General implications of chromosomal alterations in human leukemia

G E N E R A L I M P L I C A T I O N S OF CHROMOSOMAL ALTERATIONS IN H U M A N LEUKEMIA* Josd M. Trujillo, M.D.,'~ Michael J. Abeam, Ph.D.,$ and Ann Corh, M.A., M.T. (A.S.C.P.)w

Abstract Analysis o f bone m a r r o w cells is essential for tim complete cytogenetic clmracterization o f leukemia. T i m i m p o r t a n t teclmical r e q u i r e m e n t s for dealing witl~"bone m a r r o w include anticoagulation, p r o m p t processing, removal o f excessive n u m b e r s o f erythrocytes, inoculation o f optimal n u m b e r s o f cells, use o f fetal calf sernm, use o f hypotonic p h o s p h a t e buffer to swell the cells, and p h o t o g r a p h y with a camera that p r o d u c e s a large negative. Seventy-five to 90 p e r cent o f adult patients with chronic myelogenous leukemia lmve the Plfiladelplfia (PI?) c h r o m o s o m e in bone m a r r o w cells. Patients with the Pht c h r o m o s o m e Imve a better prognosis than do those who lack it. C h r o m o s o m a l studies d u r i n g the acute t r a n s f o r m a t i o n Cblastic crisis") o f chronic myelogenous leukemia have shown a high incidence o f a b n o r m a l karyotypes in which o n e or m o r e PI? cltromosomes or otlter markers are present, suggesting tlmt a process o f clonal evolution has p r o d u c e d clones tlmt are m o r e resistant to t h e r a p y . In a series o f 170 patients with acute leukemia who had sequential bone m a r r o w cytogenetic studies, we have f o u n d the incidence o f a n e u p l o i d y to be 40.5 per cent. Most o f these patients lind acute myelogenous leukemia. Fortysix per cent o f the a n e u p l o i d karyotypes were hyperdiploid, 25 per cent were hypodiploid, and 29 p e r cent were pseudodiploid. C o n t r a r y to general opinion, certain o f tlm aneuploid cytogenetic profiles had a t e n d e n c y to occur in repetitive fashion in genetically unrelated patients. Certain profiles also carried prognostic implications. Patients witlt diploid and hyperdiploid karyotypes survived for 49 weeks (mean survival time), whereas those with pseudodiploidy and hypodiploid)' survived for only 36 and 13 weeks, respectively. O u r studies lmve shown repeatedly tlmt a favorable response to t h e r a p y is m a r k e d by early disappearance o f the aneuploid or pseudodiploid clone and that this same a b n o r m a l clone r e a p p e a r s when the leukemia relapses. By con-

*Tile cytogenetic data included in this study were obtained through tile support of grant CA 12687 from the National Cancer Institnte, National Institutes of ttealth, U.S. Public Health Service. tProfessor of Pathology, Chief, Section of Cytogenetics, and Head, Department of Clinical Chenlistr)" and Laboratory Medicine, The University of Texas S)'stem Cancer Center, M. D. Anderson Hospital and Tumor Institute, Hottston, Texas. +Associate Professor, Department of Clinical Chemistry and Laboratory MediCine,The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, ttouston, Texas. w Assistant, Section of Cytogenetics, Department o]" Clinical Chemistry and Laboratory Medicine, The University of Texas System Cancer Center, M. D. Anderson ttospital and Tumor Institute, Houston, Texas.

675

IIUMAN PATIIOLOGY-VOLUME 5, NUMi',ER 6 November197.t trast, in patients with chronic myelogenous leukemia irl remission, the l'h t ciirolllOSonle persists in tile bone marrow at its initial frequency. Electron microscopic studies on aliquots of the bone marrow samples examined for cytogenetic study Imve shown a high incidence of nuclear "blebs" in aneuploid leukemic bone marrows. These "blebs" appear to be derived fi'om excessive proliferation of the nuclear membrane, disappear along with the aneuplaid clone during remission, and reappear at the time of relapse. Cytogenetic analysis has provided the clinician with a usefid new tool to characterize the leukemias.

676

Studies of the chromosomal changes in hulnan leukemia have been underway for the past 15 )'ears. The karyotypic aherations commonly associated with the neoplastic disorders of hematopoietic tissue have been described in numerous pal)ers) -r' Altiiough the nulnber of reported patients with leukemia who have undergone cytogenetic studies is probably over 1000, the significance of the chromosomal ahcrations in this disease is still obscure. Altltough cytogenetic studies have not blougllt new solutions for the problems of etiology, prevention, or cure of imman leukemia, these studies have contributed to a better clinical characterization of the disease. Cytogenetic studies of human leukemia have followed two different approaches. The first work was basic cancer research performed in the early 1960's, and more recent studies have been pertormed primarily by clinicians. The initial stt,dies of leukemias were centered around the Boveri ID'pothesis, which considered chromosomal.clmnges to be essential for the development of the neoplastic process. Later workers emphasized the possible usetidness of cytogenetic parameters for characterization of clinical disorders. Althougll tile Boveri ID'pothesis was finally disproven, it is not reasonable to believe that cllromosomal changes are without meaning in understanding the leukemic or neoplastic cell. Chromosomes play a central role in the organization of the cell, and any structtn'al or nutnerical alteration in the chromosomal apparatus must Imve lliological implications. In fact, as BaikiC 3 Ires emphasized, "If changes in the genetic al)paratus of the cell play an essential part in the induction of leukemia, then the clwomosomes are t h e m o r e likely site of such clmnges."

hi tiffs t-epol't we shall review the contributions of cytogenetics in the study of hunmn leukemia. The clinical aspects of these studies are enaphasized, since cytogenetic parameters are now used widely in the clinical evahmtion of leukemia. In discussing the acute leukemias we shall follow the usual classification into myelogenous and lymphocytic types. For the chronic nweloproliferative disorders we shall follow the classification of Dameshek into chronic myelogenous leukelnia, polycythemia vera, inyelofibrosis with myeloid metaplasia, and thrombocythenfia.

TECHNICAL REQUIREMENTS Standard methods for the cytogenetic analysis of peril)heral blood are used witli success in many laboratories. In our experience these methods have not been adequate for the study of bone marrow, which is essential for any complete cytogenetic characterization of leukemia. Two useful methods are available. The tirst is the direct method, which uses a short incubation time of two hours. Colcetnid is used in order to arrest mitosis in metaphase. With the second technique the bone marrow samples are incubated for 2,t hotws or more. The two methods are complementary, and both should be used in order to recover more cells in metaphase than is possible with either method alone. We have found the tollowing technical features to be important in obtaining satisfactory preparations of leukemic tissue: BONE ~|ARROW PREPARATION FOR CYTOGENETIC STUDY. The specimen

should be obtained in a separate 10 ml. syringe, which should be prepared by aspirating a small amount of liquid heparin

CHROMOSOMAL ALTERATIONS IN HUMAN LEUKEMIA--TROJILLO ET AL. and then expressing it, leaving only about 0.1 ml. o f heparin. Approximately 1.5 ml. of bone nmrrow should be aspirated into the heparinized syringe. ANTICOAGULATION. It is imperative tllat the bone marrow not clot. Heparin shoukl be used as just directed. T o o much heparin may interfere witll growth of the bone marrow cells in short term culttu'e. Etlwlenediamine tetra-acetic acid (EDTA) should not be nsed for tiffs same reason. PROMPT PROCESSING. T h e b o n e m a r r o w santple silonld be processed witllin one hotlr.

RE.XIOVAL OF EXCESSIVE ERYTtlROCVTZS. Excessive contamination of the bone marrow sample with erythrocytes prevents good growth in cuhure, and the red blood cells are difficult to lyse and remove froln the preparation subsequently. Tile bone marrow sample is therefore diluted in 5 to 10 ml. of culture m e d i u m (see following) and centrifuged at 800 to 1000 rpm for five minutes at room temperature in o r d e r to remove as many red blood cells as possible. INOCULATION OF OPTIMAL NUMBER OF BONE MARROW CELLS. Centrifugation

produces a buff)' coat, which is introduced into anotltel" aliquot o f culttu'e medinm. If the bone ntarrow is hypocellular, all the buff)' coat should be inoculated. For a normal bone marrow, about 0.5 ml. of buff)' coat sllonld be inoculated. If tile bone Inarrow is Ilypercelhflar (and the antount of buff)' coat large), only part of it sltould be inoculated. Wllen the bone marrow is markedly layper~zellular, e.g., in chronic myelogenous leukentia, only 0.1 nil. o f the bttffy coat should be used, because inoculation of too ninny cells inllibits g r o w t h in the sllort term culture. CULTURE MEDIUM. We llaveobtained good results by using 8 ml. of F-10 culture mediuni supplemented witlt 2 ml. o f fetal calf serum.* Penicillin G is a d d e d at a concentration of 100 units per ml. GLASSWARE. We have f o u n d it advantageous to use Owens bottles (2 oz.) because tile material is cheap e m m g h to be disposable. Betore use tlle bottles are rinsed with distilled water attd autoclaved.t *Both obtainable from Grand Island Biological Company, 3175 Stale)" Road, Grand Island, New York 14072. tThis glassware is obtainable from McKesson and Robbins Drug Co., Inc., 3122 I.eeland, ttouston, Texas.

DURATION OF CULTURE.

A two llour

culture is usually satisfactory for bone marrow study, but better resuhs are obtained if cnltures are allowed to incubate for 24 hours. HVI'OTONIC SOLUTION. A t f i l e end of the incubation period the c n h u r e m e d i u m is centrifilged, tlle liquid decanted, and tile pellet waslled once with 0.9 per cent saline in o r d e r to remove protein. A hypotonic sohltion is tllen a d d e d in o r d e r to swell the cells. Althougli 0.075 M potassium clfloride is suitable for working witll lympllocytes fiont the periplleral blood, we h a v e obtained superior results with bone marrow by using hypotonic pilosp h a t e bult'er: l'ho.q~hate buffer (ptl 7.3) stock solution NaCI NazHPO~ Nalt_,I'O~ * tt20 Distilled H.,O

90.0 13.6 " 2.14 1000.0

gm. gin. gin. nil.

T h e stock sohltion is diluted witll distilled water for use. Tlle pellet is susp e n d e d in and then exposed to the hypotonic bnffer for 10 minntes. T h e cells are then centrifltged, tlte buffer decanted, new buffer a d d e d for anotller 10 minutes, centrifilged, and the bnffer decanted completely. FIXATION, STAINING, AND SPREADING.

Fixation, staining, and spreading of the cells on slides for examination may be p e r f o r m e d by either the sqttasll or the air-dry teclmiqne. We prefer to make squash preparations. Tire cell pellet is fixed by exposure to 50 per cent acetic acid for at least one-half llour. T h e acid is discarded, and the cells are then resusp e n d e d in 2 per cent aceto-orcein (2 per cent natural orcein m a d e tip in 50 per cent acetic acid).* T h e preparations are sealed witll wax and examined wet with the phasecontrast microscope.t Many laboratories prefer tile air-dry teizlutiqne, in which the cells are fixed in glacial acetic acid-metllanol (1 vohtme: 3 voluntes). Tlle freslfly prepared fixative mttst be a d d e d to tile cells d r o p by d r o p and nfixed well. T h e cells suspended in *Natural orcein is obtainal~le tu George T. Gt~rr, Ltd., Esbe Laboratory Supp!y, 3431 Bathurst St., Toronto, Ontario, Canada. ~'We have obtained good results with Sealing Wax-Deckglaskitt nach Kr6nig, Pfahz and Bauer, Inc., 126-02 Northern Blvd., Flushing, N.Y. 11368.

677

HUMAN I'ATHOLOGY--VOLUME 5, NUMBER 6 fixative may be transferred to glass slides by either of two techniques: Tile cell suspension may be dropped onto a dry slide from a Pasteur pipette h e l d a b o u t 6 inches above tile slide and dried quickly, or a drop of tile cell suspension may be added to a slide tlmt has been immersed in cold water; this causes the cells to spread readily. The slide is then allowed to air-dry and is stained with Giemsa stain. Tile advantages of this teclmique are that the preparations are permanent and that no phase-contrast mmroscope is required. PHOTOGRAPHY. It is advantageous to photograph cells in metaphase with a camera tlmt produces a large negative so that tile images of the individual chromosomes are sharp after enlargement. CHRONIC MYELOPROLIFERATIVE DISEASES

678

A defective small G chromosonm in the peripheral blood and bone marrow of patients with chronic myelogenous leukemia is still the most consistent and most specific clmnge associated with a human neoplastic condition. Since its discovery by Nowell and Hungerford ~5 in 1961, tiffs altered chromosomal element has been known as the Philadelplfia chromosome (Pli'). By use of such new teclmiques as fluorescence microscopy after staining witfi qu!nacrine mustard and enzymatic digestion with trypsin before Giemsa staining, the Ph I chromosome has been identified as a member of the number 22 pair instead of the 21 pair as was formerly believed, t6 Tiffs marker chromosome Ires represented an important tool in the cytological and clinical evahmtion of the myeloproliferative disorders, and it has the following implications: 1. The Pht chromosome is found in 75 to 90 per cent of the typical cases of chronic myelogenous leukemia."" 17-',-,Althougli a phi-like chromosome has occasionally been described in other leukemic disorders, the presence of the Ph I chromosome is now considered to be pathognomonic of chronic myelogenous, leukemia. The Plat chromosome is a more reliable diagnostic tool than the level of neutrophil alkaline phosplmtase, which can be normal or even elevated in patients with chronic

November 1 9 7 4

myelogenous leukemia when tile)' have bacterial infections, when they are in remission, or when the disease undergoes blastic transformation. In addition, the level of leukocyte alkaline phosphatase can be very low in diseases other than chronic myelogenous leukemia. 2. Tim Phi-negative patient with chronic myelogenous leukenfia has a poorer prognosis than does the Pl?Zpositive patient. A clear understanding of the l'h'-negative group o f patients requires separate consideration of children and adults. Cytogenetic investigations of children with chronic myelogenous leukemia were first carried out by Reisman and Trujillo, -~ who studied nine patients. In four of them no I'h 1 chromosome was found. Clinical and hematological studies of these children showed timt the PI?negative group differed fi'om th'e Phi-positive patients in certain respects; i.e., they frequently had earl)' thrombocytopenia with bleeding tendencies, they were less responsive to treatment with busulfan, and their disease ran a shorter conrse.

The poor prognosis of some children with chronic myelogenous leukemia had been noted previously by other workers, who therefore made a distinction between the juvenile and the adult forms of tim disease in children. Hardisty, Speed, and Till 24 confirmed the absence of the Ph ~ chromosome in children who had the juvenile form, and reported that "Our findings lead to the conclusion that the presence or absence of tim Ph ~ chromosome provides the surest means of distinguishing between the adult and juvenile types of chronic granulocytic leukemia and the strongest for regarding them as two different entities." Adults with Phi-negative chronic myelogenous leukemia present several hematological differences fi'om Pl?-positive patients. Tjio et al.-~~ found median values for tile white blood cell and platelet counts of 41,500 per cu. mm. and 180,000 per cu. mm., respectively, in 13 Ph'-negative patients. Tim comparable median values in 60 Pht-positive patients were 133,700 p e r cu. mm. and 388,000 per cu. nun., respectively. More importantl y , tim phi-negative patients responded poorly to treatment. Their median snr-

CHROMOSOMAL ALTERATIONS IN HUMAN LEUKEMIA--TRuJILLOET AL. viral from the time of diagnosis was only 18 months, compared to 45 months for the Phi-positive group. Ahhough absence of the Ph ~ chromosome in chronic myelogenous leukemia therefore has therapeutic and prognostic implications, it is not yet clear whether these differences indicate the existence of two separate nosological entities. 3. The presence of the Ph ~ chromosome in the myeloid, erythroid, and megakaryocytic cells of tile bone marrow was demonstrated in 1963 and later confirmed. "5-27 This finding h a s a dual significance. First, it provides strong evidence for the existence of a common hematopoietic precursor cell (stem cell) distinct from lymphocytes, which appears not to be involved'in chronic myelogenous leukemia. Second, it indicates tlmt chronic myelogenous leukemia should be considered as a condition affecting not only the myeloid cells, as the name implies, but tile entire hematopoietic system. In patients with chronic myelogenous leukemia who respond well to therapy, tile clinical and hematological parameters (i.e., palpable spleen, high white blood cell cotmt, low hemoglobin level, and increased myeloid:erythroid ratio) all return to normal. Microscopic examination of the bone marrow fails to demonstrate an)' evidence of residual disease. Serial chromosomal studies in patients undergoing therapy have shown a decrease in the number of Phi-positive cells in the peripheral blood (coincident with the gradual disappearance of immature granulocytes fiom the circulation), which thus marks die initial effect of treatment. m'8-3~ During periods of complete remission, no I'l?-positive cells can be recovered from cultures of the peripheral blood. However, during even the best remissions the PI? persists in the bone marrow at its initial frequency. This finding suggests that present methods of treatment of chronic myelogenous leukemia are only palliative."9 4. T h e most common cause of death in chronic myelogenotls leukemia is a transformation to a more acute phase, known as the "blastic crisis," which occurs in about two-thirds of the patientsY Chromosomal studies during this acute phase have demonstrated a high inci-

dence of karyotypic abnormalities other than tile I'17 chromosome, a~-3~ Often these are multiple clones of aneuploid cells in which one or more PI? chromosomes or other markers are present? 2"34"3z T h e presence of identical markers in several aberrant clones indicates that muhiple lines of cells, karyotypically different but related, are evolving one from another through successive steps of misdivision, thereby creating an evolutionary trend for the appearance of more successful and probably more therapy-resistant clones. Bent Pedersen 36-3s has emphasized this idea of clonal evolution and has postulated that the resulting Phi-positive clones with additional karyotypic aherations are most resistant to cytostatic agents. We lmve obtained evidence from serial cytogenetic studies of a patient, who received toxic doses o f busulfan for several weeks that suggest that Pl?-positive clones may develop increased drug resistance. 7 More recent studies of a series of patients with chronic myelogenous leukemia in blastic crisis indicate that many of the Pl?-negative patients develop aneuploidy when their disease undergoes acute transformation.35 Ahhough the blastic crisis is not always'accompanied by additional karyotypic aberrations, the appearance of bone marrow cells with riew karyotypic abnormalities in Pi?-positive or Pl?-negative patients with chronic myelogenous leukemia almost invariably signals the development of tile acute phase of the disease. T h e s e late-developing aneuploid clones usually are resistant to the drug therapy that was formerly effective. The significance ofcytogenetic studies in the other myeloproliferative disorde!'s is less clear-cut. 7 T h e variability of the results can be attributed to several factors: (I) These disorders, including some atypical cases of chronic myelogenous leukemia, constitute an ill defined group of conditions that have overlapping clinical and hematological aspects. (2) Transitions between the various disorders occur, and mixed or borderline cases exist. (3) The clinical and hematological criteria for the classification of these disorders are variable. In general, the typical cases of a given condition can be distinguished easily, as is tile case with chronic myelo-

679

HUMAN PATHOLOGY--VOLUME 5, NUMISER 6 November 1974 genous leukemia. In contrast, ntyelofibrosis poses a more diflicuh problem, because polycythenfia vera may terminate with clinical and histological features indistinguishable fi'om those of myelofibrosis, and a few patients with chronic myelogenous leukemia have been known to develop marked fibrosis of the bone marrow. Kay, l.awler, and Millard a'~ have cartied out the best cytogenetic studies of polycythentia vera. In a study of ,t3 patients these workers found that the presence of aneuploid clones long after t r e a t m e n t with 3.,p was of value in predicting thedevelolnnent of acute leukenfia. Tiffs finding is of special significance, since the incidence of acute leukemia in patients,.xvith polycythemia vera is much ifigher after 3ap treatment. More recent studies by these same workers have confirmed their initial findings and have reemphasized the observation that abnormalities of the F group chrontos01ncs, which occur rarely in acute leukemia, m-e not infrequent in polycythemia vera? ~ There are a few reports of abnormalities involving the C group chromosomes in patients with myelofibrosis,r'4t However, most typical cases show no chromosomal almorxnalities. There have also been reports of atypical or acute myelofibrosis with aneuploid karyotypes. Possibly these cases belong in a categor X closet- to, if not identical with, acute leukemia.r.a.-, The km-yotypes of patients with thrombocythemia have been found to be diploid.

ACUTE LEUKEMIA

680

T h e reported incidence of aneuploidy in acute leukemia has varied fi'om 29 to 100 per cent. t'3-v' The lower figure may be explained by the inclusion of treated patients who have lost the aneuploid cell line. '~ The higher incidence was given in early reports that dealt with a limited ntnuber of patients who were mostly children? Larger studies have sllown all incidence of aneuploidy in acute leukemia of 40 to 50 per cent. 6-t" In a series of 170 patients with acute leukemia who underwent .sequential cytogenetic studies, we have found the incidence of aneuploidy to be ,t0.5 per cent. v' Most of the patients

in tiffs series had acute myelogenous leukemia, in contrast to the larger number of patients with acute l)'mphocytic leukemia and stem cell leukemia reported by other investigators. Most reports support the contention of Sandberg et a13' n that aneuploidy in acute leukemia shows considerable variability from patient to patient, u In their study of 219 cases of acute leukemia these workers observed: (1) stability of the initial karyotype during the phases of renfission and relapse and (2) lack of hypodiploidy in patients with acute lymphocytic leukentia compared to those with acute myelogenous leukexnia, in whom only hyper- and hypodiploidy were observed. 6 More recently Fitzgerald et al. u have inade similar observations, ahhough they reported one patient who seemed to be the exception to the rule that hypodiploidy is not found in acute lymphocytic leukemia. From an analysis of the 170 patients with acute leukemia who had sequential bone nmrrow cytogenetic studies carried out in our laboratory (ntostly adults with acute ntyelogenous leukemia), we have made the following observatmns: 9 v,1. The incidence of aneuploidy was 40.5 per cent. Forty-six per cent of the karyotypes were hyperdiploid, 25 pet" cent were hypodiploid, and 29 per cent were pseudodiploid, l'seudodiploidy in acute leukemia has not been emphasized before, probal)ly because of the ditlicuhies involved in the meticulous analysis that is necessary for proper identitication. 2. Contrary to general opinion, certain aneuploid cytogenetic profiles have a tendency to occur in a repetitive fashion in genetically urn'elated patients. 3. When the three main categories of aneuploidy were considered separately, patients with hyperdiploidy and those with diploidy both survived fox"49 weeks (mean survival time), whereas patients with pseudodiploidy and hypodiploidy survived folonly 36 and 13 weeks, respectively. 4. Certain cytogenctic profiles carried prognostic implications. A group of 15 patients who had an extra C chromosome (+C) had a mean survival time of 68 weeks, and a group of 12 patients who had a complex cytogenetic profile (-C, +D, +E, - G ) also had a long mean survival time of 80 weeks. In contrast, groups of patients who (Text continued on tmtze 68"t.)

CIIROMOSOMAI.

A I . ' I ' E R A T I O N S IN I l U M A N

I.EUKEMIA--TRtJJILLO ETAL

B

A

S JO

d~

~A

a

I

~I

o'F

#A

IS

D

,

IL~

,, .

?.9 G

ii * X

Y

Figure 1. Top. Immature myeloid cell fi'mn B. W., a patient with acute myelogenous leukemia in tile initial disease state. There is a "bleb" on the surface of tim nucleus. Stlch "blebs" are identified a s lllenlb.ratle-l)ot!!ld pockets containing cytoplasm that extend beyond the normal contour of the tmclet,s and are thereby differentiated from various nuclear invaginatious that may be obse,ved in cells. (Xl0,000.) Bottom, idiogram prepared from an aliquot o f the same bone marrow sample, demonstrating a 45,XY,--C,+D,+E,--2G clone.

681

HUMAN I'ATHOLOGY--VOLUME

5, N U M B E R 6

November 1974

A

B

ll/~ ~S ~i~ ~~ C

D

-

682

-

F

E

G

X Y

Figure 2. Top, Typical Womyelo'cyte, illustrating tile absence oF tile nuclear bleb in tile remission state in patient B.W. (• Bottom, Normal diploid (46,XY) idiogram prepared from the same sample following remission induction.

CHROMOSOMAL

ALTERATIONS

IN HUMAN

I . E U K E M IA--TRUJILLO ETAL.

p. " o,-jil~ ~ -:

eL:';/'

ltJI A

B

:~ S# al XS C

/ ? 4 ~, & S ,

AA

D

F - -

a4

E

G ~

X

Y

Figure 3. Tol,, Myeloblast with ntlclear bleb frolll a |)()lie marrow sample of patient B.W. at the time of relapse. (x 10,250.) Bottom, hliogram demonstrating the return of the original-t5.XY,--C,+D.+E,--2(; clone in the relapse bone marrow of patient BAV.

683

IIUMAN I'ATHOLOGY--VOI.UME 5, NUMBER 6 November 197:t

684

had unstable karyotypes had a mean sur;.'ival time of only eight weeks. Confirmation of these findings will require furtimr study, but our data tend to contradict the previous assertion that ' chromosomal chafiges in huma,i leukemia are random events without major biological or clinical implications. 5. Our sequential cytogenetic studies Imve shown repeatedly that a favorable response to therapy is marked by earl)" disapl)earance of the aneuploid or pseudodiploid clone and by an increase ira the nt, mber of diploid cells. Conversely, the same abnormal clone reappears when the leukemia relapses. These findings indicate than when present, the chromosouml aherations define specifically the letikemic ceils. O u r studies also i0dicate that althougli the p r e s e n c e o f an occasional aneuploid cell in the bone marrow p,'obably does not have major significance, especially in o l d e r individuals, the existence of a well established pseudodiploid or aneuploid clone in the henmtopoietic tissue is highly indicative of a neoplastic process. 7 O u r laboratory ires also perfornaed electron microscopic studies on aliquots of tile bone marrow samples submitted for cytogenetic study. An important result of these studies is the finding in aneuploid leukemic bone marrow samples of a high frequency of an ultrastructural abnormality in the nuclei of immature granuloc)'tes? a This abnormality consists of blebs, pockets, or projections on the nuclear surface, which resemble those described I)y Achong and El)stein 44-4G as an inherent feature of the Burkitt lympholna. The nuclear abnormalities appear to be derived fiom excessive proliferation of the nuclear membrane. We have not observed such findings in the bone marrow cells of normal individuals or of patients with. solid minors studied betore therapy. In patients with acute myelogenous leukemia the presence of the nuclear blebs has ahvays been associated with the existence of an abnormal cytogenetic clone in the bone marrow cells (Fig. 1). In addition, both the nuclear bleb and the aneut)loid clone are found to disappear during clinical remission and to reappear at the time of relapse (Figs. 2, 3)? s The significance of these findings is not clear at

tile pregent time. Weber et al? r have reported the occnrl"ence of similar nuclear pockets ill pcripllcral blood cells of leukemic cattle, and in a recent study these investigators have shown that animals whose cells show an increased incidence of nuclear pockets produce C-type virus particles. 4s The belmvior of aneuploid clones in leukemic patients receiving chemotlml"apy has important clinical and therapeutic implications. Until recently the results of cytogenetic studies in hmnan leukemia have lent SUl)port to the exponential "cell kill" theory of the effect of chemotimrapeutic drugs on malignant cells? 'a This theory is valid only when applied to a cell population that has already undergone malignant transfbrmation. The cytogenetic data suggest that induction of remission in acute leukemia represents a marked re(luctio,a in tile number of malignant cells, as shown by tim inability to detect cytogenetically abnormal cells in remission.r,9, t.,,a0,a, Recurrence of the disease is always accompanied by r e a p pearance of the aneuploid leukemic cell line.r.,,, v, By contrast, treatment does not remove tim Pllt-positive cells from tim bone marrow of patients with chronic myelogenous leukemia, and the clinical improvement observed in treated patients mr, st be considered only a "pseudor e l l l i s s i o n . , , 2 , a , 50

Tim present modalities of treatment of human leukemia have been developed on tile basis of these observations, which suggest that relapse in acute leukemia results from a failure to eradicate all viable leukemic cells, which are eventually able to prolifex-ate and relmpulate the marrow. Several recent reports clmllenge tile validity of this concept. Cytogenetic studies in several patients with lmmatological malignant disorders who were treated with whole-body irradiation followed by infllsion of bone marrow fi'om an HL-A matched sibling have demonstrated the unexpected recm'rence of leukemia in tim donor type cellsY ''Sa So far, the number of reported cases is small, and tim phenomenon has been demonstrated only in cases of acute lymphocytic leukemia. However, if these observations are found to apply to other types of acute leukemia, the idea that duration of re-

CHROMOSOMAL

ALTERATIONS

fission represents the magnitude of cell ill by chemotherapeutic agents will no roger be tenable. If inalignant trans~rtnation of normal cells is a recurrent vent, the reappearance of the same aneu,loid clone during relapse becomes an ltriguing phenomenon, which suggests :rot in a particular host there is chromo~mai specificity of the oncogenic agent nd that involvement of specific areas of he genome may be a necessary requireaent for leukemogenesis.

REFERENCES !. llungerford, D. A.: Chromosome studies in human leukemia. I. Acute leukemia in children. J. Natl. Cancer..Inst., 27:983, 1961. 2. Court Brown, W. M., and Tough, I. M.: Cytogenetic studies in chronic myeloid leukemia. Adv. Cancer Res., 7:351, 1963. 3. Fitzgerald, P. II., Adams, A., and Cunz, F. W.: Chromosome studies in adult acute leukenfia. J. Natl. Cancer Inst., 32:395, 1964. '-t. Reisman, L. E., Zuelzer, W. W., and Thonll)son, R. I.: Further observation oil the role of animploidy ill acute leukemia. Cancer Res., 24:1448, 1964. 5. Krogh Jensen, M.: Cl'lromosonle stt,dies in acute lenkemk~. 111. Chronlosome constitution o[ bone marrow cells in 30 cases. Acta Med. Scand., 182:629, 1967. 6. Sandberg, A. A., Takagi, N., Sofuni, T., and Crosswhite, L. I1.: Chrolnosomes and causation of human cancer and let,kemia. V. Karyotypic aspccts of acute leukemia. Cnnccr, 22: 1668, 1968. 7. Trujillo, .]. M., Fernandez, M. N., ShuIlenberger, C. C., Rodriguez, L. !!., and Cork, A.: Cytogenetic contributions to tile study of htnnan leukemia. In l.enkemia-Lyml~homa (a collection of papers presented at the XIV Annual Clinical Confcrcnce on Cancer, 1969, at The University of Texas M. D. Anderson l lospital and Tumor Institute at l louston, Texas). Chicago, Year Book Mcdical l'nblishers, Inc., 1970, p. 105. 8. Whang-I'eng, J., Ilenderson, E. S., Knutscn, T., Freireich, E..1, and Gart, J. j.: Cytogenetic studies in acute myelocytic leukelnia with special emphasis on the occurrence of Pht chronlosome. Blood, 36:448, 1970. 9. Hart, J. S., Trujillo, J. M., Freireich, E. J., George, S. 1.., and Frei, E., II1: Cytogenctic stt,dies and their clinical correlates in aduhs with acute leukeinia. Ann. Intern. Med., 75: 353, 1971. 1O. Krogh Jensen, M.: Cytogenetic studies in acute myeloid leukemia. Acta Med. Scand., 190: 9t29, 1971. II. Fitzgerald, P. II., Crossen, I'. E., and Hamer, J. W.: Abnormal karyotypic clones in hnnmn acute lenkemiai their nature and clinical, significance. Cancer, 31:1069, I (173.

IN H U M A N

L E U K E M I A - - T R u j m n o ET AL.

12. Trujillo, J. M., Cork, A., Hart, J. S., George, S. L., and Freireich, E. J: Clinical implications of aneul)loid profiles ill adnh lettkemia. Cancer, 33:82-t, 1974. 13. Baikie, A. (;.: ('lu'omosonud aspects o1 leukemia. Ill The XI Congress of the International Society of Haematology, l'lenary Sessions, The. University of Sydney, Australia. Sydney, Victor C. N. Slight, Governnaent l'rinter, !(166, p. 198. 14. Dameshek, W., and Gunz, F.: l.euketnia and tl~e myeloproliferative disorders. In l)ameshek, W., and Culaz, F. (Editors): l.eukemia. Ed. 2. New York, Grune & Stratton, Inc., 196-t, p. 356. 15. Nowcll, P. C., and lhmgerford, D. A.: Chromosome studies in hu,nan leukemia. 11. Chronic granulocytic leukenfia, j. Natl. Cancer Inst., 27:1913, 1961. 16. Casperssnn, T., Gahrton, G., Lindsten, J., and Zech, L.: Identificatiou of the Philadellflfia chromosome as a number 22 b)" quinacrine mustard fluorescence analysis. Exp. Cell. Res., 63:238, 1970. 17. Sandberg, A. A., Ishihara, T., Crosswhite, 1- I!., and tlauschka, T. S.: Comparison of chromosome constitution in chronic myelocytic leukelnia and other m)'eloproliferative disorders. Blood, 20:393, 1962. 18. Fitzgerald, P. H., Adams, A., and Gttnz, F. X~'.: Chronic granuloc)tic leukemia and tile i'lliladelphia chromosome. Blood, 21:183, 1963. 9 19. Krauss, S., Sokal, J. E., and Sandberg, A. A.: Comparison of l'hiladelphia chronmsomepositive and -negative patients with chronic myelocytic lenkemia. Ann. Intern. Med., 61: 625, 196.t. 20. Tjio, J. H., Carbnne, P. I'., Wfiang, J., and Frei, E., III: The Philadelphia chromosome and. chronic myelogeuous leukemia.J. Natl. Cancer hast., 36:567, 1966. 21. Whang-peng, J., Canellos, G. P., Carbone, I'. 1'., and Tjio, J. II.: Clinical implication of cytogenetic variants in chronic myelocytic leukenfia (CMI.). Blood, 32:755, 1968. 22. Ezdinli, E. Z., Sokal, J. E., Crosswhite, I_, and Sandberg, A. A.: l'hiladclphia chromosomepositive and -negative chronic myelocytic leukcmia. Ann. Intern. Mcd., 72:175, 1970. 23. R,eisnmn, L. E., and Trnjillo, J. M.: ChrolfiC g~nmlocytic leukemia of childhood: clinical aud cytogenetic studies..]. I'cdiat., 62:710, 1963. 24. tlardisty, R. M., Speed, D. E., and Till, M.: Grannlocytic leukemia iu childhood. Brit. J. Haematol., 10:551, 1964. 25. Trujillo, J. M., and Ohno, S.: Chromosomal ahcration of erythropoietic cells in chronic myeloid leukemia. Acta llaematol., 29:311, 1963. 26. Whang, J., Frei, E., I11, Tjio, J. tt., Carbone, P. P., and Brecher, G.: The distribution of the i'hiladclphia chromosome in patients with chronic myelogenot, s leukemia. P,hmd, 22: 66-1, 1963. 27. Rastrick, J. 31.: A method for the positive identification of erythropoietic cells in chromosome preparations of bone marrow. Brit. J. tlaematol., 16:185, 1969.

685

HUMAN PATIIOLOGY--VOLUME

5, N U M B E R 6

28. TouglL I. M., Court Brown, W. M., Baikie, A. G., Buckton, K. E., Harnden, D. G., Jacobs, P. A., and Williams, J. A.: Chronic myeloid leukemia: cy(ogenetic studies before and after splenic irradiation. Lancet, 2:115, 1962. 29. Carbone, P. P., Tjio, J. H., Whang, J., Block, J. B., Kremei', W. B., and Fret, E., lIl: The effect of treatment in patients with chronic myelogenous leukemia: hematologic and c)togenetic studies. Ann. Intern. Med., 59:6'2'2, 1963.

30. Sandberg, A. A., Kikuchi, Y., and Crosswlfite, 9 L. H . : Mitotic ability of leukenfia leukocytes in chronic myelocytic leukemia. Cancer Res., 24:1468, 1964. 31. Kemp., N. 1t., Stafford, J. L., and Tanner, R.: Chromosome studies during earl)" and terminal chronic myeloid leukemia. Brit. Med. J., l:1010, 1964. 32. Fitzgerald, P. It.: A complex pattern of chromosome abhormalities in the acute phase of chronic granuloc) tic leukenfia. J. Med. Genet., 3:2.17, 1966. 33. Goh, K.: Cytogenetic studies in blastic crises of chronic myelocytic leukemia. Arch. Intern. Med., 120:315, 1967. 34. Spiers, A. S. D., and Baikie, A. G.: C)togenetic evolution and clonal population in acute transformation of chronic granulocytic leukenfia. Brit. J. Cancer., 22:192, 1968. 35. Vallejos, C. S., Trnjillo, J. M., Cork, A., Bodey, G. ,I'., and Freireich, E. J: Blastic crisis in cltronic granulocytic leukemia: experience in 2{9 cases. Cancer. In press. 36. Pedersen, B.: Cytogenetic structure of aneuploid blood cuhure cell populations during progression and treatment of chronic myelogenous leukemia. Acta PafltoL Microbiol. Scand., 69:192, 1967. 37. Pedersen, B.: Evolutionary trends of aneuploid blood culture cell polmlations during progression and treatment of chronic myelogenous leukemia. Acta Pafl2ol. Microbiol. Scand., 69:185, 1967. 38. l'cdcrscn, B.: lnlh, cnce of hypcrdiploidy on I'17 prevalence response to therapy in chronic myelogenous leukemia. Brit. J. tiaematol., 14: 507, 1968. ,39. Kay, It. E. M., l.awler, S. D., and Millard, R. E.: The chromosomes in pbl)cytl~enfia vera. Brit. J. Haemat01., 12:507, 1966. 40. Lawler, S. D., MiIlard, R. E., and Kay, 11. E. M.: 9 Further c)togenetical investigations in polycythaemia vera. Europ.J. Cancer, 6:223, 19709 41. Kiossoglou, K. A., Mitus, W. J., and Dameshek, W.: Cytogenetic studies in the chronic myelo-' proliferative s)'ndronte. Blood,'28:241, 1966. 42. Mitus, W. J., Coleman, N., and Kiossoglou, K.A.: Abnormal (nmrker) clsromosomes in two 15:1-

43.

44.

45.

46. 47.

48.

49.

50.

51.

52.

53.

November 1974 tients with acute myelofibrosis. Arch. Intern. Med., 123:192, 1969. Ahearn, M. J., Trujillo, J. M., Cork, A., Fowler, A., and Hart, J.: The association of nuclear blebs with aneuploidy in human acute letskemia. Cancer. In press. Epstein, M. A., and Achong, B. G.: Fine structure organization of human lymphoblasts of a tissue culture strain (EBb) from Burkitt's lymphoma. J. Natl. Cancer lns't., 34:24 l, 1966. Epstein, M. A., Barr, Y. M9 and Achong,.B.G.: The behavior and morphology of a second tissite cnhure strain (EB._,) of lymphoblasts from Burkitt's lympho,na. Brit. J. Cancer, 19:108, 19659 Achong, B. G., and Epstein, M. A.: Fine strnctore of the Burkitt tumor.J. Natl. Cancer Inst., 36:877, 1966. Weber, A., Andrews, J., Dickinson, B., Larson, V., Hammer, R., Dirks, V., Sorenson, D., and Frommcs, S9 Occurrence of smclear pockets in l)'mlfiSoc)'tes of normal, persistent lymphocytic and leukenfic adult cattle. J. Natl. Cancer Inst., 43:1307, 1969. Weber, A., Falming, M., Hammer, R9 t!., and Jessen, C.: Relationship between nuclear pockets in bovine peripheral blood lymphocytes and C-type virus particles in cuhures of these cells. J. Natl. Cancer Inst., 51:81, 1973. Skipper, tt. E., Sclmbel, F. M., Jr., and Wikox, W . S.: Experimental evah,ation of potential anticancer agents. XIV. Further study of certain basic concepts underlying chemotherapy of leukenfia. Cancer Chemother. Rep., 45:5, 1965. Hart, J. S., Shirakawa, S., Trujillo, J. M., and Fret, E., Ill.: The mechanism of induction of complete remission in acute myeloblastic letskenfia iq than. Cancer Res., 29:2300, 1969. Freireich, E.J., Bode)', G. 1'., ttart, J. S., Trujillo, J. M., ttersh, E. M., Curtis, J. E., Whitecar, J. P., and Fret, E., I I h l'otential for eradication of the cellular plmse of acute leukemia. Proceedings of the Sixth International Congress of Cllemotl~erapy. In l'rogress in Antimicrobial and Anticancer Chemotllerapy. Tokyo, Universityof Tokyo Press, 1971), Vol. 2, 15. 645. Fialkow, 1'. J., Bryant, J. I., Thomas, E. D., and Neinmn, 1'. E.: l.eukemic transfornmtion of engrafted human marrow cells in vivo. Lancet, 1:251, 1971. Thomas, E. D., Buckner, C. D., Rudolph, R. H.: Fefer, A., Storb, R., Neiman, 1'. E., Bryant, J. 1., Chard, R. L., Cliff, R. A., Epstein, R. B., Fialkow, I'. J., Funk, D. D., Giblctt, E. R., Lerner, K. G., Reynolds, F. A., and Slichter, S.: Allogeneic marrow graftiqg for hematologic malig,mncv using IIL-A matched donorreoplent ssblmg pmrs. Blood, 38:267, 1971.

M. D. Anderson Hospital and Tumor Institute 6723 Bertner Avenue ttouston, Texas 771)25 (Dr. Trujillo)

686