
Fig 3. Chronic neutrophilic leukemia (patient no. 26). Bone marrow
smears show increased granulocytic elements with predominance of segmented neutrophils and bands. (Wright-Giemsa stain, original magnification, ⴛ600)
two succumbed to cerebral bleeds. One patient developed
chloromas, and one had refractory Sweet’s syndrome.32,33
Basophilia appeared in two patients and reached levels of
11% and 31% in the bone marrow. Only one patient
developed significant marrow fibrosis, and no patient
developed significant persistent monocytosis. All but
three patients maintained a normal bone marrow blast
count. Two patients eventually showed 8% and 9% bone
marrow blasts, respectively, and one patient developed an
acute myeloid leukemic phenotype with 61% blasts. (The
latter patient had undergone allogeneic bone marrow
transplantation as therapy.) In one patient, progressive
lymphadenopathy was observed.
Fig 4. Blood smear from patient no. 26 with chronic neutrophilic
leukemia shows numerous neutrophils and occasional myelocyte. (WrightGiemsa stain, original magnification ⴛ600)
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2921
BCR REARRANGEMENT–NEGATIVE CML
Breakdown of Phenotypic Characteristics and Course by
Subcategory
Comparisons between subgroups should be interpreted
with caution because of the small number of patients in each
subgroup. Overall, the only characteristic in which there
was a statistically significant difference was karyotype
(Table 4). The majority of patients with atypical CML
showed chromosome aberrations, whereas those in the other
subgroups were usually diploid. The distribution of sex by
category reached marginal significance (P ⫽ .07). A detailed examination of the phenotype and outcome of each
subgroup is discussed below and in Table 4.
Patients with typical CML. As mentioned earlier, nine
patients (four men and five women) had disease indistinguishable from typical CML by bone marrow and peripheral-blood pathology review. The median age was 60 years.
Two patients (22%) presented with “B” symptoms. Most
(78%) had splenomegaly at the time of diagnosis.
The median initial WBC count was 57 ⫻ 109/L; hemoglobin, 13.2 g/dL; and platelet count, 263 ⫻ 109/L. Mature
neutrophils comprised a median of 58% of WBCs (range,
45% to 90%). Only two of the eight patients with available
LAP scores had low values, while three had elevated values.
All of the patients but one had a diploid karyotype. The one
exception had a translocation between the short arm of
chromosome 1 and the long arm of chromosome 13.
With regard to treatment, five of the patients received
IFN-␣, and three responded. The responses included two
CHRs (lasting 63 and 101 months) and a PHR (lasting 8
months). Five patients were treated with hydroxyurea. One
of these individuals was lost to follow-up, whereas the
others attained a PHR lasting from 3 to 20 months.
Evolution to blast crisis did not occur. To date, five of the
patients have died. Progressive disease was characterized by
worsening anemia and thrombocytopenia (n ⫽ 4), significant basophilia (n ⫽ 1), bone marrow fibrosis (n ⫽ 1), and
Sweet’s syndrome (n ⫽ 1). Two patients died of
pneumonia.
Patients with atypical CML. On pathologic review,
eight patients (seven men and one woman) were classified
as having atypical CML (Table 2).13,34 The median age was
60 years. Three patients had B symptoms on presentation.
Most (75%) had splenomegaly.
The median WBC count at diagnosis was 36 ⫻ 109/L;
hemoglobin, 11.7 g/dL; platelet count, 270 ⫻ 109/L.
Mature neutrophils comprised a median of 77% of WBCs
(range, 56% to 86%). Four patients had low LAP scores
at diagnosis, while two had very high LAP scores.
(Scores were not available in two patients.) All except
one patient had one or more karyotypic abnormalities:
Table 4.
Clinical Features by Subcategory*†
Feature
Men:women, n
Age, years
Median
Range
B symptoms (night sweats,
fever, and/or weight
loss)
No.
%
Splenomegaly
No.
%
WBC ⫻ 109/L
Median
Range
Hemoglobin, g/dL
Median
Range
Platelets ⫻ 109/L
Median
Range
Patients with low LAP
No.
%
Patients with diploid
karyotype
No.
%
IFN-␣ responders
No.
%
Total treated
Patients who evolved to
blast crisis (n ⫽ 5)
No.
%
Median survival, month
CML
(n ⫽ 9)
Atypical
CML
(n ⫽ 8)
Chronic
Neutrophilic
Leukemia
(n ⫽ 9)
P
4:5
7:1
3:6
.07‡
60
39-68
74
23-88
NS§
2
22
3
38
2
22
NS‡
7
78
6
75
3
33
NS‡
57
23-108
36
22-300
36
21-97
NS㛳
13.2
10.7-14.9
11.7
8.9-15.0
11.0
8.6-13.2
NS㛳
263
118-767
270
50-1046
188
135-711
NS㛳
2
25
4
57
6
75
NS‡
8
89
1
13
6
75
.008‡
3
60
5
1
14
7
2
100
2
NS‡
0
0
57
1
13
29
0
0
48
NS‡
60
51-78
NS¶
Abbreviation: NS, not significant.
*Subcategory was determined by pathologic review as outlined in Patients
Methods and Results. Patients labeled CML had disease indistinguishable (by
blood and bone marrow examination) from classic Ph-positive CML; patients
with atypical CML had features outlined in Table 3,13,34 and patients with
chronic neutrophilic leukemia did not meet the criteria for the other diagnoses
and showed marked blood and bone marrow neutrophilia with a shift to the
right.
†All features were recorded at time of diagnosis.
‡Statistical analysis performed by Fisher exact test.
§NS ⫽ not statistically significant (P ⬎ .1 in all cases).
㛳Statistical analysis performed by Kruskal-Wallis test.
¶Statistical analysis performed by log-rank method.
trisomy 21 (n ⫽ 2), trisomy 8 (n ⫽ 2), trisomy 14 (n ⫽
1), monosomy 17 (n ⫽ 1), 13q⫺ (n ⫽ 1), and trisomy 19
(n ⫽ 1).
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2922
KURZROCK ET AL
Fig 5. Survival of 26 patients with BCR rearrangement–negative CML.
Distributions were estimated using the Kaplan-Meier method. Tick marks
represent the point of last follow-up for patients who are still alive.
Only one patient (14%) of seven treated with IFN-␣
responded. However, this patient has remained in CHR for
100⫹ months. Four patients of five treated with hydroxyurea responded with PHRs, which were, however, short-lived
(2 to 4 months). Evolution to blast crisis occurred in one
patient. This patient developed blastic transformation 6
months after allogeneic bone marrow transplantation. Progressive disease in other patients was characterized by
anemia and thrombocytopenia (n ⫽ 5), marked hepatosplenomegaly and leukocytosis (n ⫽ 5), minimal increase in
blasts (bone marrow blasts of 8% and 9%; n ⫽ 2), severe
basophilia (bone marrow basophils of 31%; n ⫽ 1), and
chloromas (n ⫽ 1). Seven patients have died. Cause of death
included progressive disease with resistant leukocytosis,
anemia, thrombocytopenia, and hepatosplenomegaly (n ⫽
3), cerebral bleed associated with thrombocytopenia (n ⫽
2), and infection (n ⫽ 2).
Patients with chronic neutrophilic leukemia. Nine patients (three men and six women) were characterized as
having chronic neutrophilic leukemia based on blood and
bone marrow pathology review. The median age was 74
years. However, the range of ages was wide; the youngest
patient was 23 years old and the oldest patient was 88 years
old. Two patients presented with B symptoms. Three
patients (33%) presented with splenomegaly, and one had
Sweet’s syndrome at the time of diagnosis.
The median WBC count was 36 ⫻ 109/L; hemoglobin,
11.0 g/dL; and platelet count, 188 ⫻ 109/L. Mature peripheral-blood neutrophils comprised a median of 72% of
WBCs (range, 59% to 90%). Six patients had low LAP
scores. (LAP score was elevated in one patient, not per-
Fig 6. Survival of patients indistinguishable from classic CML (curve A;
n ⴝ 9), those with atypical CML (curve B; n ⴝ 8), and those with chronic
neutrophilic leukemia (curve C; n ⴝ 9). Distributions were estimated using
the Kaplan-Meier method. Tick marks represent the point of last follow-up
for patients who are still alive.
formed in one patient, and normal in one patient). Six of the
patients had a diploid karyotype; one had insufficient
metaphase for analysis, one had a deletion of the long arm
of chromosome 20, and one had a t(9;14) anomaly.
Both patients treated with IFN-␣ responded, with CHRs
lasting 1 and 84 months. (The latter patient lost her CHR but
remains in PHR after 156 months of IFN-␣ therapy.) Seven
patients were treated with hydroxyurea, and all responded
(CHR, n ⫽ 3; PHR, n ⫽ 4). The median duration of
response was 20 months (range, 1.5 to 80⫹ months). None
of the patients progressed to blast crisis. Five patients have
died. The following causes of death were found: progressive
disease characterized by hepatosplenomegaly, ascites, and
lymphadenopathy (n ⫽ 1), myocardial infarction (n ⫽ 1),
graft-versus-host disease after allogeneic transplantation (n
⫽ 1), and unknown (n ⫽ 2).
Survival
Our 26 patients had an overall median survival of 37
months (Fig 5). Their actuarial 2-year survival was 61%
(95% confidence interval [CI], 39% to 77%); 3-year survival, 51%, (95% CI, 30% to 69%); and 5-year survival,
27% (95% CI, 11% to 47%).
Although patients in the subgroup with atypical CML had
shorter median survivals compared with those in the other
subgroups, there was no statistically significant difference
in survival among the three patient subgroups (P ⫽ .7,
log-rank test) (Fig 6 and Table 4). The 3-year survival rate
was 53% (95% CI, 18% to 80%) for the patients who were
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2923
BCR REARRANGEMENT–NEGATIVE CML
indistinguishable from classic CML, 50% (95% CI, 15% to
77%) for those with atypical CML, and 53% (95% CI, 18%
to 80%) for those with chronic neutrophilic leukemia. The
5-year survival rates were 36% (95% CI, 6% to 68%), 13%
(95% CI, 1% to 42%), and 40% (95% CI, 10% to 70%),
respectively (Fig 6).
DISCUSSION
CML is a generic designation given to specific clinical
features. The best-studied form of CML has the classic
Ph-positive genotype and accounts for more than 90% of
cases. A marked granulocytic proliferation is its phenotypic hallmark, and the BCR-ABL chimeric gene is its
molecular fingerprint.
Patients with a Ph-negative CML have been described for
many years. However, the earliest cases predated the emergence of molecular techniques that eventually revealed that
some of these patients still harbored the aberrant BCR-ABL
hybrid gene. Their disease was therefore identical to Phpositive CML at the molecular (as well as the phenotypic)
level.6-11 Hence, Ph-negative and Ph-positive CML have
had to be redefined as M-bcr–negative CML versus M-bcr–
positive CML.
The percentage of patients with M-bcr rearrangement
among Ph-negative CML patients has varied from less than
10% to more than 50% in different series.12,15,19-22,38,39 The
classification of the remaining patients is less clear-cut.36
Some investigators claim that at least a portion of these
patients are reminiscent of those with M-bcr–positive disease,14,15,39 whereas other investigators have described clear
distinctions.12,13,34 To some extent, the variation in both the
rates of M-bcr rearrangement positivity and the phenotypic
descriptions of the patients may be due to the initial
selection criteria; some studies included patients who could
have been diagnosed with CMMoL, whereas others had
more strict criteria. The situation is further complicated by
the fact that M-bcr rearrangement–negative CML, Phpositive CML, CMMoL, M-bcr–negative CML, essential
thrombocytosis, myelofibrosis, and polycythemia vera seem
to be nosologically related disorders. Nevertheless, the
exuberant marrow fibrosis, expanded RBC mass, florid
megakaryocytic hyperplasia, or significant monocytosis that
are the hallmark of the non-CML myeloproliferative disorders and CMMoL were not seen in our patients, an
observation supporting the distinctness of the M-bcr rearrangement–negative subgrouping of patients with a CML
phenotype. Furthermore, pathologic review of our patients’
bone marrow and peripheral-blood findings indicated that
three morphologic subgroups existed: (1) those with disease
indistinguishable from CML, (2) those with atypical CML
(Table 2),13,34-36 and (3) those with chronic neutrophilic
leukemia.
With rare exceptions, nearly all cases of Ph-positive CML
have a p210-encoding BCR-ABL gene in which the breakpoint occurs in the central M-bcr. The M-bcr encompasses
five exons termed b1 to b5 that correspond to the 12th to
16th exon of the BCR gene.3 The resultant transcript
contains either a b2a2 (BCR exon 13 [or M-bcr exon b2]
joined to ABL exon 2) or a b3a2 junction (BCR exon 14 [or
M-bcr exon b2] joined to ABL exon 2) or both. A small
number of patients have been described who have a Ph
chromosome–positive CML, albeit with a very indolent
course, and lack M-bcr rearrangement (Southern blot of
DNA) or the classic b2-a2 or b3-a2 junction (PCR of
cDNA). These patients have been found to have yet another
junction— c3-a2 (otherwise known as e19-a2)—that joins
the 19th exon of BCR to ABL exon 2 and is translated into
a p230Bcr-Abl.25,37,40-44 Rare anecdotal reports of Ph-positive
CML with e1-a2 junctions leading to the production of
p190Bcr-Abl(in the absence of p210Bcr-Abl) have also been
reported3,40,45-48 and may be associated with a phenotype
intermediate between CML and CMMoL.45 Other junctions
occur as well, but they are extremely rare.40,49,50-54 Because
alternate breakpoints exist, it could be argued that tests for
M-bcr rearrangement alone are inadequate to rule out these
unusual junctions. However, it should be noted that, almost
without exception,40,49 alternate junctions were found in
patients bearing a Ph translocation (rather than in Phnegative patients). Furthermore, 14 of our patients had one
or more additional molecular tests performed (beyond
Southern blot). These tests included Western blot, FISH,
polymerase chain reaction, and Northern blot of mRNA,
which ruled out the presence of Bcr-Abl proteins, subchromosomal translocations between chromosomes 9 and 22,
alternate junctions, and aberrant BCR or ABL transcripts,
respectively (Table 3). Importantly, six of the nine patients
who had disease indistinguishable on a pathologic basis
from classic Ph-positive CML had molecular tests (Western
blot, n ⫽ 6; FISH, n ⫽ 3) in addition to Southern blot (Table
3). These tests eliminated the possibility that a BCR-ABL
configuration existed and resulted in production of a BcrAbl protein but was missed by Southern blot because of a
deletion within BCR or an alternative BCR-ABL junction.
Furthermore, three of the patients with pathology consistent
with chronic neutrophilic leukemia had Western blot that
ruled out the presence of a p230Bcr-Abl as well as polymerase
chain reaction (n ⫽ 2) that ruled out an e19-a2 junction. The
latter abnormalities have been associated with a chronic
neutrophilic leukemia phenotype by some investigators,25
but not by others.42
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2924
KURZROCK ET AL
Eight M-Bcr–negative patients were given a diagnosis of
atypical CML in accordance with the French-AmericanBritish (FAB) Cooperative Group guidelines.13 Interestingly, one of the 14 patients with Ph-positive CML examined by the pathologist in the blinded review also received
this diagnosis. Therefore, the FAB guidelines13 (with an
emphasis on peripheral-blood findings) as used by us (albeit
with an emphasis on bone marrow aspirate and biopsy
findings in addition to peripheral-blood morphology) confirm the uniqueness of atypical CML but are not absolute in
their ability to differentiate CML from atypical CML and its
variants. Furthermore, within the subgroup of atypical
CML, a spectrum of changes was noted, with some individuals showing all the features delineated in Table 2, while
others demonstrated only very subtle changes. One patient
was put in this category on the basis of absent basophils
alone. The FAB guidelines for atypical CML were published in 1994.13 Not surprisingly, therefore, some of the
patients previously published by our group in 1990 as
typical CML14 were now reclassified as atypical CML.
M-bcr–negative atypical CML patients were distinguished
clinically from the other M-bcr–negative categories by a
predominance of men, frequent cytogenetic abnormalities,
and relatively lower response rates to IFN-␣ (except in one
patient who attained a prolonged CHR). In keeping with
the findings of others,13,34 karyotypic changes varied
from patient to patient and included abnormalities of
chromosomes 8, 13, 14, 17, 19, and 21. This was also the
only subgroup in which evolution of disease was associated with increased blast counts. Three patients had
eventual bone marrow blast elevation to 61% (after
allogeneic bone marrow transplantation), 8%, and 9%.
Significant monocytosis was not observed with disease
progression, supporting the differentiation of these patients from those with CMMoL.
Chronic neutrophilic leukemia has been previously published in case reports by other investigators.16-21 Most
describe this entity as characterized by neutrophilia with a
shift to the right (in contrast to CML in which the granulocytic series show a shift to the left), hepatosplenomegaly,
and elevated LAP scores. The diagnosis of chronic neutrophilic leukemia should be made with considerable caution
and only after all possibilities of a leukemoid reaction and
other myeloproliferative disorders have been eliminated.
Clinical, cytogenetic, and molecular studies are critical in
this differentiation. Our nine patients with this disorder all
had marked, right-shifted, peripheral-blood, and bone marrow neutrophilia. However, only one third had splenomegaly at diagnosis, and only one patient had an elevated LAP
score (Table 4). A previous report described successful
treatment of two such patients with IFN-␣.20 Two of our
patients with this entity were treated with IFN-␣, and both
attained a CHR. In one patient, IFN-␣ had to be discontinued 1 month after CHR because of side effects. However,
the second patient remained in CHR for 84 months. After
this time, she no longer met the criteria for CHR, but her
blood counts remained controlled and her disease has
remained in PHR. She has received a total of 156⫹ months
of treatment with IFN-␣.
There were several areas of similarities and differences
between the three subcategories (Table 4). However,
these distinctions did not reach statistical significance
with the exception of the karyotype differences (P ⫽
.008) (chromosomal abnormalities seen predominantly in
atypical CML), perhaps because of the small number of
patients in each category.
In conclusion, we report that M-bcr rearrangement–
negative patients with CML may be pathologically indistinguishable from classic Ph-positive CML or may present as
atypical CML13 or as chronic neutrophilic leukemia.16-21
Most patients have a diploid karyotype except in the
subgroup with atypical CML. With rare exception, M-bcr–
negative CML patients do not progress to blast crisis.
Rather, in most patients, progression is manifested by
increasing leukocytosis and organomegaly, as well as worsening anemia and thrombocytopenia. Some patients in all
three subcategories respond to IFN-␣, although the small
number of individuals precludes convincing statistical comparisons. The median survival ranged from 29 months in the
atypical CML patients to 57 months in those indistinguishable from classic CML. Only five patients of the cohort of
23 with long enough follow-up have lived more than 5
years, and these five included four individuals who achieved
a CHR on IFN-␣. Whether or not IFN-␣ contributed to their
prolonged survival remains debatable because favorable
selection criteria may have influenced the choice to treat
with this agent. The molecular and biologic basis of these
entities remains to be explored.
REFERENCES
1. Nowell PC, Hungerford DA: A minute chromosome in human
chronic granulocytic leukemia. Science 132:1197, 1960
2. Rowley JD: A new consistent chromosomal abnormality in
chronic myelogenous leukemia identified by quinacrine fluorescence
and Giemsa staining. Nature 243:290-292, 1973
3. Kurzrock R, Gutterman JU, Talpaz M: The molecular genetics of
Philadelphia chromosome-positive leukemias. N Engl J Med 319:990998, 1988
4. Kloetzer W, Kurzrock R, Smith L, et al: The human cellular ABL
gene product in the chronic myelogenous leukemia cell line K562 has
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2925
BCR REARRANGEMENT–NEGATIVE CML
an associated tyrosine protein kinase activity. Virology 140:230-238,
1985
5. Konopka JB, Watanave SM, Witte ON: An alternation of the
human c-abl protein in K562 leukemia cells unmasks associated
tyrosine kinase activity. Cell 37:1035-1042, 1984
6. Kurzrock R, Blick MB, Talpaz M, et al: Rearrangement in the
breakpoint cluster region and the clinical course in Philadelphianegative chronic myelogenous leukemia. Ann Intern Med 105:673-679,
1986
7. Shtalrid M, Talpaz M, Blick M, et al: Philadelphia-negative
chronic myelogenous leukemia with breakpoint cluster region rearrangement: Molecular analysis, clinical characteristics, and response to
therapy. J Clin Oncol 6:1569-1575, 1988
8. Bartram CR, Kleihauer E, deKlein A, et al: c-abl and bcr are
rearranged in a Ph1-negative CML patient. EMBO J 4:683-686, 1985
9. Morris CM, Reeve AE, Fitzgerald PH, et al: Genomic diversity
correlates with clinical variation in Ph-negative chronic myeloid
leukaemia. Nature 320:281-283, 1986
10. Dreasen O, Rassool F, Sparkes RS, et al: Do oncogenes
determine clinical features in chronic myeloid leukaemia? Lancet
1:1402-1405, 1987
11. Wiedemann LM, Karhi KK, Shivji MKK, et al: The correlation
of breakpoint cluster region rearrangement and p210phl/ABL expression
with morphological analysis of Ph-negative chronic myeloid leukemia
and other myeloproliferative diseases. Blood 71:349-355, 1988
12. Martiat P, Michaux JL, Rodhain J: Philadelphia-negative (Ph-)
chronic myeloid leukemia (CML): Comparison with Ph⫹ CML and
chronic myelomonocytic leukemia. Blood 78:205-211, 1991
13. Bennett JM, Catovsky D, Daniel MT, et al: The chronic myeloid
leukemias: Guidelines for distinguishing chronic granulocytic, atypical
chronic myeloid, and chronic myelomonocytic leukemia—Proposals
by the French-American-British Cooperative Leukemia Group. Br J
Haematol 87:746-754, 1994
14. Kurzrock R, Kantarjian HM, Shtalrid M, et al: Philadelphia
chromosome-negative chronic myelogenous leukemia without breakpoint cluster region rearrangement: A chronic myeloid leukemia with a
distinct clinical course. Blood 75:445-452, 1990
15. Selleri L, Emilia G, Luppi M, et al: Chronic myelogenous
leukemia with typical clinical and morphological features can be
Philadelphia chromosome negative and “bcr negative.” Hematol Pathol
4:67-77, 1990
16. Kwong YL, Cheng G: Clonal nature of chronic neutrophilic
leukemia. Blood 82:1035-1038, 1993
17. Orazi A, Cattoretti G, Sozzi G: A case of chronic neutrophilic
leukemia with trisomy 8. Acta Haemat 81:148-151, 1989
18. You W, Weisbrot IM: Chronic neutrophilic leukemia. Am J Clin
Pathol 72:233-242, 1979
19. Storek J: Chronic neutrophilic leukemia: Case report documenting the absence of bcr-abl rearrangement. Am J Hematol 41:304, 1992
(letter)
20. Meyer S, Feremans W, Cantiniaux B, et al: Successful alpha2b-interferon therapy for chronic neutrophilic leukemia. Am J Hematol
43:307-309, 1993
21. Zittoun R, Rea D, Ngoc LH, et al: Chronic neutrophilic
leukemia: A study of four cases. Ann Hematol 68:55-60, 1994
22. Bartram CR, Carbonell F: bcr Rearrangement in Ph-negative
CML. Cancer Genet Cytogenet 21:183-184, 1986
23. Chomcyznski P, Sacchi N: Single-step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal
Biochem 162:156-159, 1987
24. Kawasaki ES, Clark SS, Coyne MY, et al: Diagnosis of chronic
myeloid and acute lymphocytic leukemias by detection of leukemiaspecific mRNA sequences amplified in vitro. Proc Natl Acad Sci U S
A 85:5698-5703, 1988
25. Pane F, Frigeri F, Sindona M, et al: Neutrophilic chronic
myeloid leukemia: A distinct disease with a specific molecular marker
(BCR-ABL with C3/A2 junction). Blood 88:2410-2414, 1996
26. Dhingra K, Talpaz M, Riggs MG, et al: Hybridization protection
assay: A rapid, sensitive and specific method for detection for Philadelphia chromosome-positive leukemias. Blood 77:238-242, 1991
27. Kurzrock R, Estrov Z, Kantarjian H, et al: Conversion of
interferon-induced, long-term cytogenetic remissions in chronic myelogenous leukemia to polymerase chain reaction negativity. J Clin
Oncol 16:1526-1531, 1998
28. Guo JQ, Lian J, Glassman A, et al: Comparison of bcr-abl
protein expression and Philadelphia chromosome analyses in chronic
myelogenous leukemia patients. Am J Clin Pathol 106:442-448, 1996
29. Seong D, Thall P, Kantarjian HM, et al: Philadelphia chromosome-positive myeloid cells in the peripheral blood of chronic myelogenous leukemia patients: Comparison with the frequency detected in
cycling cells of the bone marrow. Clin Cancer Res 4:861-867, 1998
30. Seong D, Giralt S, Fischer H, et al: Usefulness of detection of
minimal residual disease by ‘hypermetaphase’ fluorescent in situ
hybridization after allogeneic BMT for chronic myelogenous leukemia.
Bone Marrow Transplant 19:565-570, 1997
31. Kaplan EL, Meier P: Non-parametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958
32. Cohen PR, Kurzrock R: Chronic myelogenous leukemia and
Sweet syndrome. Am J Hematol 32:134-137, 1989
33. Cohen PR, Talpaz M, Kurzrock R: Malignancy-associated
Sweet’s syndrome: Review of the world literature. J Clin Oncol
6:1887-1897, 1988
34. Costello R, Sainty D, Lapage-Pochitaloff M, et al: Clinical and
biological aspects of Philadelphia-negative/BCR-negative chronic myeloid leukemia. Leuk Lymphoma 25:225-232, 1997
35. Shepherd PCA, Ganesan TS, Galton DAG: Haematological
classification of the chronic myeloid leukaemias. Baillieres Clin
Haematol 1:887-906, 1987
36. Galton DAG: Haematological differences between chronic granulocytic leukaemia, atypical chronic myeloid leukaemia, and chronic
myelomonocytic leukaemia. Leuk Lymphoma 7:343-350, 1992
37. Wada H, Mizutani S, Nishimura J, et al: Establishment and
molecular characterization of a novel leukemic cell line with Philadelphia chromosome expressing p230 BCR/ABL fusion protein. Cancer
Res 55:3192-3196, 1995
38. Dobrovic A, Morley AA, Seshadri R, et al: Molecular diagnosis
of Philadelphia-negative CML using the polymerase chain reaction and
DNA analysis: Clinical features and course of M-bcr negative and
M-bcr positive CML. Leukemia 5:187-190, 1991
39. van der Plas DC, Grosveld G, Hagemeijer A: Review of clinical,
cytogenetic, and molecular aspects of Ph-negative CML. Cancer Genet
Cytogenet 52:143-156, 1991
40. Melo JV: BCR-ABL gene variants. Baillieres Clin Haematol
10:203-222, 1997
41. Saglio G, Guerrasio A, Rosso C, et al: New type of BCR/ABL
junction in Philadelphia chromosome-positive chronic myelogenous
leukemia. Blood 76:1819-1824, 1990
42. Mittre H, Leymarie P, Macro M, et al: A new case of chronic
myeloid leukemia with c3/a2 BCR/ABL junction: Is it really a distinct
disease? Blood 11:4239-4241, 1997
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.
2926
KURZROCK ET AL
43. Yamagata T, Mitani K, Kanda Y, et al: Elevated platelet count
features the variant type of BCR/ABL junction in chronic myelogenous
leukaemia. Br J Haematol 94:370-372, 1996
44. Wilson G, Frost L, Goodeve A, et al: BCR-ABL transcript with
an e19a2 (c3a2) junction in classical chronic myeloid leukemia. Blood
89:3064, 1997 (letter)
45. Melo JV, Myint H, Galton DAG, et al: P190Bcr-Abl chronic
myeloid leukaemia: The missing link with chronic myelomonocytic
leukaemia? Leukemia 8:208-211, 1994
46. Kurzrock R, Shtalrid M, Romero P, et al: A novel c-abl protein
product in Philadelphia-positive acute lymphoblastic leukaemia. Nature 325:631-635, 1987
47. Melo JV, Myint H, Galton DA, et al: Lack of correlation
between ABL-BCR expression and response to interferon-alpha in
chronic myeloid leukemias. Br J Haematol 92:684-686, 1996
48. Saglio G, Pane F, Martinelli G, et al: BCR/ABL transcripts and
leukemia phenotype: An unsolved puzzle. Leuk Lymphoma 26:281286, 1997
49. Hochhaus A, Reiter A, Skladny H, et al: Cross NCP: A novel
BCR-ABL fusion gene (e6a2) in a patient with Philadelphia chromo-
some-negative chronic myelogenous leukemia. Blood 88:2236-2240,
1996
50. Negrini M, Tallarico A, Pazzi I, et al: A new chromosomal
breakpoint in Ph positive, bcr negative chronic myelogenous leukemia.
Cancer Genet Cytogenet 61:11-13, 1992
51. Saglio G, Guerrasio A, Tassinari A, et al: Variability of the
molecular defects corresponding to the presence of a Philadelphia
chromosome in human hematologic malignancies. Blood 72:12031208, 1988
52. Melo JV: The diversity of BCR-ABL fusion proteins and their
relationship to leukemia phenotype. Blood 88:2375-2384, 1996
53. van der Plas DC, Soekarman D, van Gent AM, et al: BCR-ABL
mRNA lacking ABL exon a2 detected by polymerase chain reaction in
a chronic myelogenous leukemia patient. Leukemia 5:457-461, 1991
54. Iwata S, Mizutani S, Nakazawa S, et al: Heterogeneity of the
breakpoint in the ABL gene in cases with BCR/ABL transcript lacking
ABL exon a2. Leukemia 8:1696-1702, 1994
Downloaded from jco.ascopubs.org on June 9, 2014. For personal use only. No other uses without permission.
Copyright © 2001 American Society of Clinical Oncology. All rights reserved.