From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Direct Correlation of Cytogenetic Findings With Cell Morphology Using In Situ
Hybridization: An Analysis of Suspicious Cells in Bone Marrow Specimens of
Two Patients Completing Therapy for Acute Lymphoblastic Leukemia
By John Anastasi, James W. Vardiman, Ruth Rudinsky, Mumtaj Patel, James Nachman, Charles M. Rubin,
and Michelle M. Le Beau
Bone marrow cells from two pediatric patients completing
therapy for acute lymphoblastic leukemia were studied using
in situ hybridization with an a-satellite DNA probe specific for
chromosome 17. Morphologic analysis of the end-therapy
specimens from each patient had shown small numbers
(7.5%. 8.5%) of cells that were suspicious for residual or
recurrent disease. These cells could not be morphologically
or immunophenotypically distinguished with certainty from
immature lymphoid cells (hematogones), which may be
present normally, sometimes in increased numbers, in the
bone marrow specimens of children. In situ hybridization
with a probe to chromosome 17 was used because the
leukemic cells from each patient had originally been shown
t o have an extra copy of this chromosome. In one patient, in
situ studies showed a population of cells (106 of 1.000 cells)
with three hybridization signals indicating trisomy 17, and
thus residualhecurrent leukemia. In the other patient trisomy 17 could not be detected. Additional hybridizations to
previously stained bone marrow aspirate smears permitted a
direct correlation of the cytogenetic findings with the suspicious cells on a cell-to-cell basis. The questionable cells were
identified, photographed, and then re-examined after hybridization. In one patient, 13 of 18 (72%) of the suspicious cells
were found t o have trisomy 17, whereas in the other patient 0
of 24 (0%) demonstrated an extra copy of this chromosome.
These cases illustrate a clinical application of interphase
cytogenetic analysis and demonstrate how this technology
can be used for direct correlation of cytogenetic findings with
cell morphology. This technique should prove useful for the
detection of minimal residual disease and for lineage studies
in leukemia and myelodysplasia.
o 1991by The American Society of Hematology.
T
differentiated
Through the use of chromosomespecific DNA probes or probes to specific regions of a
particular chromosome, it is possible to detect numerical
and even structural chromosomal abnormalities without
having to analyze the chromosomes directly. Interphase
cytogenetic analysis has the advantage of being rapid to use,
simple to interpret, and independent of the production of
high-quality metaphase spreads. Interphase cytogenetic
analysis is also possible on previously stained slides.4 This
analysis permits a direct correlation of cytogenetic findings
with cell morphology as it is routinely studied microscopically.
In this report we have applied the technique of interphase cytogenetic analysis to previously stained slides to
study small numbers of suspicious cells in the bone marrows
of two pediatric patients who were at the end of therapy for
hyperdiploid acute lymphoblastic leukemia (ALL). The
ability to correlate cell morphology directly with cytogenetic findings allowed us to identify morphologically the
suspicious cells in each patient and then to determine
whether these cells carried a cytogenetic anomaly that had
been previously demonstrated at the time of initial diagnosis. This cell-by-cell correlation of cytogenetic findings with
morphology should prove useful in the detection of minimal
residual disease and can provide a means for correlating
cytogenetic findings with cell lineage.
HE CORRELATION of cytogenetic findings with cell
morphology is not possible with conventional cytogenetic techniques. Nuclear morphology, cytoplasmic features, and membrane characteristics are all destroyed when
intact cells are first arrested in metaphase and then disrupted during the preparation of metaphase cells. Teerenhovi et all have developed techniques that permit some
correlation of cytogenetic information with cytoplasmic or
membrane characteristics. By using a combination of morphology, monoclonal antibodies, and chromosome studies
(the MAC technique), metaphase chromosomes can be
analyzed simultaneously with cytochemical or immunologic
features in intact cells. Although studies with the MAC
technique have provided important information regarding
the nature of the cell containing the cytogenetic abnormality in a number of
the technology has its
limitations. Chromosome spreading and banding are far
from optimal and morphologic assessment is severely limited because the nucleus is unrecognizable in metaphase.
Recently, molecular and cytogenetic techniques have
made it possible to derive cytogenetic information from
nonmetaphase cells, ie, from interphase and terminally
From the Depa rtments of Pathology, Pediatrics, and Medicine,
University of Chicago, Chicago, IL.
Submitted October 25, 1990; accepted February 4, 1991.
Supported in part by a grant (no. 89-44)from the American Cancer
Society, Illinois Division (J.A.),and by Public Health Service Grant
Rowley). J.A. is a Fellow and M.M.L. is a Scholar of
CA42557 (J.D.
the Leukemia Society of America; C.M.R. is a Pew Scholar in the
Biomedical Sciences.
Address reprint requests to John Anastasi, MD, Box 84, University of
Chicago Medical Center, 5841 S Maryland Ave, Chicago, IL 60637.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 I991 by The American Society of Hematoloa.
0006-4971191/77II-0020$3.00/0
2456
CASE HISTORIES
Patient 1. Patient 1 was a 7-year-old girl who presented after a
month-long illness characterized by fever, tender cervical adenopathy, progressive pallor, decreased activity, and anorexia. Physical
examination was significant for asthenia, moderately enlarged
tender cervical adenopathy, and a palpable spleen tip. Initial blood
count showed a white blood cell (WBC) count of 19.4 x 103/mm3
with 16% lymphoblasts; a hemoglobin of 7.4 g/dL, and a platelet
count of 53 x 103/mm3. Bone marrow aspirate yielded few
nucleated cells; however, the majority were lymphoid blasts (FAB
L1 morphology).
Cytochemical, immunocytochemical, and flow cytometry results
Blood, Vol77, No 11 (June 1). 1991: pp 2456-2462
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2457
CORRELATIVE INTERPHASE CYTOGENETICS
were consistent with precursor B-cell lineage leukemia (positive for
HLA-DR, CD10, CD19, and CD20; negative for myeloperoxidase,
CD13, CD33, CD7, TdT, and cytoplasmic mu). Cytogenetic analysis of bone marrow showed an abnormal hyperdiploid clone: 54,
XX,+X,+X,+6,+14,+ 17,+18,+21,+22. The patient was entered
on the Children’s Cancer Study Group 105 protocol for moderaterisk ALL and achieved remission. Maintenance therapy was
continued for 2 years.
At the end of therapy, a routine aspiration of bone marrow was
performed. The marrow aspirate was a cellular specimen that
showed adequate megakaryocytes, a normal myeloid-to-erythroid
ratio, and progressive maturation in all cell lines. Seven and
one-half percent of the marrow cells were immature, and most of
these had lymphoid features (Fig 1A). These cells ranged in size
from 10 to 15 pm in diameter, although some were larger, up to 20
to 25 km. The nuclear shapes were round or oval but, in some cells,
were indented and folded, and the nuclear chromatin structure
varied from coarse to dense and smudgy. Nucleoli were generally
absent, although one or two indistinct nucleoli were present in
some cells. These immature cells had blue cytoplasm, and occasional cells had small cytoplasmic vacuoles. These cells were
regarded as suspicious for residuaVrecurrent disease. Cytochemical and immunocytochemical studies showed that the cells demonstrated no myeloperoxidase or nonspecific esterase activity, and
possessed the antigen CDlO (CALLA). TdT analysis was not
performed. Because the suspicious cells could not be distinguished
with certainty from immature lymphoid cells (hematogones) that
have been reported to be present normally in the bone marrow of
children, a repeat bone marrow aspirate was obtained for cytogenetic analysis.
Patient 2. Patient 2, a 2 and 8/12-year-old girl, presented to her
pediatrician with pallor. After receiving a packed red blood cell
(RBC) transfusion for a hemoglobin of 2.3 g/dL, she was transferred to a tertiary care facility. At that time, the WBC count was
5.4 x lO‘/mm’ with 40% blasts; platelet count was 6 x 10‘/mm3.
c
Fig 1. Suspicious cells (lymphoid-morphology blasts) in the endtherapy bone marrow aspirates in patient 1 (A) and in patient 2 (B).
Morphologically, it is difficult to distinguish between hematogones
and residualhecurrentlymphoblastic leukemia.
Physical examination was significant for pallor and enlarged liver
and spleen (both palpable at the level of the unbilicus). A bone
marrow aspirate and biopsy showed that marrow elements had
been replaced by lymphoblasts with L1 morphology. Cytochemical,
immunocytochemical, and flow cytometric analysis of the blast
population showed precursor B-cell phenotype (positive for HLADR, CDlO, CD19, CD20, and TdT; negative for cytoplasmic mu,
surface Ig, myeloperoxidase, CD13, CD33, and CD7). Cytogenetic
analysis of peripheral blood showed an abnormal hyperdiploid
clone (55,XX,+X,+4,+6,+ lo,+ 14,+ 17,+ 18,+21,+21). Remission was induced with oral prednisone, intravenous vincristine, and
doxorubicin and intramuscular L-asparaginase, and consolidation
and reintensification therapy were administered. Maintenance
chemotherapy was given for 20 months after reintensification.
A bone marrow aspirate specimen was obtained at the completion of therapy. The aspirated marrow was very cellular. The M:E
ratio was normal. Granulopoiesis was unremarkable, erythropoiesis was normoblastic, and megakaryocytes were adequate. However, of concern was the finding of an increased number of
immature cells with lymphoid features (Fig 1B). These cells had
round to slightly irregular or indented, folded nuclei, high nuclear:
cytoplasmic ratios, and modest amounts of blue cytoplasm. The
nuclear chromatin was smudgy, although occasional cells had
reticular chromatin with indistinct nucleoli. In general, the cells
ranged in size from 10 to 15 pm in diameter, but some were larger.
These cells accounted for 8.5% of the nucleated marrow elements
and it could not be ascertained with certainty whether they were
residual or recurrent lymphoblasts or hematogones. Small lymphocytes with more “mature” chromatin were numerous. Immunophenotyping studies were not performed, but TdT analysis showed
nuclear staining in 13% of the marrow cells. A repeat bone marrow
was requested for cytogenetic studies.
MATERIALS AND METHODS
Specimens and controls. The repeat bone marrow aspirates for
cytogenetic studies were obtained in the ususal fashion from
puncture and aspiration of the posterior iliac crest. A portion of
each specimen was smeared onto glass slides and the remainder
was, anticoagulated with heparin or EDTA and submitted for
conventional cytogenetic analysis and for interphase study.
Control specimens were obtained from three women undergoing
bone marrow harvest for autologous bone marrow transplantation.
The primary malignancies of these patients were nonhematopoietic, and the bone marrows had been shown to be free of tumor.
Conventional cytogenetic analysis. Cytogenetic analysis using a
trypsin-Giemsa banding technique was performed on bone marrow
or peripheral blood cells obtained at the time of diagnosis, at the
time of therapy, or at relapse. Metaphase cells were examined from
direct preparations and from cells cultured for 24 or 48 hours
without mitogens. Chromosomal abnormalities are described according to the International System for Human Cytogenetic
Nomenclature.
Interphase cytogenetic analysis. Interphase cytogenetic analysis
was performed by two methods. In one, analysis was performed on
cells prepared in a manner similar to that for conventional
cytogenetic study excluding the short-term cultures and Colcemid
(GIBCO, Grand Island, NY) treatment. In the other method (for
correlative study), analysis was performed on cells that had been
previously stained with Wright’s stain.
In the first method, RBCs from the anticoagulated aspirate were
lysed with ammonium chloride (0.16 m o m ) . The resulting preparation was incubated with hypotonic potassium chloride (0.075
mol/L) for 8 minutes at 37°C and then centrifuged. The pellets
were resuspended and then the cells were fixed (three times) with
absolute methano1:glacial acetic acid (3:l vol/vol). The fixed cells
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2458
ANASTASI ET AL
were stored at -20°C until the time of hybridization. Immediately
before analysis the cells were resuspended and then dropped onto
glass slides and dried in an oven at 60°C for 1 to 2 hours. The
hybridization methods were similar to those described previously.”
Briefly, the DNA was denatured by immersion of the slides in 70%
formamide 2X SCC at 70°C. After denaturing, the slides were
dehydrated sequentially in 70%, 80%, and 95% ethanol and then
air dried. The 70% ethanol was ice chilled. The chromosome
17-specific probe was a biotin-labeled cy-satellite DNA probe
(Oncor, Gaithersburg, MD). The hybridization mix consisted of 2
KghL probe DNA, 50% formamide in 2X SCC, and 500 @mL of
carrier salmon sperm DNA. The probe mixture was denatured by
heating to 70 to 75°C for 10 minutes. It was then cooled quickly on
ice. After application of the denatured probe to the slides at 2 to 3
KL/cmZ,a coverslipwas added and sealed with rubber cement. The
slides were then incubated in a moist chamber for 12 to 16 hours at
37°C. After hybridization, the slides were washed at 47 to 49°C in
50% formamide 2X SCC (pH 7.0) for 30 minutes, placed in 1X
SCC for 30 minutes, and then stored in 4X SCC (room temperature). Hybridized probe was detected with fluorescein-labeled
avidin (Vector Laboratories, Burlingame, CA). In some cases
propidium iodide (1 pg/mL) was used as a nuclear counterstain.
The specimens were viewed at l00x magnification on a Reichert
Microstar IV microscope (Cambridge Instruments Inc, Buffalo,
NY) that was equipped for epifluorescence optics. Hybridization
signals were enumerated for each of 200 to 1,000cells. Photographs
were taken with Kodak Ektachrome 400 color film (ASA 400)
(Eastman Kodak, Rochester, NY), and the average exposure time
was 1to 2 minutes.
For correlative interphase studies routine smears of the bone
marrow aspirate were used. The slides were used within 1or 2 days
of the aspiration or were frozen at -70°C and used at a later time
(2 to 3 weeks). The slides were fixed with absolute methanol and
then stained with Wright’s stain. Coverslips were applied with a
dilute xylenemounting medium (Pro-Ten; American Scientific,
McGaw Park, IL) mixture. The slides were studied by routine light
microscopy, and photomicrographs of cells suspicious for lymphoblasts were taken. Because we found that the hybridization could
be more easily interpreted in areas where cells were not highly
crowded, we attempted to photograph from more dilutely smeared
areas of the slides. A microscope graduated stage was used to note
the location of each cell photographed. After the photomicroscopy,
the coverslips were carefully removed with incubation in xylene (5
minutes), and the slideswere air dried. Without destaining or other
pretreatment the slides were then hybridized as described previously. The hybridized probe was detected with fluoresceinated
avidin as described above. Using the same microscope, the cells
previously photographed were located and examined by fluorescent microscopy. The photomicrographs of the Wright-stained
cells were projected in the vicinity of the fluorescent microscope so
that simultaneous viewing of the previous light microscopic findings and the fluorescent hybridization results of the same cells was
possible. Hybridization signals were enumerated for each cell.
RESULTS
The repeat bone marrow aspiration was performed
within 1 week of the suspicious aspirate in the first patient
and within 1 month in the second. The repeat aspirate from
each patient showed a persistence of the immature lymphoid cells. In both patients the questionable cells increased slightly, from 7.5% to 10.6% of the total nucleated
marrow cells in patient 1, and from 8.5% to 9.5% in patient
2 (Table 1). Immunologic and cytochemical studies were
repeated on the specimen from the first patient, and the
cells were found to be myeloperoxidase negative, and CDlO
and CD19 positive as noted initially. Immunophenotyping
studies were not performed on the specimen from the
second patient, nor was TdT analysis repeated.
Interphase cytogenetics. In the in situ hybridization studies of lysed, hypotonically treated and fixed bone marrow
cells (ie, without correlation to cell morphology), analysis
showed that in patient 1, 106 of 1,000 interphase cells had
three hybridization signals, indicating trisomy for chromosome 17. (In a separate hybridization with probes to both
chromosome 17 and 9, the cells that showed three signals
for chromosome 17 exhibited only two signals for chromosome 9; this excluded the possibility that the cells were
triploid.) In patient 2, only 5 of 1,000 cells exhibited three
signals, and this was within the range of three normal
specimens where three signals were seen in 2 2 3 of 1,000
cells (mean and 2 SDs) (Table 1).
For correlative interphase cytogenetic analysis, photographs of 24 suspicious cells in patient 1 and 23 cells in
patient 2 were taken. After in situ hybridization, fluorescent
signals could be enumerated in 18 of the cells from patient 1
and all of the cells from patient 2. Thirteen of the 18 cells in
the first case (72%) (Fig 2A) and none (0%) of the cells in
the second case (Fig 2B) exhibited three hybridization
signals. The remainder of the evaluable cells photographed
in patient 1 showed two signals.
Conventional cytogenetic analysis. The results of cytogenetic analysis are summarized in Table 1. A t the end of
therapy, 3 of 22 metaphase cells from the bone marrow of
patient 1 had chromosomal abnormalities that represented
karyotypic evolution of the original clone. Each of these
three cells had an extra copy of chromosome 17. A normal
female karyotype was noted in the posttreatment marrow
sample from patient 2.
Follow-up. Patient 1, in whom residual or recurrent
leukemia was demonstrated, returned for a follow-up bone
marrow in 2% months. The repeat study showed an overt
relapse. Blasts accounted for 58% of the bone marrow cells
in this specimen and cytogenetic analysis revealed karyotypic evolution (see Table 1). Interphase analysis showed
three hybridization signals in 43% of 300 cells of this
marrow. The patient was reinduced, but despite the treatment she died due to infectious complications and refractory disease.
Patient 2 had a follow-up bone marrow aspiration 3
months after the second study demonstrated normal cytogenetic findings. Morphologically suspicious cells persisted
and accounted for 6% of the nucleated marrow cells.
Conventional cytogenetic analysis showed normal chromosomes and no cells (0 of 20) with trisomy for chromosome
17 were detected when studied by interphase analysis. This
patient continues in remission at 12 months after the
completion of therapy.
DISCUSSION
Immature cells in bone marrow aspirates from patients
treated for acute leukemia can present a diagnostic problem. In acute myelogenous leukemia (AML), neoplastic
myeloblasts present in small numbers can often not be
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2459
CORRELATIVE INTERPHASE CYTOGENETICS
Table 1.
Clinical Course
PATIENT 1
Diagnosis
7/87
Therapy
End-therapy
7187-10189
10189
Follow-up
10/89
Relapse
PATIENT 2
Diagnosis
1/90
9/87
Cytogenetics
Pathology
+
Correlative Interphase
Cytogenetics (chr. 17)
Interphase
Cytogenetics (chr.17)
ALL-L1 92% blasts
46,XX(33%)/54,XX, X,
+X,+6,+ 14,+ 17,+ 18,+21,
+22 (67%) (30 cells)
65% with 3 signals
(200 cells)
NA
Lymphoid-morphology
blasts (7.5%)
Lymphoid-morphology
blasts (10.6Y0)
NA
NA
NA
46,XX(86%)/52,XX,+6,
+14,+ 17,+18,+21,+22(9%)/
NCA: 55,XX,+X,+2,+6,
+14.+16,+17,+18,+21,
+22(5%) (22 cells)
46,XX(60%)/52,XX,+6,+14,
+ 17,+ 18, +21,+22(30%)/
52,XX,+6,+ 14, 17,+ 18,+21,
+22, dup(l)(q12+q32)
(10%)(20cells)
10.6% with 3 signals
(1,000 cells)
13/18 (72%) Lymphoidmorphology blasts with
3 signals
43% with 3 signals
(300 cells)
ND
46,XX(58%)/55,XX, +X,
+4,+6,+ lo,+ 14,+ 17,+ 18,
21, +21(38%)/NCA:
46,XX,t( 11;16)(q13;
q23)(4%)(24 cells)
NA
NA
NA
NA
NA
46,Xx(iOO%) (21 cells)
0.5% with 3 signals*
(1,000 cells)
46,XX(lOO%) (23 cells)
ND
0/23 (0%) Lymphoidmorphology blasts with
3 signals
0/20(0%) Lymphoidmorphology blasts with
3 signals
Overt relapse
(58% blasts)
ALL-L1
+
+
Therapy
End-therapy
9/87-1190
1/90
Follow-up
2/90
Remission
4/90
Lymphoid-morphology
blasts (8.5%)
Lymphoid-morphology
blasts (9.5%)
Lymphoid-morphology
blasts (6%)
Abbreviations: NCA, nonclonal abnormality; (no. cells), number of cells analyzed; NA, not available; ND, not done.
'Within range of three control specimens in which three signals were seen in 0.2% k 0.3% of cells (mean k 2 standard deviations).
distinguished morphologically from regenerating blasts after myelosuppressive therapy. In ALL in pediatric patients
the finding of immature cells with lymphoid morphology
can pose diagnostic difficulties. Neoplastic lymphoblasts,
especially those of L-1 morphology, may be morphologically
similar to immature lymphoid cells (hematogones), which
have been reported to be present normally in the marrow of
~hildren.'~.'~
These latter cells may not only be indistinguishable morphologically from malignant lymphoblasts, but
they can also be similar immunophenotypically and with
regard to TdT activity.I6Hematogones can express CDlO,
they can be TdT positive, and they can express surface
markers of immature lymphocytes. Because hematogones
have also been reported to be elevated in some children
after chemotherapy," it has been suggested that to differentiate neoplastic lymphoblasts from hematogones, it may be
necessary to correlate morphology, immunophenotype, and
TdT reactivity with cytogenetic findings before a final
diagnosis can be rendered.16
In this report we describe two patients in whom suspicious cells presented a diagnostic problem; ie, were they
hematogones or did they represent recurrent or residual
leukemia? Because the problem arose at the end of
therapy, its significance was more than academic; a decision
about continuing therapy had to be made. Morphologic
evaluation was not conclusive in assessing the nature of the
blasts, and TdT reactivity and immunophenotyping were of
little help. Because both patients had cytogenetic abnormalities at initial diagnosis, repeat bone marrow examinations
were performed in hopes that cytogenetic findings could
resolve the diagnostic problem. In addition to conventional
cytogenetic study, the repeat aspirates were submitted for
interphase cytogenetic analysis. These cases were well
suited for this latter type of analysis because the original
leukemic clone in each patient had numerical chromosomal
abnormalities (ie, extra copies of multiple chromosomes).
Interphase cytogenetic analysis can easily be used to detect
trisomy for a targeted chromosome through in situ hybridization with an appropriate DNA probe.I3
In specimens prepared in a manner similar to that used
for conventional cytogenetics (excluding cell culture and
Colcemid treatment), we were able to use interphase
analysis to detect rapidly a population of interphase cells
with trisomy 17 in patient 1. This analysis indicated that the
patient's neoplastic clone was present as residualirecurrent
disease. In patient 2 we could not identify such a population
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ANASTASI ET AL
2460
i
-1
t
Fig 2. Correlative interphase cytogenetic analysis in patient 1 (A) and in patient 2 (a).Resultsfrom fluorescent hybridizationwith a probe to
chromosome 17 are illustrated next to the same Wright-stained cells in each specimen. In (A), suspicious cells with three hybridizationsignals
indicatethe presence of trisomy 17 and, thus, residualhecurrentleukemia. In (B) each of the suspiciouscells has only two hybridizationsignals,
indicating that they are more likely hematogones.
and concluded that the suspicious cells were more likely to
be hematogones. We could not exclude the possibility that
the neoplastic cells had lost the extra copy of chromosome
17, but felt that this was unlikely, because it is more
common for a leukemic karyotype to undergo clonal evolution with the development of additional karyotypic changes
rather than to lose abnormalities noted initially." The
additional finding in this case that there was no population
of cells trisomic for the X chromosome (data not presented)
also supported our conclusion because trisomy for the X
chromosome had also been seen in the initial clone.
Furthermore, by performing the in situ hybridization on
previously stained slides, we were able to correlate directly
the cytogenetic findings with cell morphology. We demon-
strated that the trisomy occurred specifically in the immature cells of question in the first patient, and not in the
suspicious cells in the second. Therefore, the technique of
interphase analysis on previously stained slides allowed for
the morphologic identification of suspicious cells, and then
for the subsequent determination of whether such cells
carried a targeted cytogenetic abnormality.
Correlation of cytogenetic findings with cell morphology
as illustrated here has a number of potentially important
uses: it can be useful in the detection of residual leukemia,
and it can be employed in the study of cell lineage and
clonality in myelodysplasia and acute leukemia. In the
detection of residual disease, correlative interphase cytogenetic analysis should increase the sensitivity of identifying
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2461
CORRELATIVE INTERPHASE CYTOGENETICS
rare neoplastic cells. Our first case (patient 1) clearly
illustrates its advantage over morphologic analysis alone.
Morphologically suspicious cells could not be interpreted
unequivocally as benign or malignant, but with the added
information from interphase study the neoplastic cells
could be positively identified. Although in this case conventional cytogenetic analysis and interphase analysis without
morphologic correlation each detected the residual/
recurrent disease, we suspect that when the neoplastic cells
are fewer in number, the correlative approach should offer
a more sensitive method for the detection of rare malignant
cells. For example, when residual/recurrent blasts are fewer
than 1% or 2%, conventional cytogenetic study would
require the analysis of more than 100 metaphase spreads to
detect such a population, and noncorrelative interphase
study is close to its limit of detection. The correlative
interphase approach should increase the sensitivity of
detecting rare malignant cells because it focuses the analysis on the cells in question and obviates the need of
including in the analysis marrow elements that are obviously benign (eg, most erythroid and granulocytic precursors in a patient with ALL).
Correlative interphase cytogenetic analysis will clearly
not be as sensitive as the polymerase chain reaction (PCR)
in detecting rare neoplastic cells. The latter technique can
be used to detect cells as infrequent as 1 in lo4 to 105.19
Nevertheless, correlative interphase cytogenetic analysis is
simple to use, easy to interpret, and more widely applicable
than PCR. For example, numerical abnormalities in hyperdiploid ALL and in AML can be detected, and given new
approaches to detecting structural chromosomal abnormalities in interphase cells, the applicability of this technology
will become even broader.20
Correlative interphase cytogenetic analysis should also
have important uses in the study of cell lineage and of the
clonal origin of cells in acute leukemia and myelodysplastic
syndromes. Through the analysis of cases previously demonstrated to have cytogenetic abnormalities suitable for interphase study, it will be possible to determine quickly and
easily which cell lines are involved in the neoplastic clone,
and to what extent the involvement exists. With the development of new treatment strategies in myelodysplasia and
leukemia (eg, growth factor therapy), it may become useful
to be able to define the lineage involved in the neoplastic
condition and to monitor the involvement throughout
t herapy.21722
ACKNOWLEDGMENT
The authors thank Dr Janet D. Rowley for her support; Rafael
Espinosa, Lydia Jiminez, Leslee Snyder, Elizabeth Davis, and
Edward Asner for technical assistance; and Gordon Bowie for help
with the photography.
REFERENCES
1. Teerenhovi L, Knuutila S, Ekblon M, Rossi L, Borgstrom
GH, Tallman JK, Anderson L, de la Chapelle A: A method for
simultaneous study of the karyotype, morphology, and immunologic phenotype of mitotic cells in hematologic malignancies. Blood
64:1116, 1984
2. Knuutila S , Keinanen M: Chromosome banding techniques
for morphologically classified cells. Cytogenet Cell Genet 39:70,
1985
3. Teerenhovi L, Wasenius V-M, Franssila K, Keinanen M,
Knuutila S: A method for analysis of cell morphology, banded
karyotype, and immunoperoxidase identification of lymphocyte
subset on the same cell. Am J Clin Pathol 85:602,1986
4. Knuutila S, Teerenhovi L: Immunophenotyping of aneuploid
cells. Cancer Genet Cytogenet 41:1, 1989
5. Keinanen M, Griffin JD, Bloomfield CD, Machnicki J, de la
Chapelle A Clonal chromosomal abnormalities showing multiplecell-lineage involvement in acute myeloid leukemia. N Engl J Med
318:1153,1988
6. Knuutila S, Elonen E, Teerenhovi L, Rossi L, Leskinen R,
Bloomfield CD, de la Chapelle A: Trisomy 12 in B cells of patients
with B-cell chronic lymphocytic leukemia. N Engl J Med 314:865,
1986
7. Cremer T, Landegent J, Bruckner A, Scholl HP, Schardin M,
Hager HD, Devilee P, van der Ploeg M: Detection of chromosome
aberrations in the human interphase nucleus by visualization of
specific target DNAs with radioactive and non-radioactive in situ
hybridization techniques: Diagnosis of trisomy 18 with probe
L1.84. Hum Genet 74:346, 1986
8. Landegent JE, Jansen in de Wal N, Baan RA, Hoeijmakers
JHJ, van der Ploeg M: 2-Acetylaminofluorene-modifiedprobes for
the indirect hybridochemical detection of specific nucleic acid
sequences. Exp Cell Res 153:61, 1984
9. Pinkel D, Straume T, Gray JW: Cytogenetic analysis using
quantitative, high sensitivity, fluorescence hybridization. Proc Natl
Acad Sci USA 83:2934,1986
10. Trask B, van den Engh G, Landegent J, Jansen in de Wal N,
van der Ploeg M: Detection of DNA sequences in nuclei in
suspension by in situ hybridization and dual beam flow cytometry.
Science 220:1401,1985
11. Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC:
Delineation of individual chrosomes in metaphase and interphase
cells by in situ supression hybridization using recombinant DNA
libraries. Hum Genet 80:224, 1988
12. Lichter P, Cremer T, Tang CC, Watkins PC, Manuelidis L,
Ward DC: Rapid detection of human chromosome 21 aberrations
by in situ hybridization. Proc Natl Acad Sci USA 85:9664,1988
13. Anastasi J, Le Beau MM, Vardiman JW, Westbrook C A
Detection of numerical chromosomal abnormalities in neoplastic
hematopoietic cells using in situ hybridization with a chromosomespecific probe. Am J Pathol 136:131, 1990
14. Muehleck SD, McKenna RW, Gale PF, Brunning RD:
Terminal deoxynucleotidyl transferase (TdT)-positive cells in bone
marrow in the absence of hematologic malignancy. Am J Clin
Pathol79:277, 1983
15. Woessner S, Sans-Sabrafen J, Lafuente R, Florensa L
Asymptomatic bone marrow infiltration by blast cells. N Engl J
Med 312:1129,1985 (letter)
16. Longacre TA, Foucar K, Crago S , Chen I-M, Griffith B,
Dressler L, McConnell TS, Duncan M, Gribble J: Hematogones: A
multiparameter analysis of bone marrow precursor cells. Blood
73:543, 1989
17. Steckhoven JHS, Langenhuysen CAM, Bakkeren JAJM,
Holland R, Holler I, de Vaan GAM, Schretlen EDAM: Morphology and incidence of the “posttheraputic lymphoid cell” in the
bone marrow of children with acute lymphoblastic leukemia. Am J
Pathol 124:46,1986
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2462
18. Secker-Walker LM, Alimena G, Bloomfield CD, Kaneko Y,
Whang-Peng J, Arthur DC, de la Chapelle A, Reeves BR, Rowley
JD, Lawler SD, Mitelman F: Cytogenetic studies of 21 patients
with acute lymphoblastic leukemia in relapse. Cancer Genet
Cytogenet 40:163,1989
19. Yamada M, Wasserman R, Lange B, Reichard BA, Womer
RB, Rovera G: Minimal residual diseases in childhood lymphoblastic leukemia-Persistence of leukemic cells during the first 18
months of treatment. N Engl J Med 323:448,1990
20. Lichter P, Ward DC: Is non-isotopic in situ hybridization
finally coming of age? Nature 343:93,1990
ANASTASI ET AL
21. Vadhan-Raj S, Broxmeyer HE, Spritzer G, LeMaistre A,
Hultman S, Ventura G, Tigaud J-D, Cork MA, Trujillo JM,
Gutterman JU, Hittelman WN: Stimulation of nonclonal hematopoiesis and suppression of the neoplastic clone after treatment with recombinant human granulocyte-macrophage colonystimulating factor in a patient with therapy-related myelodysplastic
syndrome. Blood 74:1491,1989
22. Negrun RS, Haeuber DH, Nagler A, Kobayashi Y, Sklar J,
Donlon T, Vincent M, Greenberg P L Maintenance treatment of
patients with myelodysplastic syndromes using recombinant human
granulocytic colony-stimulating factor. Blood 76:36, 1990
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1991 77: 2456-2462
Direct correlation of cytogenetic findings with cell morphology using
in situ hybridization: an analysis of suspicious cells in bone marrow
specimens of two patients completing therapy for acute lymphoblastic
leukemia
J Anastasi, JW Vardiman, R Rudinsky, M Patel, J Nachman, CM Rubin and MM Le Beau
Updated information and services can be found at:
http://www.bloodjournal.org/content/77/11/2456.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.